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Summary: Purpose: Previously we demonstrated that l-(+)-β-hydroxybutyrate (L-BHB), acetoacetate (ACA), acetone, and dibenzylamine (DBA) were anticonvulsant in an audiogenic seizure–susceptible model, and that DBA was a bioactive contaminant identified in commercial lots of L-BHB. In the present study, we asked whether these effects could be mediated by ionotropic glutamate or γ-aminobutyric acidA (GABAA) receptors.
Methods: We studied the effects of both stereoisomers of BHB (as well as the racemate), ACA, and DBA on N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5methyl-4-isoxazole-proprionic acid (AMPA), and GABAA receptors in cultured rodent neocortical neurons by using whole-cell voltage-clamp recording techniques.
Results: Only L-BHB and DBA exerted a concentration- and voltage-dependent block of NMDA-evoked currents, whereas none of the tested substrates affected AMPA- or GABA-activated currents. The kinetics of whole-cell block by L-BHB and DBA were similar, providing additional evidence that DBA is responsible for the anticonvulsant activity of L-BHB.
Conclusions: BHB and ACA do not exert direct actions on GABAA or ionotropic glutamate receptors in cultured neocortical neurons. In addition, we provide additional evidence that DBA is responsible for the anticonvulsant activity of L-BHB, and that this action may be mediated in part by voltage-dependent blockade of NMDA receptors.
During a prolonged (i.e., >48 h) fast, hepatic fatty acid oxidation and ketone body production provide alternative energy sources, especially for the brain. Ketogenesis, resulting primarily in the formation of β-hydroxybutyrate (BHB; 3-hydroxybutyrate or 3-hydroxybutanoic acid), and acetoacetate (ACA), and to a minor degree acetone, also is observed during diabetic ketoacidosis and a high-fat, low-carbohydrate diet known as the ketogenic diet (KD). The KD is an effective nonpharmacologic treatment for patients with intractable epilepsy (1,2). Despite >80 years of clinical experience with the KD, the mechanisms underlying its anticonvulsant actions remain poorly understood.
Recently attention has again focused on the possibility that one or more ketone bodies may modulate neuronal excitability. Although it has been observed that ACA and acetone possess anticonvulsant properties in vivo (3–5), the mechanisms underlying these effects are unknown; whether BHB, the principal ketone body, exerts similar effects is less clear. The major unresolved question is whether ketone bodies can directly affect the excitability of neuronal membranes through actions on ion channels that are the targets of most anticonvulsant medications (AEDs). Thus far, BHB and ACA do not appear to affect synaptic activity directly, at least not in the hippocampus (6).
Previously we demonstrated that l-(+)-BHB exhibits anticonvulsant activity in audiogenic seizure–susceptible mice, but that this was likely due to dibenzylamine (DBA), a chemical contaminant contained within commercial lots of this stereoisomer (4). Further, we provided evidence that ACA and acetone also were anticonvulsant in this model. To determine whether this in vivo activity could be correlated with actions on postsynaptic ligand-gated ion channels, we studied the effects of d-(−)-BHB, l-(+)-BHB, DL-BHB, ACA, and DBA on N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA), and γ-aminobutyric acidA (GABAA) receptors in cultured mouse and rat neocortical neurons by using whole-cell voltage-clamp recording techniques. We found that L-(+)-BHB exerted a concentration- and voltage-dependent block of NMDA-evoked currents, and provided additional evidence that the in vivo anticonvulsant activity of this isomer is due to DBA.
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By using whole-cell voltage-clamp recording techniques and a rapid drug-perfusion system, we tested the actions of both stereoisomers and racemate of BHB, ACA, and DBA on NMDA, AMPA, and GABAA receptors in rodent cultured neocortical neurons. We found that both L-BHB and DBA (a contaminant in commercial lots of L-BHB) blocked NMDA-activated currents in a concentration- and voltage-dependent manner. In extending our earlier observations (4), we herein provide additional evidence that the in vivo anticonvulsant activity of L-BHB is due to DBA alone.
Given the strong (but not universal) correlation between blood ketone levels and seizure control, it is important to determine whether ketones can directly modulate neuronal excitability and/or synchronization. We previously demonstrated that BHB, the major ketone moiety, is not directly anticonvulsant, at least in an audiogenic seizure–susceptible model (4). Furthermore, we found that, in cultured neocortical neurons, neither BHB nor ACA directly interacts with either GABAA or ionotropic glutamate receptors, the principal molecular targets of many AEDs (12–14).
