Alteration of intracellular pH and activity of CA3-pyramidal cells in guinea pig hippocampal slices by inhibition of transmembrane acid extrusion

Transmembrane acid extruders, such as electroneutral operating Na(+)/H(+)-exchangers (NHE) and Na(+)-dependent Cl(-)/HCO(3)(-)-exchangers (NCHE) are essential for the maintenance and regulation of cell volume and intracellular pH (pH(i)). Both of them are hypothesised to be closely linked to the control of excitability. To get further information about the relation of neuronal pH(i) and activity of cortical neurones we investigated the effect of NHE- and/or NCHE-inhibition on (i) spontaneous action potentials and epileptiform burst-activity (induced by bicuculline-methiodide, caffeine or 4-aminopyridine) and (ii) on pH(i) of CA3-neurones. NHE-inhibition by amiloride (0.25-0.5 mM) or its more potent derivative dimethylamiloride (50 microM) and NCHE-inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 0.25-0.5 mM) induced a biphasic alteration of neuronal activity: an initial, up to 30 min lasting, increase in frequency of action potentials and bursts preceded a growing and partially reversible suppression of neuronal activity. In BCECF-loaded neurones the pH(i), however, continuously decreased during either amiloride- or DIDS-treatment and reached its steady-state (DeltapH(i) up to 0.3 pH-units) when the neuronal activity was markedly suppressed. Combined treatment with amiloride (0.5 mM) and DIDS (0.5 mM) or treatment with harmaline alone (0.25-0.5 mM), which also continuously acidified neurones via inhibition of an amiloride-insensitive NHE-subtype, induced a monophasic and partially reversible suppression of neuronal activity. As an initial excitatory period failed to occur during combined NHE/NCHE-inhibition we speculate that its occurrence during amiloride- or DIDS-treatment resulted rather from disturbances in volume- than in pH(i)-regulation. The powerful inhibitory and anticonvulsive properties of NHE- and NCHE-inhibitors, however, very likely based upon intracellular acidification - as derived from our previous findings that a moderate increase in intracellular free protons is sufficient to reduce membrane excitability of CA3-neurones.     

Intracellular acidification alters myogenic responsiveness and vasomotion of mouse middle cerebral arteries

Intracellular pH (pH_i) in the vascular wall modulates agonist-induced vasocontractile and vasorelaxant responses in mesenteric arteries, whereas effects on myogenic tone have been unsettled. We studied the role of Na⁺,HCO₃⁻ cotransporter NBCn1 in mouse isolated middle cerebral arteries and the influence of pH_i disturbances on myogenic tone. Na⁺,HCO₃⁻ cotransport was abolished in arteries from NBCn1 knockout mice and steady-state pH_i ∼0.3 units reduced compared with wild-type mice. Myogenic tone development was low under control conditions but increased on treatment with the NO-synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME). This effect of L-NAME was smaller in arteries from NBCn1 knockout than wild-type mice. Myogenic tone with L-NAME present was significantly lower in arteries from NBCn1 knockout than wild-type mice and was abolished by rho-kinase inhibitor Y-27632. The arteries displayed vasomotion, and this rhythmic contractile pattern was also attenuated in arteries from NBCn1 knockout mice. No differences in membrane potential or intracellular [Ca²⁺] were seen between arteries from NBCn1 knockout and wild-type mice. We propose that NO production and rho-kinase-dependent Ca²⁺ sensitivity are reduced at low pH_i in pressurized mouse middle cerebral arteries. This likely impedes the ability to adjust to changes in perfusion pressure and regulate cerebral blood flow.     

