Ketogenic diet treatment abolishes seizure periodicity and improves diurnal rhythmicity in epileptic Kcna1-null mice

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1 FULL-LENGTH ORIGINAL RESEARCH Ketogenic diet treatment abolishes seizure periodicity and improves diurnal rhythmicity in epileptic Kcna1-null mice *Kristina A. Fenoglio-Simeone, *Julianne C. Wilke, *Heather L. Milligan, ycharles N. Allen, *Jong M. Rho, and *Rama K. Maganti *Barrow Neurological Institute and St. Joseph s Medical Center, Neurology Research, Phoenix, Arizona, U.S.A.; and ycenter for Research on Occupational and Environmental Toxicology, OHSU, Portland, Oregon, U.S.A. SUMMARY Introduction: Seizures are known to perturb circadian rhythms in humans as well as in animal models of epilepsy. However, it is unknown whether treatment of the underlying epilepsy restores normal biologic rhythms. We asked whether: (1) seizure activity is characterized by diurnal rhythmicity, (2) chronically epileptic mice exhibit impaired rest activity rhythms, and (3) treatment with the anticonvulsant ketogenic diet (KD) improves such perturbations. Methods: Chronically epileptic Kcna1-null mice were fed either a standard diet (SD) or KD for 4 weeks and subjected to continuous video-eeg (electroencephalography) and actigraphy monitoring for 3 5 days to assess seizure activity and rest activity cycles. Results: Seizure activity in Kcna1-null mice demonstrated diurnal rhythmicity, peaking at zeitgeber (ZT)2.30 ± Rest activity rhythms of epileptic mice were significantly disrupted. Whereas locomotor activity for wild-type mice peaked at ZT15.45 ± 0.28 (ZT14:26 ZT16:51), peak activity of epileptic mice was more unpredictable, occurring over a 12.4 h range (ZT06:33 ZT18: 57). In six of nine epileptic mice, peak activity was delayed to ZT17.42 ± 0.38, whereas peak activity was advanced to ZT10.00 ± 1.26 in the remaining mice. Treatment with the KD abolished seizure periodicity and restored the rest activity rhythm to values resembling those of wild-type mice (i.e., activity peaking at ZT16.73 ± 0.67). Conclusions: Kcna1-null mice experience seizures with 24-h periodicity and impaired circadian behavior. KD reduces the number and periodicity of seizures and restores normal behavioral rhythms, suggesting that this nonpharmacologic therapy may benefit biologic rhythm disturbances in epileptic patients. KEY WORDS: Ketogenic diet, Seizures, Epilepsy, Kcna1-null mice, Circadian rhythms. Circadian rhythms are endogenous behavioral and physiologic rhythms with a 24-h cycle. The hypothalamus, specifically the suprachiasmatic nucleus (SCN), is the principal circadian pacemaker that modulates many physiologic functions, including sleep wake cycles. In addition to hypothalamic pacemakers, the circadian clock is entrained by both photic and nonphotic stimuli (i.e., zeitgebers; Rosenwasser & Dwyer, 2001). Accepted April 2, 2009; Early View publication June 1, Address correspondence to Rama K. Maganti, Barrow Neurological Institute, St. Joseph s Hospital & Medical Center, 350 W. Thomas Road, Phoenix, AZ 85013, U.S.A. rama.maganti@chw.edu Wiley Periodicals, Inc. ª 2009 International League Against Epilepsy Human and animal studies demonstrate a close relationship between seizures and the circadian-timing system. Clinical studies using scalp (Ferrillo et al., 2000) and intracranial electroencephalography (EEG) recordings (Asano et al., 2007) report that epileptiform activity (i.e., interictal spikes) tends to occur more often while patients are sleeping. Depth electrode studies involving single neuron recordings in the mesial temporal lobe of epileptic patients revealed that significantly higher firing rates, burst propensity, and synchronous discharges occur more often during slow-wave sleep than during wakefulness (Staba et al., 2002). Similar observations were made in animal models of epilepsy. For example, rats are more susceptible to amygdala kindling induced epileptogenesis during subjective night (i.e., during lights on when the 2027

