Neurons, which are connected by synapses, exhibit electrical activities in the living brain. The electrical activities in neurons are generated by ion channels and synaptic receptors. Although these electrical activities are essential for normal brain function, they become excessive in the brains of epileptic patients.
Therefore, medicines for epilepsy have to suppress the neuronal hyperexcitation, and currently-used antiepileptic drugs have been designed to act on ion channels and synaptic receptors (Meldrum and Rogawski, 2007). For example, carbamazepine and lamotrigine inhibit voltage-dependent Na+ channels, gabapentin inhibits the α2δ subunits of L-type Ca2+ channels, phenobarbital activates GABAA receptors, and perampanel modulates AMPA-type glutamate receptors (reviewed in Bialer and White, 2010).
However, these antiepileptic drugs are not effective for all epileptic patients. Approximately 1% of the world’s population suffer from epilepsy, and one-third of epileptic patients are resistant to currently-available antiepileptic drugs (Kwan and Brodie, 2000). It should be noted that the diet treatment using the ketogenic diet is effective for some patients with drug-resistant epilepsy (Freeman et al., 1998; Neal et al., 2008).
Since these reviews have introduced many antiseizure mechanisms associated with the metabolic, electrical, epigenetic and inflammatory changes induced by the ketogenic diet, we do not summarize these mechanisms thoroughly in the present review. We here summarize electrophysiological studies that focus on electrical control in neurons by the ketogenic diet.
KETOGENIC DIET FOR DRUG-RESISTANT EPILEPSY
The ketogenic diet treatment was developed in the 1920s, and its concept dates back to biblical times (Wheless, 2008). The diet treatment was originally developed by Dr. Wilder at the Mayo Clinic in 1921, and its modified version using a medium-chain triglyceride (MCT) diet was developed in the 1970s (Huttenlocher et al., 1971).
These ketogenic diets are high-fat and low-carbohydrate diets, which produce ketone bodies (β-hydroxybutyrate and acetoacetate) by the liver. Ketone bodies are then delivered to the brain and used as alternative energy sources to glucose.
The ketogenic diet not only increases ketone bodies, but also mildly decreases blood glucose levels in epileptic patients (Huttenlocher, 1976). These two metabolic changes are now recognized to exert antiseizure effects (reviewed in Rho, 2017). The direct actions of ketone bodies were recently reviewed in detail (Simeone et al., 2018).
Historically, although the ketogenic diet treatment was developed in 1921, its use in clinical setting decreased after the development of the antiepileptic drug diphenylhydantoin (phenytoin) in 1938 (Merritt and Putnam, 1938). However, the diet treatment re-attracted attention in the 1990s because it was shown to be effective for patients with drug-resistant epilepsy (Freeman et al., 1998).
MOLECULES FOR ELECTRICAL CONTROL BY THE KETOGENIC DIET
As described above, the ketogenic diet is now established as a treatment for patients with drug-resistant epilepsy. Since seizures are elicited by the hyperexcitation of electrical activities in neurons, it is mechanistically presumed that the ketogenic diet acts on the molecules generating electrical currents, such as ion channels and synaptic receptors.
This issue can be directly addressed using electrophysiological techniques in rodents. We therefore introduce electrophysiology-based studies, in which the following seven molecules have been identified as regulators of neuronal electrical activities by the ketogenic diet.
ATP-Sensitive K+ Channels
The ketogenic diet consists of high-fat and low-carbohydrate, and mainly elicits two metabolic changes in blood: increases in ketone bodies and decreases in glucose (Bough et al., 2006). In rodent studies, the ketogenic diet was shown to increase the blood plasma level of β-hydroxybutyrate to 1–8 mM (Bough and Eagles, 1999; Bough et al., 1999, 2006), which is similar to that in humans (Neal et al., 2009).
Using electrophysiological techniques, Yellen and colleagues found adenosine 5’-triphosphate (ATP)-sensitive K+ channels (KATP channels) as the molecule that ketone bodies acted on (Ma et al., 2007). KATP channels are known to be metabolic sensors that regulate electrical activities, which are blocked by intracellular ATP. Ma et al. (2007) proposed that ketone bodies increase intracellular global ATP, but decrease glycolytic ATP production, which leads to decreases in ATP near the plasma membrane, the opening of KATP channels, and reductions in the firing rate of neurons. Consistent with this, the opening of KATP channels by ketone bodies was directly demonstrated by single channel recordings (Tanner et al., 2011).
Voltage-Dependent Ca2+ Channels
Voltage-dependent Ca2+ channels (VDCCs) in presynaptic terminals are known to be essential for synaptic transmission (Zucker and Regehr, 2002; Catterall et al., 2013). By using patch-clamp recordings from hippocampal slices, Inoue and colleagues demonstrated that acetoacetate inhibited Ca2+ influx through VDCCs in CA1 pyramidal cells.
They also found that acetoacetate modulated the short-term synaptic plasticity of excitatory postsynaptic currents (EPSCs) in pyramidal cells, showing that it acted on presynaptic VDCCs. Interestingly, acetoacetate reduced EPSCs in slices exhibiting epileptiform activities, but not in normal slices (Kadowaki et al., 2017), suggesting that the effects of acetoacetate preferably emerge in the hyperexcitable state of the brain.