Critical Role of Astrocyte NAD+ Glycohydrolase in Myelin Injury and Regeneration

Western-style diets cause disruptions in myelinating cells and astrocytes within the mouse CNS. Increased CD38 expression is present in the cuprizone and experimental autoimmune encephalomyelitis models of demyelination and CD38 is the main nicotinamide adenine dinucleotide (NAD⁺)-depleting enzyme in the CNS. Altered NAD⁺ metabolism is linked to both high fat consumption and multiple sclerosis (MS). Here, we identify increased CD38 expression in the male mouse spinal cord following chronic high fat consumption, after focal toxin [lysolecithin (LL)]-mediated demyelinating injury, and in reactive astrocytes within active MS lesions. We demonstrate that CD38 catalytically inactive mice are substantially protected from high fat-induced NAD⁺ depletion, oligodendrocyte loss, oxidative damage, and astrogliosis. A CD38 inhibitor, 78c, increased NAD⁺ and attenuated neuroinflammatory changes induced by saturated fat applied to astrocyte cultures. Conditioned media from saturated fat-exposed astrocytes applied to oligodendrocyte cultures impaired myelin protein production, suggesting astrocyte-driven indirect mechanisms of oligodendrogliopathy. In cerebellar organotypic slice cultures subject to LL-demyelination, saturated fat impaired signs of remyelination effects that were mitigated by concomitant 78c treatment. Significantly, oral 78c increased counts of oligodendrocytes and remyelinated axons after focal LL-induced spinal cord demyelination. Using a RiboTag approach, we identified a unique in vivo brain astrocyte translatome profile induced by 78c-mediated CD38 inhibition in mice, including decreased expression of proinflammatory astrocyte markers and increased growth factors. Our findings suggest that a high-fat diet impairs oligodendrocyte survival and differentiation through astrocyte-linked mechanisms mediated by the NAD⁺ase CD38 and highlights CD38 inhibitors as potential therapeutic candidates to improve myelin regeneration.SIGNIFICANCE STATEMENT Myelin disturbances and oligodendrocyte loss can leave axons vulnerable, leading to permanent neurologic deficits. The results of this study suggest that metabolic disturbances, triggered by consumption of a diet high in fat, promote oligodendrogliopathy and impair myelin regeneration through astrocyte-linked indirect nicotinamide adenine dinucleotide (NAD⁺)-dependent mechanisms. We demonstrate that restoring NAD⁺ levels via genetic inactivation of CD38 can overcome these effects. Moreover, we show that therapeutic inactivation of CD38 can enhance myelin regeneration. Together, these findings point to a new metabolic targeting strategy positioned to improve disease course in multiple sclerosis and other conditions in which the integrity of myelin is a key concern.     

Stimulation of brain Na,K-ATPase by norepinephrine in vivo: prevention by receptor antagonists and enhancement by repeated stimulation

The role of alpha 1- and beta-noradrenergic receptors in the stimulation of the K+-p-nitrophenylphosphatase activity and ouabain binding associated with rat brain Na+, K+-ATPase by acute or repeated yohimbine were examined. Repeated yohimbine increased enzyme activity and ouabain binding by about 25%; this was prevented by the alpha 1-receptor antagonist prazosin and decreased by the beta-antagonist propranolol. Pretreatment with repeated yohimbine increased the stimulation of enzyme activity by acute yohimbine while the stimulation was prevented by prazosin pretreatment.     

Intracellular acidification induced by membrane depolarization in rat hippocampal slices: roles of intracellular Ca and glycolysis

To elucidate the mechanism of pH_i changes induced by membrane depolarization, the variations in pH_i and [Ca²⁺]_i induced by a number of depolarizing agents, including high K⁺, veratridine, N-methyl-

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-aspartate (NMDA) and ouabain, were investigated in rat hippocampal slices by the fluorophotometrical technique using BCECF or fura-2. All of these depolarizing agents elicited a decrease in pH_i and an elevation of intracellular calcium ([Ca²⁺]_i) in the CA1 pyramidal cell layer. The increases in [Ca²⁺]_i caused by the depolarizing agents almost completely disappeared in the absence of Ca²⁺ (0 mM Ca²⁺ with 1 mM EGTA). In Ca²⁺ free media, pH_i acid shifts produced by high K⁺, veratridine or NMDA were attenuated by 10–25%, and those produced by ouabain decreased by 50%. Glucose-substitution with equimolar amounts of pyruvate suppressed by two-thirds the pH_i acid shifts induced by both high K⁺ and NMDA. Furthermore, lactate contents were significantly increased in hippocampal slices by exposure to high K⁺, veratridine or NMDA but not by ouabain. These results suggest that the intracellular acidification produced by these depolarizing agents, with the exception of ouabain, is mainly due to lactate accumulation which may occur as a result of accelerated glycolysis mediated by increased Na⁺–K⁺ ATPase activity. A Ca²⁺-dependent process may also contribute to the intracellular acidification induced by membrane depolarization. Since an increase in H⁺ concentration can attenuate neuronal activity, glycolytic acid production induced by membrane depolarization may contribute to the mechanism that prevents excessive neuronal excitation.     

