A 1 R activation suppresses seizures. In line with our previous demonstration (6), A 1 R+/– mice, A 1 R–/– mice, and Adk-Tg mice (based on expression of a ubiquitously expressed Adk transgene on an Adk-null background) maintained on a control diet (CD) experienced regular electrographic hippocampal seizures consistent with deficient adenosine/A 1 R signaling (Figure 1). Seizure frequency and duration were similar between Adk-Tg and A 1 R–/– mice and significantly lower in A 1 R+/– mice (but still elevated above WT; Table 1). Behavioral (clinical) seizures were never observed in the mutants, and electrographic seizures were never observed in the WT group.

Figure 1 Seizure suppression by KD depends on A 1 R activation. Representative EEG recordings from the CA3 of WT and transgenic mice reflect seizure distribution over a 1-hour time span (top traces) and individual seizures at higher resolution (1 minute; bottom traces). Asterisks in top traces denote the individual seizures chosen. Beginning and end of seizures are marked by vertical arrows. Traces from CD-fed animals showed baseline seizure activity in all mutants and lack of seizures in WT. KD almost completely abolished seizures in Adk-Tg mice; rare seizures were of reduced duration, as shown. KD reduced seizure activity in A 1 R+/– mice, had no effect in A 1 R–/– mice. Treatment with glucose or DPCPX reversed KD effects. See Table 1 for quantitation.

Table 1 Seizure frequency and duration

To quantify the effects of KD, WT and transgenic mice were fed CD or KD. At 3 weeks, KD nearly abolished seizures in Adk-Tg mice and significantly reduced the duration of remaining seizures (Table 1 and Figure 1). If KD suppresses seizures in Adk-Tg mice by elevating adenosine in brain, we predicted that its seizure-suppressing potential would be reduced in A 1 R+/– mice, which have only 50% of this receptor and lowered sensitivity to adenosine (9). As expected, KD-fed A 1 R+/– mice experienced a significant, but lesser (approximately 50%), reduction in seizure frequency, although seizure duration was not changed compared with CD-fed A 1 R+/– mice (Table 1). We further predicted that spontaneous seizures in A 1 R–/– mice would be resistant to the beneficial effects of KD if its protective mechanism involves activation of A 1 Rs. Indeed, KD was completely ineffective in affecting seizures in A 1 R–/– mice. These findings demonstrate that KD suppresses seizures caused by adenosine deficiency (Adk-Tg) or reduced adenosine signaling (A 1 R+/–), but has no effect in the absence of A 1 Rs (A 1 R–/–).

Seizures are restored with glucose or A 1 R blocker treatment. To determine the extent to which the reduced frequency of spontaneous seizures in Adk-Tg and A 1 R+/– mice was specifically the result of the low-carbohydrate nature of KD, we injected glucose into KD-fed mice of each genotype. In Adk-Tg and A 1 R+/– mice, which displayed reduced seizures after KD, glucose injection increased seizure frequency significantly within 30–90 minutes; in Adk-Tg mice, the increase did not quite reestablish the baseline CD-fed seizure phenotype (Table 1 and Figure 1).

The loss of KD-induced seizure suppression in A 1 R–/– mice, combined with the reduced efficacy of KD in A 1 R+/– compared with Adk-Tg mice, is indicative of seizure suppression via a mechanism involving A 1 Rs. To test this further, we injected a nonconvulsive dose of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1 mg/kg), an A 1 R-selective antagonist. In Adk-Tg and A 1 R+/– mice, DPCPX restored seizure frequency to control levels, whereas it had no significant effect in WT or A 1 R–/– mice (Figure 1 and Table 1). DPCPX in KD-fed Adk-Tg mice increased seizure duration significantly compared with both CD and KD conditions; however, it did not change seizure duration in A 1 R+/– mice, which suggests that its effect on duration depended on the relative abundance of A 1 Rs. As expected, DPCPX had no effect on seizures in A 1 R–/– mice. These data, coupled with the lack of KD efficacy in A 1 R–/– mice, demonstrated that A 1 Rs are a molecular target whereby KD reduces ictogenesis in vivo.

WT and A 1 R–/– mice have similar metabolic responses. To confirm that A 1 R–/– mice experienced a similar metabolic response to KD, we measured the plasma level of β-hydroxybutyrate (βHB) in A 1 R–/– compared with WT mice. There was no difference in βHB levels between genotypes when mice were fed CD (WT, 0.48 ± 0.09 mM; A 1 R–/–, 0.60 ± 0.09 mM; n = 5 per group, P > 0.05), and there was no evidence for inadequate or decreased ketones in A 1 R–/– mice fed KD for 3 weeks. βHB levels were elevated similarly and significantly in both genotypes in KD- versus CD-fed mice (WT, P < 0.001; A 1 R–/–, P < 0.002); overall, KD-fed A 1 R–/– mice demonstrated a nonsignificant trend toward higher βHB levels compared with KD-fed WT mice (WT, 1.38 ± 0.15 mM; A 1 R–/–, 1.96 ± 0.29 mM; n = 5 per group, P > 0.05).

KD reduces ADK expression. Ambient adenosine in brain is regulated largely by ADK (10). Whereas overexpression of ADK (causing adenosine deficiency) has been linked to seizures (6), reduced expression of ADK (thus increasing adeno­sine) renders the brain less susceptible to seizures (6) and might be involved in an endogenous protective mechanism of the brain in response to stress or injury (10). We therefore hypothesized that metabolic stress imposed by KD might likewise lead to reduction in brain ADK. KD- or CD-fed WT mice were sacrificed at 3 and 4 weeks of feeding, corresponding to the time span of seizure analysis above. Western blot showed that this key adenosine-regulating enzyme was downregulated significantly in the KD group (Figure 2), which suggests a possible mechanism for KD’s seizure suppression.