2 2028 K. A. Fenoglio-Simeone et al. animals are least active, Weiss et al., 1993). In animal models of limbic epilepsy (induced by electrical stimulation of the hippocampus), spontaneous seizures are more frequent during subjective night (Bertram & Cornett, 1994; Quigg et al., 2000), a rhythm that persists under conditions of constant darkness (Quigg et al., 2000). These data suggest that the periodicity of the seizures is possibly regulated by an intrinsic mechanism that is maintained in the absence of an exogenous zeitgeber (i.e., light). Additional studies have shown that seizures themselves may act as internal zeitgebers and can thus influence circadian rhythms. For example, rats with electrically induced hippocampal seizures have phase-shifted temperature circadian rhythms (Quigg et al., 2000). Phase delays in diurnal locomotor activity rhythms are prevalent in animals with lithium-pilocarpine induced status epilepticus (Stewart & Leung, 2003). Whether treatment of the underlying epilepsy (i.e., a reduction in seizure activity) reverses these perturbations in circadian rhythms has yet to be determined and is a major question addressed in the current study. We chose to examine circadian dysfunction in epileptic Kcna1-null mice and determine whether treatment with the anticonvulsant ketogenic diet (KD, a high fat and low carbohydrate diet, see Muller-Schwarze et al., 1999) restores diurnal activity rhythms. The Kcna1 gene encodes the delayed-rectifier voltage-gated potassium channel a subunit protein, Kv1.1 (Smart et al., 1998), and mutations in the human homolog are associated with epilepsy (Zuberi et al., 1999). By the end of the first postnatal month, Kcna1-null mice exhibit recurrent spontaneous seizures (Rho et al., 1999b). Based on previous studies, we hypothesized that: (1) seizures in this animal model of chronic epilepsy have 24-h periodicity; (2) chronic seizure occurrence in Kcna1-null mice disturbs the normal diurnal rest activity cycles; and (3) treatment with the anticonvulsant KD restores rest activity patterns. Methods Animals Kcna1-null mice were bred at the Barrow Neurological Institute (BNI) vivarium. Mice were born and reared in a quiet, temperature-controlled room and entrained to a 12- h light dark cycle; lights on at zeitgeber time (ZT) 00:00. Tail clips were taken at P10 P15 and sent to Transnetyx Inc. for genotyping (Cordova, TN, U.S.A.). After weaning at P18 P20, mice were placed on either a standard diet (SD) or a KD (6:1, fat to carbohydrates plus proteins (Bio- Serv F3666, Frenchtown, NJ, U.S.A.). The diet and water were provided ad libitum. Mice on the KD become ketotic almost immediately; there are no differences in weight between KD- and SD-fed mice (see Szot et al., 2001; Rho et al., 2004; Sullivan et al., 2004). Activity and video-eeg recordings were conducted in a separate animal facility room with similar temperature and light dark cycles as the colony room. Mice were brought into the recording room and allowed to habituate for 3 4 h prior to the start of the recording session. All protocols were in accordance with National Institutes of Health (NIH) guidelines and approved by Barrow Institutional Animal Care and Use Committee. EEG electrode implantation surgery and seizure scoring Electrical and behavioral seizures were recorded using Stellate video-eeg technology and HARMONIE software (Stellate, Quebec, Montreal, Canada). Animals were anesthetized with isoflurane (5% induction, 2% maintenance) prior to transmitter implantation. A wireless, PhysioTel telemetry transmitter (Data Sciences international, St. Paul, MN, U.S.A.) was implanted in a subcutaneous pocket along the dorsal flank. The biopotential leads were implanted bilaterally on the dura, 2 mm lateral of the midsagittal suture and 1 mm caudal to Bregma, with the ground implanted in the occipital bone; the transmitter contains an internal reference electrode. Following a 3-day recovery period, animals underwent continuous video-eeg monitoring for three to five consecutive days. Behavioral seizures were scored on a modified Racine scale (Tasker et al., 1991) and correlated with EEG interictal and ictal activity. Generalized tonic clonic seizures typically began with tonic arching and tail extension, followed by forelimb clonus, or rearing, and forelimb clonus, and then generalized synchronous forelimb and hind-limb clonus, after which there is postictal depression. Only seizures with behavioral manifestations were included in subsequent analyses. The time of onset and severity was recorded for each seizure event. The number of seizures observed during each hour was subsequently collapsed into the following 4-h time bins for each animal: ZT00:00 (this time-point includes seizures that occurred between ZT00:00 3:59); ZT04:00 (ZT04:00 07:59); ZT08:00 (ZT08:00 11:59); ZT12:00 (ZT12:00 15:59); ZT16:00 (ZT16:00 19:59); and ZT20:00 (ZT20:00 23:59). For all studies, statistical significance (p < 0.05) among experimental groups was determined using analysis of variance (ANOVA) with Bonferroni s post hoc test (PRISM; GraphPad, San Diego, CA, U.S.A.) unless otherwise noted. Cosinor analyses for seizure periodicity IGOR software was used to determine the least square approximation of the time series using the following cosine function: 2pðt Acrophase) CðtÞ ¼Mesor þ A cos P where Mesor (an acronym for midline estimating statistic of rhythm) is the mean of the oscillation; A is the ampli-

