Seizure susceptibility of neuropeptide-Y null mutant mice in amygdala kindling and chemical-induced seizure models

Epilepsy Research 61 (2004) 49–62

Seizure susceptibility of neuropeptide-Y null mutant mice in amygdala kindling and chemical-induced seizure models Harlan E. Shannon∗ , Lijuan Yang Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA Received 16 October 2003; received in revised form 10 February 2004; accepted 4 June 2004 Available online 27 July 2004

Abstract Neuropeptide Y (NPY) administered exogenously is anticonvulsant, and, NPY null mutant mice are more susceptible to kainate-induced seizures. In order to better understand the potential role of NPY in epileptogenesis, the present studies investigated the development of amygdala kindling, post-kindling seizure thresholds, and anticonvulsant effects of carbamazepine and levetiracetam in 129S6/SvEv NPY(+/+) and NPY(−/−) mice. In addition, susceptibility to pilocarpine- and kainate-induced seizures was compared in NPY(+/+) and (−/−) mice. The rate of amygdala kindling development did not differ in the NPY(−/−) and NPY(+/+) mice either when kindling stimuli were presented once daily for at least 20 days, or, 12 times daily for 2 days. However, during kindling development, the NPY(−/−) mice had higher seizure severity scores and longer afterdischarge durations than the NPY(+/+) mice. Post-kindling, the NPY(−/−) mice had markedly lower afterdischarge thresholds and longer afterdischarge durations than NPY (+/+) mice. Carbamazepine and levetiracetam increased the seizure thresholds of both NPY (−/−) and (+/+) mice. In addition, NPY (−/−) mice had lower thresholds for both kainate- and pilocarpine-induced seizures. The present results in amygdala kindling and chemical seizure models suggest that NPY may play a more prominent role in determining seizure thresholds and severity of seizures than in events leading to epileptogenesis. In addition, a lack of NPY does not appear to confer drug-resistance in that carbamazepine and levetiracetam were anticonvulsant in both wild type (WT) and NPY null mutant mice. Published by Elsevier B.V. Keywords: Neuropeptide-Y; Null mutant mice; Amygdala kindling; Kainate-induced seizures; Pilocarpine-induced seizures; Rapid kindling

1. Introduction Neuropeptide Y (NPY) is a 36-amino acid peptide member of the pancreatic polypeptide family (Tatemoto, 1982), and is thought to modulate neuronal excitability, particularly in the hippocampus, a region implicated in the generation and modulation ∗ Corresponding author. Tel.: +1-317-276-4360; fax: +1-317 276-5546. E-mail address: [email protected] (H.E. Shannon).

0920-1211/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.eplepsyres.2004.06.002

of seizure activity (e.g., see review by Palmiter et al., 1998). In vitro, NPY inhibits excitatory neurotransmission in hippocampal (e.g., Klapstein and Colmers, 1993, 1997; Baraban et al., 1997; Ho et al., 2000) and frontal cortex (Bijak, 2000) slice preparations. In vivo, hippocampal NPY content was increased after acute seizures in rats (Sperk et al., 1992; Gruber et al., 1994), and, overexpression of NPY resulted in decreased seizure susceptibility in rats (El Bahh et al., 2001; Vezzani et al., 2002). Moreover, the exogenous administration of NPY suppressed seizure

