All procedures used in this study were approved by the Institutional Animal Care and Use Committee of Johns Hopkins University.

Hippocampal slices were prepared from Sprague-Dawley rats (Envigo International, Indianapolis, IN, USA) aged 8–16 days, when they are most susceptible to epileptiform bursting activity in slice electrophysiology experiments (Swann and Brady, 1984). Rat pups were deeply anesthetized by isoflurane inhalation and decapitated. Their brains were quickly removed and placed in pre-chilled and oxygenated low Ca²⁺/high Mg²⁺ cutting solution containing (in mM): 125 NaCl, 3 KCl, 1.25 NaH₂PO₄, 25 NaHCO₃, 0.25 CaCl₂, 10 MgSO₄ and 11 glucose, for ~1–2 min. One hemisphere was glued onto the platform of a specimen syringe and embedded with 1.6% low-melting point agarose (type I-B), and placed into a buffer tank filled with cutting solution. Coronal hippocampal slices (350 μm) were cut using a VF-300 compresstome (Precisionary Instruments Inc., Greenville, NC, USA), and transferred to a storage chamber filled with a holding solution (Feldmeyer et al., 2006) containing (in mM): 125 NaCl, 3 KCl, 1.25 NaH₂PO₄, 25 NaHCO₃, 0.5 CaCl₂, 5 MgSO₄ and 11 glucose, and continuously bubbled with 95% O₂ and 5% CO₂. Slices were allowed to recover for ~2 h, first at 34°C for ~45 min and thereafter at room temperature.

For recording, hippocampal slices were transferred to a submerged chamber and perfused with oxygenated artificial cerebrospinal fluid (aCSF) containing (in mM): 125 NaCl, 3 KCl, 1.25 NaH₂PO₄, 25 NaHCO₃, 1.3 CaCl₂, 1.3 MgSO₄ and 11 glucose, at 2 ml/min. For experiments eliciting epileptiform bursts and NMDA currents (see below), Mg²⁺ was omitted from aCSF and Ca²⁺ was elevated to 2 mM. All recordings were conducted at 32–33°C through a digital temperature control system (Scientifica, East Sussex, UK). To visualize and approach individual neurons, a PatchPro 6000 patch-clamp recording system (Scientifica Instruments, East Sussex, UK) was used. This system is comprised of a custom designed motorized microscope with infrared differential interference contrast (IR-DIC) components, light-emitting diode (LED) IR and fluorescence light sources, 40× water immersion objective and CCD camera, and two motorized micromanipulators sitting on top of a motorized movable base plate. The entire visualized patch-clamp procedure was monitored on a computer screen and operated remotely through a control panel and LinLab software (Scientifica Instruments, East Sussex, UK). Electrophysiological signals were acquired by a MultiClamp 700B amplifier, a Digidata-1550 digitizer, and Clampex 10.4 software (Molecular Devices, Sunnyvale, CA, USA). Single or dual whole-cell recordings, extracellular field-potential recording, or simultaneous field potential-whole cell recordings were conducted in hippocampal CA3. In some field-potential experiments, two slices were recorded simultaneously. Synaptic stimulation was delivered by a concentric bipolar electrode (FHC, Bowdoin, ME, USA) placed in the CA3 stratum radiatum 100–200 μm lateral to the recording electrodes, through a digital stimulus generator (STG 4002, Multichannel Systems, Reutlingen, Germany).

Recording pipettes were pulled from filamented thick-walled (outer diameter 1.5 mm, inner diameter 0.86 mm) borosilicate glass through a P-1000 pipette puller (Sutter Instruments, Novato, CA, USA). The electrodes typically have a resistance of 4–9 MΩ. For whole-cell current-clamp experiments, pipettes were filled with K-gluconate-based internal solution containing (in mM): 130 K-gluconate, 10 HEPES, 5.5 EGTA, 0.5 CaCl₂, 1 NaCl, 2 KCl, 1 MgCl₂, 10 phosphocreatine-tris, 0.5 Na-GTP, 2 Mg-ATP. The pH was adjusted to 7.25 with 5 M KOH. For whole-cell voltage-clamp experiments, pipettes were filled with Cs-methanesulfonate-based internal solution containing (in mM): 120 Cs-methanesulfonate, 10 HEPES, 5.5 EGTA, 0.5 CaCl₂, 5 NaCl, 10 phosphocreatine-tris, 0.5 Na-GTP, 4 Mg-ATP supplemented with Na⁺ channel blocker QX 314 (5 mM). The pH was adjusted to 7.25 with CsOH. The estimated liquid junction potentials between the bath medium and the K-gluconate-based or Cs-methanesulfonate-based pipette solution were 7 mV and 10 mV, respectively (calculated using junction potential calculator in PClamp 10, Molecular Devices, Sunnyvale, CA, USA), and were not corrected in the values presented in this article. For field-potential recordings, pipettes were filled with aCSF.