3 2029 Ketogenic Diet and Diurnal Rhythms tude, P is the prefixed period, and Acrophase is the timing of the cosine maximum (Smolensky et al., 1976). Actigraphy Actigraphy is a noninvasive method of monitoring human sleep wake and animal rest-activity cycles. Behavioral rest activity cycles were assessed using the Vital View data acquisition system, which integrates radio telemetry technology and switch-closure activity monitoring (Mini Mitter Company, Inc; Bend, OR, U.S.A.). Mice were placed in an transparent Plexiglas arena and allowed to habituate for 3 4 h. The activity was monitored in 3-min epochs and scored on an activity scale (0 50) over a 4 7 day period. Data were analyzed with ACTIVIEW Biological Rhythm Analysis software (Mini Mitter Company, Inc.). The time of peak activity was determined by the maximum value of a fitted cosine function. A chi-square periodogram method was used to determine the diurnal activity period. Results Daily periodicity of seizures in epileptic Kcna1-null mice Electroclinical seizures of kcna1-null mice were scored and the time of each seizure was recorded. The number of seizures per 4-h time bin was determined (as described in the Methods) and the percentage total number of seizures per bin was calculated for each animal [Fig. 1 shows the mean standard error of the mean (SEM) of these values]. Significantly fewer seizures occurred during the dark phase, when mice were more active, between ZT16:00 and 23:59 (p < 0.05). Subsequent cosinor analyses indicated that seizures occurred with diurnal periodicity with an amplitude of %. The highest percentage of seizures occurred at ZT Epileptic mice have disrupted diurnal rest activity patterns Wild-type mice entrained to the light dark phases exhibited typical rest activity patterns, that is, mice were significantly more active during the dark phase when compared to the light phase (Fig. 2A). In contrast, activity patterns of Kcna1 null mice were markedly disrupted and had atypical diurnal rhythmicity, that is, the mean diurnal peak activity was reduced and trough activity was elevated compared to that of wild-type mice (Fig. 2B). Rest activity data were collapsed across days into a 24- h period and fit to a cosine function (Fig. 3A). Peak activity for wild-type mice occurred at ZT (ranging from ZT14:26 16:51). In contrast to this 2.4-h range, peak activity for chronically epileptic mice was more unpredictable, occurring throughout a 12.4-h range (ZT6: 33 18:57). Variability was reduced after mice were further Figure 1. Cosinor analyses indicate that seizures in chronically epileptic kcna1-null mice exhibit diurnal rhythmicity. Seizure incidence for each animal was tallied and binned into 4-h periods (i.e., the ZT00:00 time-point includes seizures that occurred from ZT00:00 03:59). The datapoints in this figure reflect the number of seizures that occurred during each 4-h bin relative to the total number of seizures for each animal. Seizure scores were calculated for individual mice and are expressed as the mean percentage total ± standard error of the mean (SEM). The period (P) of the cosinor analysis was fixed to 24 h. *p < 0.05 when compared to ZT00:00 using a one-factor analysis of variance (ANOVA). ZT, zeitgeber time. sorted based on whether their peak activity was greater or less than twice the standard error of the wild-type mean. In six of nine Kcna1-null mice, peak activity was phase delayed occurring at ZT The remaining three mice were phase-advanced (peak activity at ZT ). The phase shifts in peak activity also accounted for variability in the activity period. Compared to wildtype mice ( h) all epileptic mice exhibited an extended activity period ( h); however, those that were phase-advanced had a significantly longer activity period compared to phase-delayed epileptic mice ( h and h, respectively; Fig. 3B). Superimposition of seizure periodicity and the rest activity rhythm of epileptic mice showed an inverse relationship, that is, Kcna1-null mice had fewer seizures during times they were most active and had a greater number of seizures during periods of rest (Fig. 3C). Ketogenic diet treatment abolishes seizure periodicity and restores circadian function Treatment with the KD significantly reduced the number of seizures and the diurnal rhythmicity of seizure