Figure 2 KD leads to downregulation of ADK. (A) Representative Western blot from brain extracts of WT mice fed CD or KD for 3 and 4 weeks. Note the 2 different splice variants of ADK in the ADK-reactive bands. Anti-tubulin immunoreactivity was used to normalize for equal loading. Lanes were run on the same gel but were noncontiguous (white line). (B) Brain ADK from mice fed CD or KD for 3–4 weeks, expressed relative to CD (n = 4 per group). Data are mean ± SEM. **P < 0.01 vs. CD.

ADK overexpression in human epilepsy. We have demonstrated previously that astrogliosis, ADK overexpression, and resulting adenosine deficiency are implicated in seizure generation in rodent models of epilepsy (6, 10). Therefore, reconstitution of normal adenosine signaling by KD might be of therapeutic value for epileptic patients with refractory temporal lobe epilepsy (TLE). To investigate whether adenosine dysfunction is likewise implicated in the pathology of human epilepsy, we studied the expression of ADK in hippocampus of patients with hippocampal sclerosis (HS) and histologically normal human hippocampus. ADK was upregulated within specific hippocampal regions in HS specimens compared with control hippocampus (Figure 3, A–D). Abundant ADK-immunopositive cells with typical astroglial morphology were observed in areas with prominent gliosis in all HS specimens; double-labeling confirmed ADK expression in glial fibrillary acidic protein–positive (GFAP-positive) reactive astrocytes (Figure 3, B and D). Western blot analysis confirmed a greater than 3-fold increase in ADK levels in hippocampus of TLE patients (Figure 3, E and F), which suggests that adenosine deficiency could contribute to the epileptic phenotype in those patients.

Figure 3 ADK immunoreactivity in hippocampus of control and TLE patients with medial temporal HS. (A–D) Sections were counterstained with hematoxylin. Shown are representative CA1 (A and B) and hilus (C and D) from the same sample. (A and C) Control hippocampus showed weak ADK immunoreactivity. Histologically normal surgical hippocampus displayed an immunoreactivity pattern similar to that in control autopsy hippocampus (not shown). (B and D) The HS specimen demonstrated increased ADK expression in both residual pyramidal and hilar neurons (arrows and B, top inset) and in reactive astrocytes (arrowheads and B, bottom inset). Insets in D show expression of ADK (red) in a reactive astrocyte (GFAP, green). Scale bars: 160 μm (A and B); 80 μm (C and D); 40 μm (A, inset, and B, top inset); 15 μm (B, bottom inset, and D, insets). (E and F) Western blot analysis of ADK of total homogenates from control autopsy hippocampus and HS specimens. (E) Representative immunoblots. (F) Densitometric data, expressed relative to optical density of β-actin (n = 5 per group). Data are mean ± SEM. *P < 0.05 vs. control.

Using transgenic mice and complementary pharmacology, we demonstrated that KD-induced seizure control was dependent on A 1 Rs: KD virtually abolished seizures in Adk-Tg mice (with reduced endogenous adenosine and intact A 1 Rs), reduced seizures partially but significantly in A 1 R+/– mice, and had no effects in A 1 R–/– mice or in the presence of A 1 R blockade. We showed overexpression of ADK (which should reduce A 1 R activation) in human epilepsy and KD-induced reduction of ADK in mouse brain, thus providing a mechanism for KD-induced increase in adenosine and anticonvulsant effects of KD via A 1 Rs. Whereas altered adenosinergic signaling might not be the underlying cause for seizures in all epilepsies, adenosine has stopped seizures in every seizure model tested to date, including models of pharmacoresistant epilepsy. Thus, no matter the cause of the seizure, adenosine can help to resolve it, provided — as shown here — that A 1 Rs are present. Subclinical, brief electrographic seizure events (undetectable with scalp electrodes), as exhibited in all 3 strains of transgenic mice in the present study, are now increasingly recognized and likely play an important role in early epileptogenesis (11).

Acute reversal of seizure suppression by glucose confirmed the specificity of KD’s metabolic effects and comported with clinical observations and animal studies highlighting the importance of low glucose to the effects of KD (12). Indeed, we recently demonstrated in vitro that metabolic consequences of KD — reduced extracellular glucose and increased intracellular ATP — produce A 1 R-dependent inhibition of cornu ammonis region 3 (CA3) pyramidal cells (3). Of interest, glucose-induced seizure reversal in Adk-Tg mice was not complete (which indicates that the specific enzymatic activity of ADK might have been increased based on rapid glucose-dependent restoration of the energy charge), although ADK levels were still quantitatively reduced (Figure 2). Additional mechanisms might also contribute to KD-induced seizure suppression: in vitro studies demonstrated direct acute effects of ketones (13, 14), although the relative importance of these mechanisms to the chronic in vivo effects of KD administration in humans remains to be determined.

When relating these findings to human epilepsy, we found that brain tissue resected from humans with intractable epilepsy showed increased ADK — and therefore likely a relative adenosine deficiency. Previously, adenosine deficiency has been demonstrated directly in microdialysis samples from epileptogenic hippocampus in human patients with TLE (15). These results and our current data indicate that alteration of adenosine signaling is relevant to human epilepsy. Specifically, at least some forms of human epilepsy replicate key features of our adenosine-deficient model, increasing the translational potential of these results. Together, these data delineate a clinically relevant relationship among KD, adenosine, and epilepsy, which could lead to less-restrictive diets, alternate pharmaceutical approaches, and broader applications of metabolic strategies to different medical conditions.