Blockade of Na+/H+ exchanger type 3 causes intracellular acidification and hyperexcitability via inhibition of pH-sensitive K+ channels in chemosensitive respiratory neurons of the dorsal vagal nucleus in rats

Extracellular pH (pH_e) and intracellular pH (pH_i) are important factors for the excitability of chemosensitive central respiratory neurons that play an important role in respiration and obstructive sleep apnea. It has been proposed that inhibition of central Na⁺/ H⁺ exchanger 3 (NHE-3), a key pHi regulator in the brainstem, decreases the pH_i, leading to membrane depolarization for the maintenance of respiration. However, how intracellular pH affects the neuronal excitability of respiratory neurons remains largely unknown. In this study, we showed that NHE-3 mRNA is widely distributed in respiration-related neurons of the rat brainstem, including the dorsal vagal nucleus (DVN). Whole-cell patch clamp recordings from DVN neurons in brain slices revealed that the standing outward current (I _so) through pH-sensitive K⁺ channels was inhibited in the presence of the specific NHE-3 inhibitor AVE0657 that decreased the pHi. Exposure of DVN neurons to an acidified pH_e and AVE0657 (5 μmol/L) resulted in a stronger effect on firing rate and I _so than acidified pH_e alone. Taken together, our results showed that intracellular acidification by blocking NHE-3 results in inhibition of a pHsensitive K⁺ current, leading to synergistic excitation of chemosensitive DVN neurons for the regulation of respiration.     

Evaluation of the electrophysiological consequences of GABA removal from the synaptic cleft by Na ion transport-coupled neuronal uptake

The pre- and postsynaptic electrophysiological consequences of a carrier-mediated, Na+ ion transport-coupled removal of gamma-aminobutyric acid (GABA) from the relevant synaptic clefts are discussed. Assuming for the GABA internalization process a stoichiometry like GABAo + 3NA+o + K+i in equilibrium GABAi + 3Na+i + K+o and a synaptic cleft GABA maximal concentration of 100 microM we calculated the presynaptic depolarization associated with GABA removal between 11.5 and 38.2 mV. At the postsynaptic level the effect appears to be less marked.     

Lowering of the potassium concentration induces epileptiform activity in guinea-pig hippocampal slices

Extra- and intracellular recording techniques were used to study the epileptiform activity generated by guinea-pig hippocampal slices perfused with low potassium containing artificial cerebrospinal fluid. Extracellular field potentials were recorded in CA1 and CA3 regions along with intracellular recordings in CA3 subfield. Reduction of the extracellular potassium concentration [K(+)](o) from 4 to 2 mM caused a transient neuronal hyperpolarisation which was followed by a repolarisation and subsequent depolarisation period. Paroxysmal depolarisation shifts occurred during the transient hyperpolarisation period while epileptic field potentials (EFP) appeared in the late repolarisation or early depolarisation phase. EFP elicited by reduction of [K(+)](o) were neither affected by blockade of N-methyl-D-aspartate (NMDA) glutamate-subreceptor or gamma aminobutyric acid receptor, nor by application of the organic calcium channel blocker nifedipine or the anticonvulsant drugs carbamazepine and valproic acid. Upon application of non-NMDA glutamate-subreceptor blocker the EFP were abolished in all trials, while application of the organic calcium channel blocker verapamil only suppressed the EFP in some cases. The results point to a novel mechanism of epileptogenesis and may provide an in vitro model for the development of new drugs against difficult-to-treat epilepsy.     

Stiff person syndrome associated anti-amphiphysin antibodies reduce GABA associated [Ca]i rise in embryonic motoneurons

Autoantibodies to the synaptic protein amphiphysin play a crucial pathogenic role in paraneoplastic stiff-person syndrome. Impairment of GABAergic inhibition is the presumed pathophysiological mechanism by which these autoantibodies become pathogenic. Here we used calcium imaging on rat embryonic motor neurons to investigate whether antibodies to amphiphysin directly hinder GABAergic signaling. We found that the immunoglobulin G fraction from a patient with stiff-person syndrome, containing high titer antibodies to amphiphysin and inducing stiffness in rats upon passive transfer, reduced GABA-induced calcium influx in embryonic motor neurons. Depletion of the anti-amphiphysin fraction from the patient's IgG by selective affinity chromatography abolished this effect, showing its specificity for amphiphysin. Quantification of the surface expression of the Na⁺/K⁺/2Cl²⁻ cotransporter revealed a reduction after incubation with anti-amphiphysin IgG, which is concordant with a lower intracellular chloride concentration and thus impairment of GABA mediated calcium influx. Thus, anti-amphiphysin antibodies exert a direct effect on GABA signaling, which is likely to contribute to the pathogenesis of SPS.     