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activity produced by kainate (Woldbye et al., 1997), pentylenetetrazole (Woldbye, 1998), and electrical stimulation of the hippocampus (Woldbye et al., 1996; Reibel et al., 2003; Klemp and Woldbye, 2001). In addition, NPY null transgenic mice lacking NPY had a reduced threshold for pentylenetetrazole-induced seizures (Erickson et al., 1996), and were unable to terminate kainate-induced seizures, resulting in a high mortality rate (Baraban et al., 1997; De Prato Primeaux et al., 2000). Taken together, these data strongly implicate a role for NPY in suppressing seizures and raise the question of whether a lack of inhibitory control by NPY could have a role in epileptogenesis. One method for investigating the role of an endogenous neurotransmitter such as NPY in epileptogenesis is to determine the rate of kindling development in null mutant animals lacking the neurotransmitter. Kindling refers to a process whereby repeated application of an initially subconvulsive electrical or chemical stimulus results in progressive intensification of stimulus-induced epileptic activity, potentially culminating in a generalized seizure (Goddard et al., 1969). Recently, Reibel et al. (2003) demonstrated that a 7-day continuous infusion of NPY in the hippocampus delayed the progression of hippocampal kindling in the rat, whereas anti-NPY immunoglobulins enhanced kindling. Reibel et al. (2003) suggested that NPY constitutes an endogenous mechanism counteracting excessive hippocampal excitability. If NPY plays a role in suppressing epileptogenesis, then it might be expected that kindling would occur more rapidly in NPY null mutant mice than in wild-type control mice. The major purpose of the present studies was to investigate the rate of development of amygdala kindling in NPY knockout (KO) (null mutant, −/−) mice (Erickson et al., 1996). In an initial experiment, NPY knockout and wild type (WT) mice were stimulated once daily in the amygdala until 10 stage 5 (Racine, 1972) seizures were evoked. Afterdischarge thresholds were subsequently determined once per week for 4 weeks. In order to determine if the lack of NPY conferred drug resistance to anticonvulsant drugs, the efficacy of carbamazepine and levetiracetam in raising afterdischarge threshold were determined in these amygdala-kindled animals. In a second experiment, the rate of development of amygdala kindling in knockout and wild type mice was determined using

a rapid kindling protocol in which the mice received 12 stimulations approximately 30 min apart on two separate days. Generalized seizure thresholds were subsequently determined once per week for 4 weeks. The efficacy of carbamazepine and levetiracetam was also determined in these rapidly amygdala kindled animals. In addition, the doses of kainate and pilocarpine required to produce limbic seizures were determined in NPY knockout and wild type mice.

2. Methods 2.1. Animals The subjects were male mutant [Npy(−/−)] mice and wild-type (129S6/SvEv) mice. Mice were housed in a temperature-controlled (22 ◦ C) environment with a 12-h light and 12-h dark (1800–0600 h) diurnal cycle. All mice had ad libitum access to food and water. Wild-type 129S6/SvEv mice were purchased from Taconic Farms, Inc., Germantown, NY; Npy(−/−) mice, originally provided to us by Dr. Richard Palmiter, were raised at Taconic Farms, Inc. All experiments were conducted in accordance with the “Principles of laboratory animal care” (NIH publication No. 85-23, revised 1985) and were approved by the Eli Lilly Institutional Animal Care and Use Committee. 2.2. Electrode implantation Mice were anesthetized with ketamine (75 mg/kg) and xylazine (10 mg/kg) and a twisted bipolar electrode (0.25 mm diameter; MS333/1-B, Plastics One, Roanoke, VA) was stereotaxically implanted into the right hemisphere aimed at the basolateral amygdala (1.3 mm posterior and 3.0 mm lateral to bregma, and 4.6 mm below the dorsal surface of the skull) and anchored with dental acrylic to three jeweler’s screws placed in the skull. A skull screw over the frontal cortex served as the indifferent reference electrode. 2.3. Kindling development with once-daily stimulations Approximately 2 weeks after surgery, mice were stimulated at a suprathreshold intensity of 500 ␮A

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(all electrical stimuli were 60 Hz, 1 s train of 1 ms monophasic square wave pulses) daily (Monday– Friday) until 10 stage 5 seizures (Racine, 1972) were evoked. After all mice had met this criterion, afterdischarge thresholds were determined four times at intervals of 1 week. The afterdischarge threshold was determined using an ascending stairstep procedure. The initial current intensity was 10 ␮A, and the current intensity was increased in steps of approximately 0.2 log units (10, 16, 25, 40, 65, 100, 160, 250, 400, and, 500 ␮A) at intervals of 1 min until an afterdischarge of at least 5 s duration was elicited. Mice in which an afterdischarge was not evoked by 500 ␮A were assigned a value of 501 ␮A. Electrographic seizure activity was monitored from the amygdala electrode using a Model 15RX Physiodata Amplifier System with PolyView software (Grass Instruments Division, Astro-Med, Inc., W. Warwick, RI). Behavioral manifestations of seizures were classified according to the scale described by Racine (1972): 1, eye blinking or chewing; 2, head nodding; 3, unilateral forelimb clonus; 4, bilateral forelimb clonus and rearing; 5, bilateral forelimb clonus and rearing plus loss of postural control. 2.4. Rapid kindling development Approximately 2 weeks after surgery, an initial (prekindling) threshold for producing an electrical stimulus afterdischarge was determined using the ascending stairstep procedure described above. On the following day, and again 2 days later, mice were stimulated at a suprathreshold intensity of 500 ␮A at 30-min intervals for a total of 12 stimulations on each of the 2 days, with 1 day intervening between the two sessions. Afterdischarge thresholds were subsequently determined four times, at intervals of 1 week. Electrographic and behavioral seizure activity were monitored as described above. 2.5. Anticonvulsant drug testing On the day before drug testing (“baseline” day), an afterdischarge threshold was determined in each animal. On the day of drug testing, carbamazepine (30 mg/kg i.p.), levetiracetam (100 mg/kg i.p.) or vehicle was administered and the afterdischarge threshold and duration determined 30 min later.