Population epileptiform bursts in CA3 were induced by Mg²⁺-free medium containing 50 μM 4-aminopyridine (4-AP), and recorded extracellularly in non-clamp mode (I = 0) with a gain of 100–200. Intrinsic firing of CA3 neurons was recorded in the whole-cell current-clamp mode and in the presence of AMPA-, NMDA- and GABA_A- receptor antagonists DNQX (10 μM), AP-5 (50 μM) and SR95531 (10 μM), to eliminate synaptic influences. Glutamatergic synaptic currents were evoked by electrical stimulation and recorded in whole-cell voltage-clamp mode holding at −65 mV. Stimuli were delivered at 0.05 Hz at an intensity of 150–300 μA, 100 μs, which was determined as 2–3 times threshold stimulation, defined as the minimal current stimulation (~50–75 μA) to evoke an EPSC. AMPAR-mediated excitatory postsynaptic current (EPSC_AMPA) was recorded in the presence of SR95531 (10 μM) and AP-5 (50 μM), while NMDAR-mediated excitatory postsynaptic current (EPSC_NMDA) was evoked in Mg²⁺-free medium and in the presence of SR95531 (10 μM) and DNQX (10 μM). GABA_AR-mediated current was evoked by local stimulation in the CA3 pyramidal cell layer to directly activated GABAergic neurons, and recorded in the presence of DNQX (10 μM) and AP-5 (50 μM) and held at 0 mV. Voltage-activated calcium current (I_Ca) was recorded in whole-cell voltage-clamp mode in the presence of SR95531 (10 μM), DNQX (10 μM) and AP-5 (50 μM). A ramp voltage command (−70 to +20 mV, 200–500 ms) and a series of stepwise voltage commands (−70 to +20 mV, 200–500 ms, step: 10-mV) were used to characterize voltage-dependent activation kinetics. For steady-state inactivation kinetics, the membrane potential was first clamped at different levels (−70 to +20 mV) for 1 s (i.e., pre-pulses) and then stepped to −10 mV (200 ms, test pulses). High voltage-activated I_Ca was evoked by a single voltage step from −70 mV to −10 mV (200–500 ms), repeated at 0.1 Hz. The series resistance (Rs) was compensated by 55%–75% and I_Ca was leak subtracted online using a P4 subtraction paradigm in Clampex 10 (Molecular Devices, Sunnyvale, CA, USA). In a subset of experiments, neurons were loaded with F1,6BP (5 mM) intracellularly via recording pipettes by adding F1,6BP to the internal solution. All signals were acquired at 10 kHz and low-pass filtered at 2 kHz. For analysis, field-potential population bursts were high-pass filtered at 1 Hz offline.

Qualitative and quantitative data analyses were conducted using Clampfit 10.4 (Molecular Devices, Sunnyvale, CA, USA), SigmaPlot 11 (SPSS Inc., Chicago, IL, USA), and Excel (Microsoft Corp., WA, USA) programs. The mean amplitude of epileptiform population bursts, EPSC_AMPA and EPSC_NMDA, GABA_A current and I_Ca and the mean frequency of neuronal firing were calculated from a 5-min recording before and after application of F1,6BP, and averaged across the recorded cells/slices in their respective groups. One-way analyses of variance (ANOVA) was used for statistical analysis across multiple groups, followed by the Holm-Sidak test for comparison between each two groups. Student’s t-test was used for comparisons between two groups. Data are presented as means ± SEM with statistical significance set at p < 0.05.

All chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA), except DNQX (di-sodium salt), which was obtained from Abcam (Cambridge, MA, USA).