Our data are consistent with the study by Thio et al. (6), who applied standard cellular electrophysiologic techniques to evaluate the direct effects of ketone bodies in hippocampal synaptic transmission. In their hands, short-term application of BHB and ACA did not affect (a) excitatory postsynaptic potentials (EPSPs) and population spikes in CA1 pyramidal neurons after Schaffer collateral stimulation; (b) spontaneous epileptiform activity in the hippocampal–entorhinal cortex slice seizure model; and (c) whole-cell currents evoked by glutamate, kainate, and GABA in cultured hippocampal neurons.
The major ketone BHB is structurally related to GABA; thus it has been speculated that BHB (and possibly ACA) may exert direct modulatory effects on GABA receptors. The evidence thus far fails to support this notion. This is not surprising, given that BHB does not contain important amine moieties critical for GABA-receptor binding (15). Nevertheless, Niesen et al. (16) presented preliminary data supporting the direct anticonvulsant actions of BHB and ACA. In their hands, BHB (150 mg/kg), administered in the short term, was effective in blocking pentylenetetrazol (PTZ)-induced seizures in adolescent male Wistar rats (16). In acute and cultured hippocampal slices, 1–3 mM BHB or ACA decreased the amplitude and number of multiple CA1 population spikes induced by four different proconvulsant conditions: 8 mM extracellular KCl, 100 μM 4-aminopyridine, 50 μM bicuculline, and a “rapid kindling” paradigm (17). Finally, in intracellular recordings of immature CA1 neurons from cultured rat hippocampal slices, BHB (0.03–3 mM) potentiated evoked early inhibitory postsynaptic potentials (IPSPs), suggesting an action on postsynaptic GABAA receptors (18).
In the current study, we found that neither isomer of BHB (through a concentration range of 30 μM to 30 mM) nor ACA changed the amplitude of whole-cell currents evoked by 3 μM GABA, consistent with the observations of Thio et al. (6). It is possible that BHB may preferentially modulate specific molecular isoforms of GABAA receptors found in CA1 hippocampal neurons but not in neocortex, but this is highly unlikely because pyramidal cells in both neocortex and hippocampus exhibit a similar spectrum of GABAA-receptor subtypes (19,20). In the studies reported by Niesen's group, it is unclear which stereoisomers of BHB were used. It is possible that the effects of BHB seen by these investigators may be due to block of presynaptic K+channels by DBA, as suggested by Doepner et al. (21,22), resulting in increased presynaptic release of neurotransmitter (i.e., GABA). This possibility, however, has yet to be confirmed. Doepner et al. (23) recently demonstrated that DBA blocked voltage-dependent K+ currents in mouse cardiac myocytes, specifically inhibiting the slow component of the recovery from inactivation.
For the current investigations, we chose not to study the effects of acetone on GABAA and ionotropic glutamate receptors because its extreme volatility renders its use in electrophysiologic experiments challenging at best. This is despite reports of solvents such as dimethylsulfoxide (DMSO) affecting GABA- and glutamate-activated currents in cultured neurons (24,25). Although we cannot exclude the possibility of direct effects of acetone on membrane-bound ion channels, no data support this hypothesis. The anticonvulsant efficacy of acetone has been recently confirmed in animal models (4,5), but its underlying mechanisms have yet to be established.
DBA is a benzaldehyde derivative whose clinical import stems from its use as a pharmacologic vehicle for certain antibiotics (26) and as a biologically active contaminant identified in commercial preparations of l-(+)-β-hydroxybutyrate (4,22). Although it is a novel compound with promising anticonvulsant properties, it is labeled as a toxic, potentially carcinogenic substance in the Environmental Protection Agency (EPA) inventory under the Toxic Substances Control Act (TSCA), and thus is a poor candidate for further preclinical and clinical development. Nevertheless, our finding that DBA is a biologically active contaminant in L-BHB is not the first (nor likely the last) instance of an unexpected compound possessing anticonvulsant properties. It is well known that the anticonvulsant activity of valproic acid (VPA) was serendipitously discovered after it was used as a vehicle to dissolve investigational compounds (27).
In summary, we have shown that DBA, a contaminant in L-BHB, is a voltage-dependent blocker of NMDA receptors. Consistent with what has been previously reported in hippocampus (6), we also demonstrated that BHB and ACA do not directly modulate GABAA or ionotropic glutamate receptors in cultured neocortical neurons. In extending our earlier work (4), we provided additional evidence that the in vivo anticonvulsant activity of L-BHB is due to DBA alone. The role of ketone bodies as anticonvulsant effectors and/or mediators of the KD remain to be clarified. Given the prominent role of ketone bodies in intermediary metabolism, there are likely other novel mechanisms through which they may exert an anticonvulsant, and potentially neuroprotective, effect (28–30).