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2.6. Kainate- and pilocarpine-induced seizures Groups of 10 mice were administered a dose of 20 mg/kg of kainate or 100 mg/kg of pilocarpine i.p. every 20 min until the onset of limbic seizures. Limbic seizures were defined as repetitive head movements, forelimb clonus, and rearing. The time until the onset of limbic seizures, as well as the number of animals which died within 4 h, were recorded. 2.7. Drugs Kainate and pilocarpine (Sigma Chemical Co., St. Louis, MO) were dissolved in distilled water. Carbamazepine (Sigma) was dissolved in 25% 2-hydroxypropyl-␤-cyclodextrin. Levetiracetam (Lilly Research Laboratories) was dissolved in distilled water. All drugs were administered i.p. in a volume of 10 ml/kg. 2.8. Data analysis Data are expressed as means + S.E.M. Treatment groups were compared to appropriate control groups using repeated measures ANOVA and Dunnett’s t-test. Pairs of means were compared using Student’s t-test. Statistical analyses were performed using JMP v4.04 statistical software (SAS Institute Inc., Cary, NC). A probability of P < .05 was taken as the level of statistical significance.

3. Results 3.1. Kindling development with once daily stimulations The development of amygdala-kindled seizures in wild type and knockout mice is presented in Fig. 1. There was a progressive increase in both the seizure severity scores and in the afterdischarge duration across kindling days. Although the differences between the two groups were not large in magnitude on any given day, overall, the severity scores were higher and the afterdischarge duration longer in the knockout mice than in the wild type mice. In the ANOVAs for seizure severity and afterdischarge duration, the main effect for days was significant for both variables

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H.E. Shannon, L. Yang / Epilepsy Research 61 (2004) 49–62 Table 1 Number of stimulations to achieve amygdala kindling stagesa Genotype

Number of stimulations Stages 1 and 2

Wild type NPY knockout

1.6 ± 0.2 2.4 ± 0.5

Stages 3 and 4 10 ± 1.0 7.9 ± 1.0

Stage 5 12.4 ± 1.0 11.1 ± 1.1

a Each number represents the mean ± S.E.M. of the number of stimulations required to achieve the indicated stage in 14 mice.

Post-kindling afterdischarge thresholds averaged approximately 75 ␮A in the NPY knockout mice and approximately 150 ␮A in the wild type mice (Fig. 2, upper panel). Thus, afterdischarge thresholds were consistently lower in the NPY knockout mice compared to the wild type mice, but the main effect

Fig. 1. Amygdala kindling development of seizure stage (upper panel) and afterdischarge duration (lower panel) in NPY null mutant (knockout, KO) mice and wild type (WT) 129SvEv mice. Mice received one stimulation per day, 5 days per week, until 10 stage 5 seizures occurred; only the first 20 days are presented for purposes of clarity. Each point represents the mean of one observation in each of 14 animals. Vertical lines represent ± S.E.M. and are absent when less than the size of the symbol.

[F(19, 476) = 122.9, P < .0001; F(19, 476) = 42.3, P < .001, respectively], but the main effect for genotype was not [F(1, 26) = 1.2, P > .05; F(1, 26) = 3.1, P > .05, for severity and afterdischarge, respectively]. However, the genotype × days interaction term was not significant for seizure severity but was significant for afterdischarge duration [F(19, 476) = 0.74, P > .05; F(19, 476) = 2.5, P = .0004, respectively]. Table 1 presents the mean number of stimulations required to achieve various seizure stages. There were no significant differences between the wild type and knockout mice in the number of stimulations required to achieve any of the stages.