Glial contribution to seizure: K activation of (Na, K)-ATPase in bulk isolated glial cells and synaptosomes of epileptogenic cortex

Glial and neuronal (Na⁺, K⁺)-ATPase have dissimilar apparent affinities for K⁺ ions. Glial (Na⁺, K⁺)-ATPase is maximally activated by 20 mM K⁺ while neuronal (Na⁺, K⁺)-ATPase is maximally stimulated by 3–5 mM K⁺. Because this glial property may play an important role in the clearance of [K⁺]₀ during seizures, we investigated the K⁺ activation of (Na⁺, K⁺)-ATPase within bulk isolated glial cells and synaptosomes isolated from epileptogenic brains. The primary focus (F), the homolateral brain area around the focus (PF) and the mirror (M) or secondary focus induced by freezing lesions were studied.Results show that both glial and synaptosomal enzyme activities in the epileptogenic state change in comparison with controls, i.e. sham-operated cats. In F and M., glial enzyme decreased reaction velocities between 3 and 18 mM K⁺. In PF, maximum velocities increased in glial (Na⁺, K⁺)-ATPase. These results indicate that in actively firing epileptogenic tissue (F, M), glial (Na⁺, K⁺)-ATPase decreased rate reactions while the catalytic activity was increased in cortical tissues surrounding the focus. These phenomena appeared early, i.e. 1–3 days after production of the freezing lesion, and was associated with a sharp decrease in absolute levels of enzyme activity.Synaptosomal (Na⁺, K⁺)-ATPase from controls always exhibited a saturation curve at 3–6 mM K⁺. Synaptosomal enzyme activities in the primary (F) lesion increased slightly 24 h after lesion production, then progressively decreased 3 days after lesion production. No significant changes were seen in M and PF.     

Slight impairment of Na+,K+-ATPase synergistically aggravates ceramide- and beta-amyloid-induced apoptosis in cortical neurons

Dysfunction of the Na(+),K(+)-ATPase (Na(+),K(+)-pump), due to reduced energy supply or increased endogenous ouabain-like inhibitors, likely occurs under pathological conditions in the central nervous system. In cultured mouse cortical neurons, we examined the hypothesis that a mild non-toxic inhibition of the Na(+),K(+)-ATPase could synergistically sensitize the vulnerability of neurons to normally non-lethal apoptotic signals. Ouabain at a low concentration of 0.1 microM slightly lessened the Na(+),K(+)-pump activity measured as an ouabain-sensitive current, yet did not affect K(+) homeostasis and viability of cortical neurons. Co-exposure to 0.1 microM ouabain plus non-lethal C(2)-ceramide (5 microM) or beta-amyloid 1-42 (5 microM), however, induced marked intracellular K(+) loss, caspase-3 cleavage, DNA laddering, and synergistically triggered neuronal death. The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) predominantly blocked the caspase activation and neuronal death. These results suggest that slight impairment of Na(+),K(+)-pump activity may amplify the disruption of K(+) homeostasis in the presence of a non-lethal apoptotic insult, leading to activation of apoptotic cascade and substantial neuronal injury.     

Pharmacological modulation of brain Nav1.2 and cardiac Nav1.5 subtypes by the local anesthetic ropivacaine

Objective

The present study was aimed to investigate the pharmacological modulatory effects of ropivacaine, an amide-type local anesthetic, on rat Nav1.2 (rNav1.2) and rNav1.5, the two Na⁺ channel isoforms heterologously expressed in Xenopus oocytes and in HEK293t cell line, respectively.

Methods

Two-electrode voltage-clamp (TEVC) and whole-cell patchclamp recordings were employed to record the whole-cell currents.

Results

Ropivacaine induced tonic inhibition of peak Na⁺ currents of both subtypes in a dose- and frequency-dependent manner. rNav1.5 appeared to be more sensitive to ropivacaine. In addition, for both Na⁺ channel subtypes, the steady-state inactivation curves, but not the activation curves, were significantly shifted to the hyperpolarizing direction by ropivacaine. Use-dependent blockade of both rNav1.2 and rNav1.5 channels was induced by ropivacaine through a high frequency of depolarization, suggesting that ropivacaine could preferentially bind to the 2 inactivated Na⁺ channel isoforms.