F1,6BP has previously been shown to exhibit a prominent anticonvulsant effect in several animal models of acute seizures (Lian et al., 2007). However, no study to date has studied F1,6BP effects on epileptiform activity in vitro. We first tested the hypothesis that F1,6BP blocks epileptiform activity in the CA3 area of hippocampal slices. Epileptiform population bursts were consistently induced in 0 Mg²⁺ aCSF with 50 μM 4-AP. Similar to our previous study (Shao and Stafstrom, 2017), these bursts were typically “interictal-like” discharges consisting of 1–4 short bursts, lasting for 100–500 ms and occurring at variable frequencies (Figures 2A,B). Bath application of F1,6BP (2.5–5 mM) markedly suppressed epileptiform bursts in majority (13 of 19, 68%) of the slices (Figures 2A–D). This dosage of F1,6BP is similar to previous in vitro studies (3.5 mM, Vexler et al., 2003); 10 mM, (Izumi et al., 2003) and comparable to (but slightly lower than) in vivo studies (0.25–1 g/kg, (Lian et al., 2007); 1 g/kg, (Rogido et al., 2003)). More specifically, F1,6BP caused two types suppression: partial and complete. In partial suppression, epileptiform population bursts were minimized in amplitude by F1,6BP but did not disappear, and recovered quickly when F1,6BP was washed out from bathing medium (Figure 2A). In complete suppression, epileptiform bursts were fully abolished by F1,6BP and recovered much more slowly (up to 35 min; Figure 2B). Interestingly, single-unit activity in the slices was not blocked by F1,6BP (Figure 2B, middle inset). Overall, F1,6BP effectively inhibited epileptiform bursts activity in brain slices, corroborating its anticonvulsant effects in vivo (Lian et al., 2007). However, the rapid time to blockade with F1,6BP (4.2 ± 0.7 min, Figures 2A,B) was surprising, because it occurred faster than expected for an action mediated via an intracellular metabolic pathway (for comparison, glycolytic inhibition with 2-DG blocks similar epileptiform activity in CA3 in 14.2 ± 1.6 min; Shao and Stafstrom, 2017). Thus, we asked whether the quick F1,6BP effect might be mediated by an acute extracellular mechanism.

To explore cellular mechanisms of F1,6BP on neuronal excitability and seizure susceptibility, we next tested its action on intrinsic firing of CA3 pyramidal neurons, which are known to fire action potentials spontaneously (Wong and Prince, 1978; Hablitz and Johnston, 1981; Traub and Wong, 1983). To minimize synaptic influence on intrinsic neuronal firing due to the dense interconnections between CA3 neurons, particularly at young ages (Miles and Wong, 1986; Shao and Dudek, 2009), we blocked AMPA-, NMDA- and GABA_A receptors with DNQX (10 μM), AP-5 (50 μM) and SR95531 (10 μM), respectively. The average resting membrane potential (RMP) of the recorded CA3 neurons was −66 ± 1.3 mV (n = 9). Most of these neurons (6/9) fired action potentials spontaneously at their RMP; the other neurons (3/9) were manually depolarized to ~60 mV via current injection to fire action potentials. Under these experimental conditions, the neurons fired action potentials continuously at an average frequency of ~2–3 Hz, mostly in bursts (Figures 3A–D), persisting for prolonged periods (up to >60 min). Bath application of F1,6BP did not stop neuronal firing (Figures 3A–C); in fact, it tended to increase the firing frequency in some neurons (Figures 3B,C). Overall, F1,6BP did not significantly change the frequency of intrinsic firing of CA3 neurons (Figures 3B,D; 118 ± 28 spikes/min vs. 161 ± 33 spikes/min, p > 0.05, n = 9), suggesting that F1,6BP does not block epileptiform population bursts through inhibition of intrinsic firing mechanisms.

Next, we asked whether F1,6BP blocked glutamatergic transmission, and tested its effect on EPSC_AMPA and EPSC_NMDA separately. EPSC_AMPA was evoked in the presence of AP-5 (50 μM) and SR95531 (10 μM) to block NMDA and GABA_A receptors. The EPSC_AMPA was typically fast (~20 ms) with an amplitude of ~200 pA (Figure 4A). Addition of F1,6BP (5 mM) seemed to slightly reduce the amplitude of the EPSC_AMPA but did not cause overall significant reduction (Figures 4A–C,G; 206 ± 23 pA vs. 173 ± 31 pA, p > 0.05, n = 9). EPSC_NMDA was evoked in Mg²⁺-free solution in the presence of DNQX (10 μM) and SR95531 (10 μM) to block AMPA and GABA_A receptors, and held at −65 mV. The extracellular Ca²⁺ concentration was modestly elevated to 2 mM (from 1.3 mM) to partially compensate for membrane surface charge of Mg²⁺ (Hille et al., 1975) and reduce multi-synaptic responses. Compared with EPSC_AMPA, the EPSC_NMDA was usually larger and much slower (>100 ms; Figure 4D). Application of F1,6BP tended to decrease the amplitude of EPSC_NMDA but the reduction was not significant overall (Figures 4E–G, 248 ± 45 pA vs. 186 ± 24 pA, p > 0.05, n = 8). These data suggest that it is unlikely that F1,6BP blocks epileptiform bursts through reduction of glutamatergic transmission.