Fig. 2. Post-kindling afterdischarge thresholds and afterdischarge durations in NPY knockout (KO) and wild type (WT) mice after the once daily stimulation procedure presented in Fig. 1. Thresholds were determined at weekly intervals for 4 weeks. Each point represents the mean of one observation in each of 14 animals. Vertical lines represent ± S.E.M.

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for genotype only approached being statistically significant [F(1, 26) = 3.1, P = .09]. Moreover, the afterdischarge thresholds decreased over time in a similar manner in both genotypes, as indicated by a significant main effect for week [F(3, 78) = 6.5, P < .0005] but not a significant genotype × week interaction [F(3, 78) = 2.2, P > .05]. In comparison, the afterdischarge durations averaged approximately 50 s in the NPY knockouts and 40 s in the wild type mice (Fig. 2, lower panel). The afterdischarge durations were consistently and significantly longer in the NPY knockout mice than in the wild type mice [F(1, 26 = 11.9, P = .0019]. In order to determine if the NPY knockout and wild type mice differed in their sensitivity to representative antiepileptic drugs, the effects of vehicle, carbamazepine (30 mg/kg) and levetiracetam (100 mg/kg) were determined. The afterdischarge threshold of the NPY knockout mice continued to be significantly lower than in the wild type mice across the three baseline tests [main effect for genotype: [F(1, 16) = 7.6,

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P = .014)], and the thresholds were unchanged across the baseline days [main effect for days: [F(2, 10) = 0.21, P > .05] (Fig. 3, upper panels, columns above Baseline). Thirty minutes after the administration of vehicle, afterdischarge thresholds were unchanged on the test day relative to the value determined on the prior baseline day for either genotype of mice (Fig. 3, upper left panel; Student’s t-test, P > .05). Afterdischarge durations were similarly unchanged after vehicle in the wild type mice, but were approximately 10 s longer after vehicle in the knockout mice (t(6) = 3.5, P = .013). Thirty minutes after the administration of 30 mg/kg of carbamazepine i.p., afterdischarge thresholds were increased to approximately 315 ␮A in both the wild type and knockout mice (Fig. 3, upper middle panel), which was statistically significant for both the genotypes compared to their respective baseline values [t(8) = 2.8, P = .02 and t(6) = 2.7, P = .04, respectively]. However, the afterdischarge duration at the elevated threshold value was virtually identical to the duration on the baseline day in the wild type

Fig. 3. Anticonvulsant effects of carbamazepine (30 mg/kg, i.p.) and levetiracetam (100 mg/kg i.p.) in amygdala-kindled NPY knockout (KO) and wild type (WT) mice. Baseline thresholds were determined the day before drug testing, and Test thresholds were determined 30 min after vehicle or drug administration. Each point represents the mean of one observation in each of four to seven animals. Vertical lines represent ± S.E.M. ∗ P < .05 vs. baseline in same genotype, Student’s t-test.

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mice [[t(8) = 0.3, P > .05]. In the knockout mice, the afterdischarge duration was of shorter duration, but this difference did not achieve statistical significance [t(6) = 2.1, P = .08]. Similarly, thirty minutes after the administration of 100 mg/kg of levetiracetam i.p., afterdischarge thresholds were increased to approximately 300–400 ␮A in the wild type and knockout mice (Fig. 3, upper right panel), which was statistically significant for both the genotypes compared to their respective baseline values [t(12) = 5.2, P = .0002 and t(12) = 4.2, P = .0001, wild type and knockout, respectively]. In contrast with the results with carbamazepine however, the afterdischarge duration at the elevated threshold value was shorter in duration on the test day in the wild type mice [t(12) = 2.7, P = .02], but unchanged in the knockout mice [t(12) = 0.8, P > .05] by the administration of levetiracetam. The difference in the wild type mice was due to the fact that an afterdischarge was evoked in only two of the seven mice even at the maximum stimulus intensity of 500 ␮A while an afterdischarge was evoked in five of the seven knockout mice. When only the mice that had an afterdischarge were included in the analysis, the mean afterdischarge threshold and duration obtained one hour after levetiracetam in the wild type (N = 2) and knockout (N = 5) mice were, respectively, 157.5 and 263 ␮A, and 58.0 and 70.2 s. 3.2. Rapid kindling development Prior to rapid kindling, the afterdischarge thresholds in the knockout mice were lower than in the wild type