4 2030 K. A. Fenoglio-Simeone et al. A B Figure 2. Kcna1-null mice have disrupted rest activity rhythms. Representative double-plotted activity graphs reflecting activity of (A) one wild-type and (B) one kcna1-null mouse during five consecutive days. The open bars above the activity graphs indicate the light phases, during which the mice are more restful; the solid bars indicate dark phases, during which the animals are more active. The activity pattern of wild-type mice predictably has the highest density of activity during the dark phases (arrows), whereas the onset of rest activity phases of Kcna1-null mice is more random. These data are presented in the line graphs below. Each peak trough cycle reflects one 24-h period (demarked by the vertical hashes). The horizontal blue dashed lines drawn from the peaks and troughs of the wildtype highlight the abnormal rest activity cyclicity of the epileptic mice. ZT, zeitgeber time. occurrence in Kcna1-null mice. KD reduced the total number of seizures by 54% (Fig. 4A). A majority of seizures (135 of 175 or 77%) in SD-fed mice occurred during the light phase. In contrast, seizures of KD-treated mice were more evenly distributed across time, with approximately half (43 of 81) occurring during the light phase and half during the dark phase (Fig. 4A). The average number of seizures per animal per day was plotted for each group (Fig. 4B). Significantly more seizures occurred during subjective night (between ZT0:00 and ZT7:59) than during subjective day (between ZT16:00 and ZT23:59) in kcna1-null mice fed the SD; this diurnal rhythm of seizure occurrence did not occur in mice fed the KD (two-factor ANOVA, p < 0.05). KD treatment significantly reduced the number of seizures, specifically those experienced between ZT0:00 and ZT04:00 (Fig. 4B, two-factor ANOVA, p < 0.05).

5 2031 Ketogenic Diet and Diurnal Rhythms A A B B C Figure 4. Ketogenic diet (KD) treatment reduces seizure frequency. (A) The total number of seizures that occurred during light (ZT00:00 11:59) and dark phases (ZT12:00 23:59) were examined. KD-treated mice had fewer seizures than those fed a standard diet (SD). (B) Further analyses examining the average number of seizures per animal per day per 4-h time bin indicated that KD treatment specifically reduced seizures, specifically during ZT00:00 04:00. In addition, the number of seizures in KD-treated mice was similar at all times of day. *Differs significantly from KD; **SD ZT16:00 and 20:00 differ significantly from SD ZT0:00 and 04:00; p < ZT, zeitgeber time. Figure 3. (A) Activity scores were collapsed across days into a 24-h period and fit to a cosine function (n = 9 wildtype, n = 9 Kcna1-null mice). The peak activity for wildtype mice occurred at ZT15:28 ± 0:18, whereas activity of Kcna1-null mice was either phase-delayed or phase-advanced. (B) Kcna1-null mice had extended activity periods when compared to wild-type mice. Epileptic mice with advanced peak activity had a significantly longer activity period. Delayed and advanced labels below the bar graph indicate the population of mice whose peak activity was either phase-advanced or phase-delayed. (C) Seizure occurrence and activity rhythms were inversely correlated. Activity scores were collapsed across days into a 24-h period and averaged across animals in 2-h bins (n = 9). Seizure scores are expressed as the mean percentage total ± standard error of the mean (SEM) (described in Methods and Fig. 1 legend). WT, wildtype mice; ZT, zeitgeber time; p < The rest activity patterns of KD-treated epileptic mice were not statistically different from normal wild-type mice (see Fig. 5A for representative activity graphs). Peak activity occurred at ZT and the 12-h peak activity range of epileptic mice was markedly narrowed to a 4.6 h period in those treated with the KD (ZT14:15 18:51, n = 7, Fig. 5B). Discussion The principal findings of the current study are: (1) seizures in epileptic Kcna1-null mice exhibit diurnal periodicity; (2) the rest activity cycles are abnormal in epileptic mice with an inverse relationship between seizure occurrence and locomotor activity; (3) treatment with the anticonvulsant KD significantly reduces seizure frequency and diminishes seizure diurnal periodicity; and (4) KD-treated Kcna1-null mice have restored diurnal function. Our findings support the notion of impaired circadian function in epileptic brain, and indicate that KD treatment may be beneficial to epileptic patients, not only with respect to seizure control, but also to either directly or indirectly correcting comorbid circadian abnormalities. Seizures manifest circadian periodicity Seizures in epileptic patients can occur with 24-h periodicity. Continuous video-eeg studies of epileptic patients show that frontal lobe seizures peak between 4:00