Further, we examined whether F1,6BP affects GABAergic transmission. GABA_AR-mediated currents were consistently evoked in the presence of DNQX (10 μM) and AP-5 (50 μM) by holding membrane voltage at 0 mV. Unlike its effect on glutamatergic synapses, bath application of F1,6BP reduced GABA current by ~50% (Figure 5A–D, 311 ± 27 pA vs. 161 ± 15 pA, p < 0.001, n = 6), suggesting that F1,6BP causes synapse-specific effects.

Given that two of the main contributors to epileptiform bursts, intrinsic neuronal firing and synaptic transmission, were not significantly altered by F1,6BP, it was reasonable to speculate that F1,6BP somehow acts through I_Ca, which is known to be essential for the generation of epileptiform bursts (Schwartzkroin and Wyler, 1980; Kohling et al., 2000; Siwek et al., 2012). To test this hypothesis, we evoked I_Ca in CA3 neurons using depolarizing voltage protocols (Figures 6A,E). I_Ca began to be activated at ~−40 mV and peaked at ~−10 mV (Figures 6E,F). I_Ca appeared to comprise a prominent inactivating component followed by a smaller steady-state component, and was completely abolished by the non-specific Ca²⁺ channel blocker, CdCl₂ (100 μM; Figures 6A,E). These data are consistent with a previous study that showed that immature CA3 neurons have significantly more prominent inactivating I_Ca than adult neurons (Thompson and Wong, 1991). The peak amplitude of I_Ca ranged from 400 pA to 1400 pA (on average: 996 pA, n = 9). Application of F1,6BP (5 mM) quickly (2–3 min) and consistently attenuated I_Ca by ~40% (Figures 6A–D, 996 ± 97 pA vs. 600 ± 62 pA, p < 0.001, n = 9). To test the whether F1,6BP can block I_Ca intracellularly, a subset of CA3 neurons were loaded with F1,6BP (5 mM) intracellularly through recording pipettes. In these neurons, the amplitude of I_Ca activated by the same protocol was similar to that in non-loaded neurons (Figure 6D, 938 ± 91 pA, n = 6; vs. 996 ± 97 pA, n = 9; p > 0.05). Moreover, further extracellular application of F1,6BP caused similar reduction of I_Ca in the F1, 6BP-loaded neuron (~40%, Figure 6D, 938 ± 91 vs. 598 ± 64, p < 0.001, n = 6). These data suggest that F1,6BP most likely blocks I_Ca extracellularly rather than intracellularly. F1,6BP did not seem to alter the voltage dependence of I_Ca as it was activated at the same voltage range before and during F1,6BP application (Figure 6F). Steady-state inactivation, assessed by the amplitude of I_Ca after 1 s-long pre-pulses holding at different voltage levels (Figure 6G), was also not altered by F1,6BP. In fact, the inactivation kinetics of I_Ca before and during F1,6BP were almost identical (Figure 6H). Altogether, these data suggest that F1,6BP blocks a significant portion of I_Ca, which may account for its anticonvulsant action on epileptiform bursts.

If it were true that F1,6BP blocks epileptiform bursts by reducing I_Ca, it would be expected that Ca²⁺ channel antagonists would also block epileptiform bursts as F1,6BP did. To confirm this, we applied CdCl₂, a non-selective Ca²⁺ channel antagonist. As expected, a low concentration of CdCl₂ (20 μM) completely and reversibly blocked epileptiform bursts (Figures 7A–C). Similar to F1,6BP, CdCl₂ did not block the single-unit activities in the slices (Figures 2B, 7A, middle insets). Taken together, these data confirm that the epileptiform bursts in CA3 are Ca²⁺-dependent and can be blocked by Ca²⁺ channel blockers or modulators (such as F1,6BP).