mice (Fig. 4, left panel); this difference approached statistical significance [t(16) = 1.9, P = .08]. In addition, the afterdischarge duration was longer in the knockout than the wild type mice (Fig. 1, middle level panel), but again this difference was not statistically significant [t(16) = 1.5, P = .16]. Similarly, the seizure severity score was higher, but not statistically significantly so [t(16) = 0.9, P = .39], in the knockout relative to the wild type mice (Fig. 1, right panel) determined on the day before initiating kindling development. During the first day of rapid kindling (Fig. 5, left panel), the seizure severity score and the afterdischarge duration generally increased as additional stimulations were presented, and, the NPY knockout mice typically had higher seizure severity scores and longer afterdischarge durations. In the ANOVA for seizure severity, the main effect for genotype was highly statistically significant [F(1, 168) = 16.3, P < .0001] and the main effect for stimulation number approached statistical significance [F(11, 168) = 1.79, P = .06]. The genotype × stimulation number interaction term was not significant [F(11, 168) = 1.1, P > .05]. In the ANOVA for afterdischarge duration, the main effect for genotype was highly statistically significant [F(1, 168) = 15.2, P < .0001], as was the main effect for stimulation number [F(11, 168) = 2.7, P = .003]. However, the genotype × stimulation number interaction term was not significant [F(11, 168) = 1.0, P > .05]. During the second day of rapid kindling (Fig. 5, right panels), the seizure severity score and the afterdischarge duration generally started at elevated values

Fig. 4. Baseline amygdala afterdischarge threshold, and the afterdischarge duration and seizure severity score at that threshold stimulation intensity, in NPY knockout (KO) and wild type (WT) mice which had not previously received any electrical stimulation. Each point represents the mean of one observation in each of nine animals. Vertical lines represent ± S.E.M.

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Fig. 5. Amygdala kindling development of seizure stage (upper panel) and afterdischarge duration (lower panel) in NPY knockout (KO) mice and wild type (WT) 129SvEv mice in the rapid kindling procedure. Mice were stimulated at 30 min intervals for a total of twelve stimulations on each of 2 days, with 1 day in between. Each point represents the mean of one observation in each of eight animals. Vertical lines represent ± S.E.M. and are absent when less than the size of the symbol.

Fig. 6. Post-kindling afterdischarge thresholds and afterdischarge durations in NPY knockout (KO) and wild type (WT) mice after the rapid kindling procedure presented in Fig. 5. Thresholds were determined at weekly intervals for 4 weeks. Each point represents the mean of one observation in each of eight animals. Vertical lines represent ± S.E.M. and are absent when less than the size of the symbol.

and were unchanged across the session. The NPY knockout mice occasionally had higher seizure severity scores and longer afterdischarge durations, but this was not consistent across stimulations. In the ANOVA for seizure severity, the main effects for genotype approached statistical significance [F(1, 168) = 3.0, P = .09] as did the main effect for stimulation number [F(11, 168) = 1.58, P = .11]. The genotype × stimulation number interaction term was not significant [F(11, 168) = 1.1, P > .05]. In order to gain information on generalized seizure thresholds as compared with afterdischarge thresholds in NPY knockout mice, post-kindling generalized seizure thresholds were determined in the rapidly kindled mice. Prior to kindling, a generalized seizure was

evoked in none of the mice of either genotype, and thus the threshold was greater than 500 ␮A. In addition, none of the mice had an afterdischarge and thus the afterdischarge duration at generalized seizure threshold was zero seconds, in both the wild type and knockout mice (Fig. 6, points above Pre). Approximately 1 week after the second day of kindling stimulations, a generalized seizure was evoked in six of eight mice of both genotypes, and the generalized seizure threshold was approximately 450 and 350 ␮A averaged across the eight mice of the wild type and knockout groups, respectively (Fig. 6); however, this difference between the groups only approached significance [t(14) = 1.57, P = .14]. Interestingly, the generalized seizure thresholds were markedly lower in both groups when tested approximately 2 weeks after the second day of kindling, averaging approximately 190 and 150 ␮A in