6 2032 K. A. Fenoglio-Simeone et al. A B Figure 5. Rest activity patterns are restored in epileptic mice fed the ketogenic diet (KD). (A) Representative activity and line graphs of one KD-treated epileptic mouse to show restored diurnal rest activity patterns. (B) There were no differences in peak activity or activity period of wild-type and KD-treated Kcna1-null mice. ZT, zeitgeber time. and 7:00 h, whereas mesial temporal lobe seizures have biphasic peaks, occurring more frequently between 7:00 and 10:00 h in the morning and then peaking later in the early evening between 16:00 and 19:00 h (Herman et al., 2001). These differences in peak occurrence of seizures in humans are also observed in animal models of epilepsy. In our model, seizures maintained a diurnal periodicity, with most seizures occurring during the light (resting) phase. Similarly, rats that underwent electrical stimulation induced status epilepticus have a higher percentage of seizures (87%) occurring during periods of rest (Bastlund et al., 2005). These data contrast with those reported by Quigg et al. (1998), who reported limbic seizures preferentially peaking at 16:48 h, with a nadir at 06:00 h. The seizures are likely intrinsically rhythmic (Quigg et al., 2000); however, the physiologic and anatomic mechanisms underlying such seizure periodicity and its close relation to circadian rhythms remain unclear. Altered circadian rhythms in epilepsy Circadian rhythms are homeostatic processes that partly control sleep wake cycles (BorbØly, 1982) and are modulated by a number of zeitgebers. In animal models, nonphotic cues can cause phase shifts in circadian rhythms. Locomotor activity (van Esseveldt et al., 2000) or seizures can alter the timing system, in turn delaying or advancing peak activity. Alterations in circadian rhythms have been reported in humans with epilepsy including altered melatonin (Schapel et al., 1995) and cortisol cycles (Bazil et al., 2000) as well as altered temperature and rest activity rhythms (Laakso et al., 1993; Quigg et al., 2000, 2001; Stewart & Leung, 2003). In the current study, we found that epileptic mice have altered diurnal rest activity patterns. Kcna1-null mice had reduced activity during the dark phase, increased activity during the light phase, and either an advance or delay in peak activity. Previous studies by Stewart et al. (2001) reported that severity of the seizures follows diurnal variation, with tonic clonic features occurring more frequently during the dark phase and facial-forelimb clonus and rearing being more prominent during the light phase. Therefore, seizure severity and timing may influence whether some kcna1-null mice have an advanced and others a delayed shift in peak activity.

7 2033 Ketogenic Diet and Diurnal Rhythms Stewart and Leung (2003) reported that pilocarpineinduced seizures were associated with postictal increases in bouts of exploratory behavior, rearing, and grooming. These data support the notion that seizures occurring while the animal is at rest may promote inappropriate transitions in rest and activity states. Similarly, we found that the intra-rest activity cycles of Kcna1-null mice were significantly disrupted with qualitatively shorter durations of rest and frequent bouts of activity. These observations resemble the disrupted sleep architecture of epileptic mice reported by Bastlund et al. (2005). In this study, epileptic mice spent more time awake, less time in paradoxical sleep, and had more awakenings during sleep (Bastlund et al., 2005). These animals showed significant cell loss in the dorsomedial hypothalamus (DMH), a region that when lesioned disrupts circadian sleep wake regulation and increases wakefulness (Chou et al., 2003). Future studies will examine whether neurons in the DMH of Kcna1-null mice are dysfunctional. Effects of the ketogenic diet KD treatment reduces seizure frequency in humans (Hallbççk et al., 2007a) and animal models of epilepsy (Rho et al., 1999a). KD also improves sleep quality in children with epilepsy (Hallbççk et al., 2007b). Consequently, we hypothesized that the KD would improve or restore behavioral rhythms in epileptic mice. In the current study, a distinct improvement in seizure frequency following administration of the KD was noted. To our knowledge, this is the first study simultaneously demonstrating that the KD reduces seizure activity and normalizes behavioral rhythms in epileptic animals. Further studies are needed to understand the physiologic basis of altered biologic rhythms and why seizures are distributed in a circadian manner. Moreover, it remains unclear how the treatment of epilepsy may improve or restore normal circadian rhythms. 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