Our data in hippocampal slices corroborates the pioneering work showing that F1,6BP inhibits seizures in animal models (Lian et al., 2007). As in that study (Lian et al., 2007), we initially hypothesized that the anti-seizure activity of F1,6BP was mediated by an intracellular metabolic effect of shifting glycolysis to PPP (Figure 1), thus promoting the production of the endogenous antioxidant and anticonvulsant, GSH (Lian et al., 2007). Yet, we noticed that the time to block epileptiform bursts by F1,6BP (4.2 ± 0.7 min) was much quicker than epileptiform bursts blocked by the glycolytic inhibitor 2-DG in our previous study (14.2 ± 1.6 min; Shao and Stafstrom, 2017). The short time to blockade suggested that F1,6BP might have a direct extracellular effect independent of its assumed intracellular effect and prompted us to examine its effect on extracellular mechanisms that contribute to neural excitability. However, F1,6BP did not stop neuronal firing nor did it significantly reduce excitatory synaptic transmission, two of the major contributors to epileptiform bursting (Traub and Wong, 1983). Finally and unexpectedly, we discovered that F1,6BP consistently blocks a significant portion of voltage-activated I_Ca extracellularly. Since epileptiform bursts in CA3 are Ca²⁺-dependent and readily blocked by Ca²⁺ channel blockade (CdCl₂), these data strongly suggest that F1,6BP achieves its anticonvulsant activity in part by attenuating Ca²⁺ influx, at least in our in vitro seizure model.

Epileptiform bursts may be mediated by both presynaptic and postsynaptic Ca²⁺-dependent mechanisms. Presynaptic Ca²⁺ influx is important for transmitter release. Postsynaptic (somatic) I_Ca is known to be essential for the genesis and maintenance the plateau potentials of epileptiform bursts (Schwartzkroin and Wyler, 1980; Kohling et al., 2000; Siwek et al., 2012). Our data that F1,6BP did not cause significant reduction in EPSCs suggest that F1,6BP blocks epileptiform bursts mainly by reducing postsynaptic (somatic) rather than presynaptic Ca²⁺ influx; and that a moderate reduction (40%) of postsynaptic Ca²⁺ influx is sufficient to block seizures_. The 40% reduction of Ca²⁺ influx is derived from somatic high-voltage activated (HVA) Ca²⁺ currents. Whether presynaptic Ca²⁺ entry is similarly reduced by F1,6BP is unclear. Possibly, Ca²⁺ channels at presynaptic terminals of glutamatergic synapses may be less sensitive to F1,6BP or F1,6BP might block certain types Ca²⁺ channels that are not coupled to glutamate release (Turner et al., 1992; Dunlap et al., 1995). In contrast, Ca²⁺ influx at the GABAergic presynaptic terminals is likely affected by F1,6BP as indicated by the reduction of GABA_A current. These data suggest that F1,6BP differentially affects glutamatergic and GABAergic transmission. At the developmental period in this study (P8–16), the action of GABA on CA3 neurons is believed to have switched from excitatory (P0–P6) to inhibitory (Ben-Ari et al., 1990). However, the roles of GABAergic inhibition in seizures are complex. Recent studies have demonstrated that selective activation of perisomatic targeting interneurons (such as parvalbumin-expressing neurons) leads to initiation of seizures (Toyoda et al., 2015; Yekhlef et al., 2015; Khoshkhoo et al., 2017), while activation of axonic targeting interneurons (or axon-axonic synapses) inhibits seizures (Wang et al., 2014). In this study, we stimulated the CA3 pyramidal layer, where the axons of perisomatic interneurons are located, thus the resulting IPSCs are mostly derived from perisomatic synapses. A reduction in the strength of this type of inhibition by F1,6BP might therefore contribute to the reduction of epileptiform activity. Whether F1,6BP exerts additional effects on intracellular metabolic pathways in our model remains to be determined. Apparently, the mechanisms underlying the anticonvulsant action of F1,6BP in animal models (Lian et al., 2007) are likely to be more complex than in brain slices. The extent to which this extracellular mechanism contributes to F1,6BP’s in vivo anticonvulsant action is uncertain. Arguably, administration of antioxidants does not consistently block seizures in the same animal models (Xu and Stringer, 2008), which suggests that F1,6BP may act on other targets in addition to PPP activation and GSH production. Nonetheless, the present study revealed a novel extracellular mechanism of F1,6BP that may explain its anticonvulsant activity, at least in this in vitro model of seizures.