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the wild type and knockout mice, respectively. Moreover, the generalized seizure thresholds were further reduced to approximately 100 ␮A at week 3, where they remained at week 4. In the ANOVA for the generalized seizure threshold for the 4 post-kindling weeks, the main effect for genotype was not significant [F(1, 56) = 2.4, P > .05] although the main effect for week was highly significant [F(3, 56) = 30.9, P < .0001]. The genotype × week interaction term was also not significant [F(3, 56) = 0.55, P > .05]. In contrast, the afterdischarge durations at the generalized seizure threshold were relatively constant across all 4 weeks, averaging approximately 35 s in the wild type mice and 45 s in the NPY knockouts (Fig. 6, lower panel). In the ANOVA for the afterdischarge duration for the generalized seizure threshold for the 4 post-kindling weeks, the main effect for genotype was significant [F(1, 56) = 4.7, P = .035] although the main effect for week was not significant [F(3, 56) = 0.44, P > .05], nor was the genotype × week interaction term [F(3, 56) = 0.06, P > .05]. The effects of carbamazepine and levetiracetam on afterdischarge thresholds were also determined in the rapidly kindled mice. In the rapidly kindled knockout mice (Fig. 7, upper left panel), carbamazepine approximately doubled the afterdischarge threshold, but this increase only approached significance relative to the baseline threshold in this genotype [t(12) = 2.0, P = .07]. In the wild type mice, carbamazepine produced approximately a three-fold increase in afterdischarge threshold in the wild type mice (Fig. 7, upper left panel), an increase that was statistically significant [t(2.3), P = .04)]. On the other hand, carbamazepine (30 mg/kg) had no significant effect on afterdischarge durations at the elevated threshold in either genotype (Fig. 7, lower left panel). In comparison, levetiracetam (100 mg/kg) produced approximately three- to four-fold increases in the afterdischarge threshold in both the knockout and wild type mice (Fig. 7, upper right panel). For the knockout mice, this increase approached statistical significance [t(12) = 2.1, P = 0.06], and for the wild type mice the increase was significant [t(12) = 5.3, P < .0001]. Somewhat surprisingly, afterdischarge durations were significantly increased after levetiracetam in both the knockout and wild type rapidly kindled mice [t(12) = 2.4, P = .04 and t(12) = 2.5, P = .02, respectively].

Fig. 7. Anticonvulsant effects of carbamazepine (30 mg/kg, i.p.) and levetiracetam (100 mg/kg i.p.) in massed stimulation amygdala-kindled NPY knockout (KO) and wild type (WT) mice. Baseline thresholds were determined the day before drug testing, and Test thresholds were determined 30 min after drug administration. Each point represents the mean of one observation in each of seven or eight animals. Vertical lines represent ± S.E.M. ∗ P < .05 vs. baseline in same genotype, Student’s t-test.

3.3. Kainate- and pilocarpine-induced seizures Threshold doses for producing kainate- and pilocarpine-induced limbic seizures were determined by administering 20 mg/kg of kainic acid or 100 mg/kg of pilocarpine i.p. every 20 min until the onset of limbic seizures. The threshold doses for kainate to produce seizures was significantly lower in the knockout mice than in the wild type mice (approximately 25 and 40 mg/kg, respectively; Fig. 8, left panel). Similarly, the latency to onset of limbic seizures was significantly shorter in the knockout mice than in the wild type mice (approximately 17 and 27 min, respectively; Fig. 8, right panel). In addition, although the knockout mice received significantly less kainate, 7 of the 10 knockout mice died within 4 h, but only 1 of the 10 wild type mice died within this time frame. As with kainate, the threshold dose of

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Fig. 8. NPY knockout (KO) mice were more susceptible to kainate-induced limbic seizures than were wild type (WT) mice. Ten mice were used in each group. Vertical lines represent ± S.E.M. Numbers in parentheses indicate the number of mice that died within 4 h (numerator)/number tested (denominator). ∗ P < .05, Student’s t-test.

pilocarpine required to produce limbic seizures in knockout mice was significantly lower than in the wild type mice (approximately 275 and 425 mg/kg, respectively; Fig. 9, left panel). Similarly, the latency to onset of limbic seizures was significantly shorter in the knockout mice than in the wild type mice (approximately 40 and 80 min, respectively; Fig. 9, right panel). In contrast to the results with kainate, 7 of the 10 wild type mice (which received the higher total dose of pilocarpine) died within 4 h, but only 1 of the 10 knockout mice died within this time frame.