Conclusion

The results will be helpful in understanding the pharmacological modulation by ropivacaine on Nav1.2 subtype in the central nervous system, and on Nav1.5 subtype abundantly expressed in the heart.     

Regulation of intracellular pH in vertebrate central neurons

The regulation of intracellular pH (pHi) was investigated in reticulospinal neurons of the lamprey using ion-selective microelectrodes. Steady-state pHi in 23 mM HCO-3-buffered Ringer was 7.44 +/- 0.03 with a membrane potential of 54 +/- 4 mV (mean +/- S.E.M., n = 6). In nominally HCO-3-free solutions, pHi recovery from acid loading was blocked by 10(-3)M amiloride. Recovery was stimulated by transition to HCO-3-containing solutions. Results suggest that pHi regulation in lamprey reticulospinal neurons is mediated by a Na+-H+ exchanger. The presence of a distinct, HCO-3-dependent pHi regulatory mechanism is postulated.     

Effects of ethanol on Na-dependent amino acid uptake: dependence on rat age and Na, K-ATPase activity

Acute effects of ethanol on Na(+)-dependent transport of gamma-aminobutyric acid (GABA) and glutamic acid (GLU) were investigated in crude synaptosomal preparations from rat cerebral cortex. In experiments with 30-40-day-old (peripubertal) rats, the overall dose responses of the GABA and GLU transport systems to ethanol were biphasic. Stimulation was observed at ethanol concentrations (40-160 mM) relevant to intoxication. Inhibition was observed at higher concentrations of ethanol. The stimulatory phase of the dose response was not observed in 60-100-day-old (adult) rats. In preparations from peripubertal rats, other alcohols also had biphasic dose response curves with stimulation at low alcohol concentrations. The relative efficacy of the different alcohols appeared to correlate with the relative membrane-buffer partition coefficient. In synaptosomal membrane vesicles, where artificial ion concentration gradients rather than Na+,K(+)-ATPase activity provide the driving force for uptake, ethanol did not stimulate GABA uptake. In direct measures of Na+,K(+)-ATPase activity, both Rb+ uptake and ATP hydrolysis were enhanced by 80 mM ethanol. We conclude that stimulation of Na(+)-dependent uptake of amino acids by ethanol was secondary to enhanced Na+,K(+)-ATPase activity and may be associated with a specific developmental stage in the rat.     

The ouabain-induced [Ca]i increase in leech Retzius neurones is mediated by voltage-dependent Ca channels

In leech Retzius neurones the inhibition of the Na⁺–K⁺ pump by ouabain causes an increase in the cytosolic free calcium concentration ([Ca²⁺]_i). To elucidate the mechanism of this increase we investigated the changes in [Ca²⁺]_i (measured by Fura-2) and in membrane potential that were induced by inhibiting the Na⁺–K⁺ pump in bathing solutions of different ionic composition. The results show that Na⁺–K⁺ pump inhibition induced a [Ca²⁺]_i increase only if the cells depolarized sufficiently in the presence of extracellular Ca²⁺. Specifically, the relationship between [Ca²⁺]_i and the membrane potential upon Na⁺–K⁺ pump inhibition closely matched the corresponding relationship upon activation of the voltage-dependent Ca²⁺ channels by raising the extracellular K⁺ concentration. It is concluded that the [Ca²⁺]_i increase caused by inhibiting the Na⁺–K⁺ pump in leech Retzius neurones is exclusively due to Ca²⁺ influx through voltage-dependent Ca²⁺ channels.     

Alterations of cerebromicrovascular Na, K-ATPase activity due to fatty acids and acute hypertension

Acute hypertension, induced in rats by intravenous injection of angiotensin II, previously has been shown to increase cerebrovascular permeability to macromolecules. The purpose of this study was to examine the effect of acute hypertension on Na⁺, K⁺-ATPase, the enzyme responsible for controlling ionic permeability of the cerebromicrovascular endothelium. The K⁺-dependent p-nitrophenylphosphatase activity of the cerebromicrovascular Na⁺, K⁺-ATPase was determined using microvessels prepared from hypertensive and normotensive rats. When compared to controls, a 70% decrease (P < 0.02) in the maximum rate (V_max) of the Na⁺, K⁺-ATPase from hypertensive rats was evident with no change in the Michaelis constant (K_M). In contrast, γ-glutamyltranspeptidase, a marker enzyme for cerebral endothelic cells, was not significantly affected. Sodium arachidonate (1–100 μM) inhibited the phosphatase activity of the Na⁺, K⁺-ATPase in microvessels isolated from both normotensive and hypertensive rats in a dose-dependent manner. Furthermore, poly-unsaturated fatty acids (sodium linoleate and arachidonate) evoked the greatest inhibition of the enzyme, while sodium oleate and sodium palmitate inhibited the Na⁺, K⁺-ATPase to lesser extents. This regulation of enzyme activity by fatty acids was comparable in control and hypertensive groups. In summary, the data indicate that the cerebromicrovascular Na⁺, K⁺-ATPase was altered as a consequence of acute hypertension and that poly-unsaturated fatty acids can modulate this enzyme in microvessels derived from hypertensive or control rats     