CA3 neurons in the developing hippocampus possess both HVA and low-voltage activated (LVA) Ca²⁺ channels (Thompson and Wong, 1991). It is generally recognized that Ca²⁺ influx through HVA channels (particularly L- and R-types) are critically involved in the genesis of the plateau potential of epileptiform bursts (Straub et al., 2000; Siwek et al., 2012), while Ca²⁺ influx through LVA channels (i.e., T-type) contributes to rebound bursting and pacemaking activity (Siwek et al., 2012). The major component of I_Ca in the present study resembles the HVA inactivating currents (R, N-, P/Q) with an activation threshold of −40 mV and fast decay kinetics (Yamakage and Namiki, 2002; Lacinová, 2005; Catterall, 2011), mixed with a smaller component of non-inactivating current (L-type). This is consistent with a previous study showing that inactivating I_Ca contributed to a significantly larger percentage of the total I_Ca in immature than adult CA3 neurons (Thompson and Wong, 1991). F1,6BP reduces the amplitude of I_Ca without altering channel activation and inactivation kinetics, suggesting that F1,6BP does not bind to HVA Ca²⁺ channels and works in a way that is different from conventional Ca²⁺ channel blockers. We did not further differentiate which specific subtypes of HVA Ca²⁺ channel are blocked by F1,6BP nor did we evaluate the effect of F1,6BP on LVA (T-type) I_Ca, which is beyond the main focus of the present study and may be addressed in future studies. Alternatively, F1,6BP may reduce Ca²⁺ influx by chelating Ca²⁺ ions because of its negative charges (Hassinen et al., 1991). A reduction of Ca²⁺ influx may also affect neuronal firing (by reducing Ca²⁺-activated K⁺ currents). Our data show that F1,6BP tends to increase neuronal firing but this effect did not reach statistical significance.

F1,6BP has previously been shown to be neuroprotective against hypoxic-ischemic injury (Farias et al., 1990; Kelleher et al., 1995; Sola et al., 1996) and excitotoxic injury (Rogido et al., 2003) by stabilizing intracellular Ca²⁺ mediated by activation of phospholipase C (PLC; Donohoe et al., 2001; Fahlman et al., 2002; Rogido et al., 2003). Whether this mechanism occurs in seizures is not known. Also, we do not completely rule out the possibility that F1,6BP acts through an intracellular metabolic pathway (i.e., PPP), in addition to its action on Ca²⁺ channels. Theoretically, with its negative charge and lack of known transporter, it may be difficult for exogenous F1,6BP to enter cells, but several studies have shown a specific membrane permeability for F1,6BP (Hardin and Roberts, 1994; Wheeler et al., 2004; Wheeler and Chien, 2012).

Manipulation of neurometabolism exerts broad action and mechanisms influencing neural excitability and seizures. For instance, the anti-seizure effects of the KD may involve activation of adenosine receptors (Kawamura et al., 2010, 2016) and K_ATP (Ma et al., 2007; Lutas and Yellen, 2013), production of ketone bodies that inhibit transmitter release (Juge et al., 2010) or alteration of mitochondrial activity (Kim et al., 2015). Glycolytic inhibition with 2-DG not only blocks acute epileptiform activity in brain slices and animal models (Stafstrom et al., 2009; Forte et al., 2016; Shao and Stafstrom, 2017), but also interrupts the chronic process of epileptogenesis (Garriga-Canut et al., 2006; Stafstrom et al., 2009). Other metabolic interventions include disruption of the glial-neuronal energy coupling by targeting the so-called “astrocyte-neuron lactate shuttle” (Belanger et al., 2011) which also effectively blocks seizure activity (Sada et al., 2015). The efficacy of anticonvulsant activity of F1,6BP first reported by Stringer and colleagues and Lian et al. (2007) was quite remarkable in that it not only blocked seizures in all three animal models tested (KA, pilocarpine, PTZ), but also exceeded the treatment efficacy of 2-DG, valproic acid and KD. Therefore, F1,6BP appears to be a promising antiepileptic agent. The present study reveals an unexpected extracellular effect of F1,6BP, effectively blocking seizures by attenuating I_Ca without disrupting normal physiological function (neuronal firing and excitatory synaptic transmission), which is desirable for clinical anti-seizure treatment. Moreover, its neuroprotective effect against hypoxic-ischemic injury would be additionally beneficial to patients, as seizures are often accompanied by or caused by hypoxic-ischemic injury, particularly in neonates (Kang and Kadam, 2015). Also, it is important to note that as a natural intracellular glucose metabolite, F1,6BP exerts very little toxicity even at a dose eight times larger than its effective dose (Vexler et al., 1999). All these features of F1,6BP makes it an attractive candidate for an effective and safe antiepileptic agent.

In summary, metabolic interventions represent a novel strategy for treatment of drug-refractory seizures. We have identified a novel mechanism of the promising metabolic anticonvulsant, F1,6BP, which may provide new insight into its anti-seizure and/or neuroprotective activity.