Fig. 9. NPY knockout (KO) mice were more susceptible to pilocarpine-induced limbic seizures than were wild type (WT) mice. Ten mice were used in each group. Vertical lines represent ± S.E.M. Numbers in parentheses indicate the number of mice that died within 4 h (numerator)/number tested (denominator). ∗ P < .05, Student’s t-test.

4. Discussion NPY administered exogenously is anticonvulsant (e.g., Woldbye et al., 1996; Woldbye, 1998; Reibel et al., 2003; Klemp and Woldbye, 2001) and NPY null mutant knockout mice had a reduced threshold for pentylenetetrazole-induced seizures (Erickson et al., 1996) and were unable to terminate kainate-induced seizures, resulting in a high mortality rate (Baraban et al., 1997; De Prato Primeaux et al., 2000). Further, the intra-hippocampal infusion of NPY delayed hippocampal kindling, whereas the infusion of anti-NPY immunoglobulins enhanced kindling (Reibel et al., 2003). Taken together, these findings strongly implicate a role for NPY in suppressing seizures and suggest that a lack of inhibitory control by NPY could have a role in epileptogenesis. In order to address this question, the present studies sought to characterize the seizure susceptibility of NPY knockout mice in amygdala kindling and chemical-induced seizure models. When the mice were stimulated once daily in the amygdala in the present studies, NPY knockout mice did not differ from wild type mice in the number of stimulations required to achieve different seizure stages, in the afterdischarge duration, nor was there a significant genotype × days interaction term for either seizure severity or afterdischarge duration. Thus, NPY knockout mice did not kindle more rapidly than wild type mice. The present results with amygdala kindling in the mouse differ somewhat from the results of

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Reibel et al. (2003) on the effects of intra-hippocampal infusion of NPY or anti-NPY immunoglobulins on hippocampal kindling in the rat. Reibel et al. (2003) found that the infusion of NPY during the second week of hippocampal kindling delayed the progression of seizure severity scores but had little effect on the duration of the afterdischarge. On the other hand, the infusion of anti-NPY immunoglobulins during the second week of hippocampal kindling increased the seizure severity scores and also increased the duration of hippocampal and cortical afterdischarges in rats (Reibel et al., 2003). In the present studies, the NPY knockout mice showed nonsignificant increases in seizure scores and afterdischarge durations during amygdala kindling, with more marked effects after kindling in the form of lower thresholds and longer afterdischarge durations. The differences between the present results and those of Reibel et al. (2003) could be related to species differences (rat vs. mouse) and/or differences in the kindling site (amygdala vs. hippocampus). Further, Reibel et al. (2003) infused anti-NPY immunoglobulins during the second week of kindling only, whereas with NPY knockout mice, NPY is absent throughout the kindling process and the possibility of compensatory changes cannot be ruled out. Future studies are needed to address the potential influences of these differences. However, both studies are in agreement that a lack of NPY can contribute at some point to more severe seizures. NPY and its receptor subtypes have been shown to exhibit considerable plasticity during and after kindling epileptogenesis in rats. In hippocampal slices from electrically kindled rats, potassium-induced release of NPY was enhanced both during and after hippocampal kindling acquisition (Rizzi et al., 1993). Hippocampal kindling also produced an increase in NPY immunoreactivity in the hilus as well as in presumed GABA-containing pyramidal shaped basket cells in the subgranular region (Schwarzer et al., 1996). Rapid hippocampal kindling, where 40 stimulations were given 5 min apart, produced an upregulation of NPY mRNA levels at 2–4 h post-seizures (Kopp et al., 1999). Moreover, changes in Y1-, Y2and Y5-receptor mRNA also have been observed (Kopp et al., 1999; Bregola et al., 2000). These changes in NPY systems have generally been interpreted as compensatory changes in response to seizures. Changes in NPY systems have also been