In vitro dopamine biosynthesis in the median eminence of rat hypothalamic slices: Involvement of voltage-dependent Ca channels

Equimolar replacement of Na⁺ in medium with choline chloride or sucrose and experimental manipulations known to increase [Na⁺]_i, such as ouabain addition and K⁺ deprivation from medium, caused a marked increase in in vitro DOPA synthesis in the median eminence of rat hypothalamic slices in a Ca²⁺-dependent manner. These results suggest that a Na⁺−Ca²⁺ exchange mechanism is closely involved in the regulation of dopamine biosynthesis in tuberoinfundibular neurons.     

Late expression of Na+/H+ exchanger 1 (NHE1) and neuroprotective effects of NHE inhibitor in the gerbil hippocampal CA1 region induced by transient ischemia

Although acidosis may be involved in neuronal death, the participation of Na⁺/H⁺ exchanger (NHE) in delayed neuronal death in the hippocampal CA1 region induced by transient forebrain ischemia has not been well established. In the present study, we investigated the chronological alterations of NHE1 in the hippocampal CA1 region using a gerbil model after ischemia/reperfusion. In the sham-operated group, NHE1 immunoreactivity was weakly detected in the CA1 region. Two and 3 days after ischemia/reperfusion, NHE1 immunoreactivity was observed in glial components, not in neurons, in the CA1 region. Four days after ischemia/reperfusion, NHE1 immunoreactivity was markedly increased in CA1 pyramidal neurons as well as glial cells. These glial cells were identified as astrocytes based on double immunofluorescence staining. Western blot analysis also showed that NHE protein level in the CA1 region began to increase 2 days after ischemia/reperfusion. The treatment of 10 mg/kg 5-(N-ethyl-N-isopropyl) amiloride, a NHE inhibitor, significantly reduced the ischemia-induced hyperactivity 1day after ischemia/reperfusion. In addition, NHE inhibitor potently protected CA1 pyramidal neurons from ischemic damage, and NHE inhibitor attenuated the activation of astrocytes and microglia in the ischemic CA1 region. In addition, NHE inhibitor treatment blocked Na⁺/Ca²⁺ exchanger 1 immunoreactivity in the CA1 region after transient forebrain ischemia. These results suggest that NHE1 may play a role in the delayed death, and the treatment with NHE inhibitor protects neurons from ischemic damage.     

Rapid adaptation of rat brain and liver metabolism to a ketogenic diet: an integrated study using 1H- and 13C-NMR spectroscopy

The ketogenic diet (KD) is an effective alternative treatment for refractory epilepsy in children, but the mechanisms by which it reduces seizures are poorly understood. To investigate how the KD modifies brain metabolism, we infused control (CT) and 7-day KD rats with either [1-¹³C]glucose (Glc) or [2,4-¹³C₂]β-hydroxybutyrate (β-HB). Specific enrichments of amino acids (AAs) measured by ¹H- and ¹³C-NMR in total brain perchloric acid extracts were similar between CT and KD rats after [1-¹³C]Glc infusion whereas they were higher in KD rats after [2,4-¹³C₂]β-HB infusion. This suggests better metabolic efficiency of ketone body utilization on the KD. The relative rapid metabolic adaptation to the KD included (1) 11%-higher brain γ-amino butyric acid (GABA)/glutamate (Glu) ratio versus CT, (2) liver accumulation of the ketogenic branched-chain AAs (BCAAs) leucine (Leu) and isoleucine (ILeu), which were never detected in CT, and (3) higher brain Leu and ILeu contents. Since Glu and GABA are excitatory and inhibitory neurotransmitters, respectively, higher brain GABA/Glu ratio could contribute to the mechanism by which the KD reduces seizures in epilepsy. Increased BCAA on the KD may also contribute to better seizure control.