Although its VDCC types remain unclear, it is probably mediated by P/Q-type (Cav2.1) or N-type (Cav2.2) Ca2+ channels, because these Ca2+ channels mainly contribute to synaptic transmission. A previous study reported that β-hydroxybutyrate inhibited N-type Ca2+ channels in rat sympathetic neurons of the peripheral nervous system.
Vesicular Glutamate Transporters
Vesicular glutamate transporters (VGLUTs), which fill synaptic vesicles with glutamate, critically regulate excitatory synaptic transmission in the brain (Fremeau et al., 2004). Thus, VGLUTs are also electrical regulators that indirectly affect postsynaptic glutamate receptors.
Using a proteoliposome containing purified VGLUTs, Moriyama and colleagues demonstrated that acetoacetate was a specific inhibitor of VGLUTs (Juge et al., 2010). Although VGLUTs were activated by Cl− ions, the elevated VGLUT activities were inhibited by acetoacetate: therefore, they proposed that VGLUTs are oppositely regulated by Cl− ions and acetoacetate. Functionally, acetoacetate inhibited miniature EPSCs in CA1 pyramidal cells of hippocampal slices and suppressed acute seizures in rats in vivo (Juge et al., 2010).
Adenosine A1 Receptors
Adenosine is an inhibitory neuromodulator, which acts on four types of adenosine receptors in the brain (A1, A2A, A2B and A3; Dunwiddie and Masino, 2001). Among them, the activation of adenosine A1 receptors (A1Rs) was shown to suppress chronic seizures in a mouse model of pharmacoresistant mesial temporal lobe epilepsy (Gouder et al., 2003).
Boison and colleagues found that the antiseizure effects of the ketogenic diet were not observed in knockout mice of adenosine A1Rs (Masino et al., 2011). Furthermore, the antiseizure mechanism is presumably attributed to the decreases in glucose by the ketogenic diet.
By using patch-clamp recordings from hippocampal slices, Masino and colleagues revealed that the decreases in glucose hyperpolarized hippocampal pyramidal cells, and this was mediated by adenosine A1Rs and KATP channels (Kawamura et al., 2010). They further found that the decreases in glucose reduced the neural excitability in mice fed the ketogenic diet, and this was also mediated by adenosine A1Rs and KATP channels (Kawamura et al., 2014).
AMPA-Type Glutamate Receptors
The MCT ketogenic diet is a modified version of the classical ketogenic diet (Huttenlocher et al., 1971), and increases not only ketone bodies but also two fatty acids (octanoic and decanoic acids) in the blood of epileptic patients (Haidukewych et al., 1982; Sills et al., 1986). Octanoic and decanoic acids are straightchain saturated monocarboxylic acids with 8 and 10 carbons, respectively.
These medium-chain fatty acids directly penetrate the blood-brain barrier (Oldendorf, 1973). Walker, Williams, and colleagues reported that decanoic acid, but not octanoic acid, suppressed in vitro epileptiform activity in entorhinal cortexhippocampus slices (Chang et al., 2013). They also elucidated the underlying mechanism, in which decanoic acid reduced EPSCs via AMPA-type glutamate receptors (Chang et al., 2016).
By using electrophysiological recordings from an oocyte expression system, they showed that decanoic acid inhibited glutamateinduced currents derived from various types of AMPA receptor subunits (GluA1, GluA1/2 and GluA2/3), and also showed that it was the most sensitive to GluA2/3 (Chang et al., 2016). BCL-2-Associated Agonist of Cell Death BCL-2-associated agonist of cell death (BAD) is a member of the BCL-2 family, which is known to regulate cellular metabolism (Giménez-Cassina and Danial, 2015).
Glucose is directly transported into neurons and used as an energy source. As an alternative metabolic pathway, glucose is transported into astrocytes and converted to lactate, which is then released to extracellular spaces and transported into neurons: this metabolic pathway is called the astrocyte-neuron lactate shuttle (Bélanger et al., 2011). Lactate is suggested to be a preferred energy source over glucose in the brain (Larrabee, 1995; Smith et al., 2003). This astrocyte-derived lactate regulates electrical activities in neurons (Rouach et al., 2008; Parsons and Hirasawa, 2010).
Based on these backgrounds, Inoue and colleagues demonstrated that the astrocyte-neuron lactate shuttle contributes to neuronal inhibition and seizure suppression by the ketogenic diet (Sada et al., 2015). By using slice patch-clamp recordings, they found that ketogenic-like metabolic changes induced hyperpolarization in neurons, which was recovered by the activation of the lactate shuttle.
DRUG DEVELOPMENT BASED ON THE KETOGENIC DIET
The ketogenic diet treatment requires strict dietary control. Since medicines are markedly easier to administer, antiepilepticdrugs based on the ketogenic diet will be useful for epileptic patients.
Historically, the ketogenic diet treatment developed in the 1920s shifted to drug therapy using phenytoin in the late 1930s (Wheless, 2008). Several approaches have been reported toward the development of ketogenic diet-based antiepileptic drugs. The first approach is ketone supplementation without dietary control.
Author: Nagisa Sada and Tsuyoshi Inoue