observed in resected tissue from patients with temporal lobe epilepsy where the total length of NPY immunoreactive fibers was increased in patients with hippocampal sclerosis, suggesting an upregulation of NPY systems in these patients (Furtinger et al., 2001). If an upregulation of NPY systems also occurs in the mouse during amygdala kindling, then the lower seizure thresholds and longer afterdischarge durations observed in the present studies in the NPY knockout mice may be due to the inability of NPY knockout mice to respond to the kindling stimulations and/or seizures by increasing expression and/or release of NPY in order to suppress seizures. Taken together, the data are consistent with the hypothesis that NPY appears to play a more predominant role in suppressing seizures than in suppressing the rate of epileptogenesis. The present results with the rapid kindling protocol are generally consistent with the results from the once-daily stimulation procedure. In the present studies, while the seizure severity scores and afterdischarge durations were significantly larger in magnitude in the knockout mice, the interaction terms were not significant for either variable, indicating that the rate of change of these two variables did not differ between the two genotypes. The present results indicate that rapid kindling with the massed stimulations procedure, as with the once-daily procedure, did not occur significantly more rapidly in the knockout than in the wild type mice and provide further evidence to suggest that NPY plays a more prominent role in decreasing seizure severity than in decreasing epileptogenesis. In the weeks after completion of either kindling procedure, several observations were apparent. Following the once-daily stimulation procedure, afterdischarge thresholds were lower, and afterdischarge durations longer, in NPY knockout mice than in the wild type mice. Thus, while the rates of kindling development were not appreciably different, the post-kindling differences observed in afterdischarge thresholds suggest that the cellular events which occur during and after kindling which lead to these differences are greater in magnitude in the knockout mice than in the wild type mice, an interpretation which would imply that NPY can be important in suppressing epileptogenesis. Post-kindling changes were more readily observed after the rapid kindling procedure where the generalized seizure threshold

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was observed to change markedly over the first 2 weeks after kindling, even though the only stimulations presented were the weekly threshold testing. These results suggest that cellular changes continued to occur even after the completion of the rapid kindling procedure. However, the rapid decreases in the generalized seizure threshold were not different in the knockout and wild type mice, indicating that a lack of NPY did not alter the rate of change of the cellular events underlying the change in post-rapid kindling generalized seizure threshold. Moreover, the afterdischarge durations did not change over time following the rapid kindling procedure, and the NPY knockout mice had significantly longer afterdischarge durations at each post-kindling test than did the wild type mice. Thus, while the majority of the evidence from the rapid kindling procedure is supportive of the interpretation that NPY plays a more prominent role in seizure severity than in epileptogenesis, the data also suggest the possibility that there are post-kindling changes in the NPY system which may contribute to the suppression of seizures and/or epileptogenesis. In may also be noted that in the present studies, both afterdischarge and generalized seizure thresholds decreased over time in the weeks post-kindling. This decrease was observed in both the wildtype and knockout mice, and the changes occurred at similar rates in both genotypes. As noted above, seizures produce an up-regulation of NPY (e.g., Vezzani et al., 1994; Schwarzer et al., 1996; Kopp et al., 1999), and post-kindling decreases in threshold could be due to decreases in NPY expression over the weeks after completion of the kindling protocol. The present findings that thresholds decreased over time post-kindling at essentially equal rates in wildtype and knockout mice indicates, however, that these time-dependent changes in threshold are not related to changes in NPY protein expression, but rather that changes in some other factor, or factors, lead to time-dependent changes in thresholds after kindling. If NPY plays a prominent role in suppressing seizures, then a lack of NPY might contribute to a lack of efficacy of antiepileptic drugs. To address this question, we determined the efficacy of two antiepileptic drugs with dissimilar mechanisms of action, carbamazepine and levetiracetam. The mechanism of action of carbamazepine is considered to be the use-dependent blockade of sodium channels (e.g.,

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Rogawski and Porter, 1990; Meldrum, 1996). The mechanism of action of levetiracetam is unknown, but is clearly not blockade of sodium channels e.g., see review by Schachter (2000). In the present studies, both carbamazepine and levetiracetam were efficacious in increasing afterdischarge seizure thresholds in both the NPY knockout and wild type mice in both kindling models used here, with the exception that the increase produced by carbamazepine only approached significance in the knockout mice after rapid kindling. Thus, the present findings suggest that a lack of NPY does not appear to confer resistance to the efficacy of antiepileptic drugs. However, during the testing of carbamazepine and levetiracetam, several NPY knockout mice were observed to have secondary seizures 5 to 30 min after threshold testing was completed and the mice had been returned to their home cage (unpublished observations). Moreover, five of the knockout mice went into status and subsequently died following a single threshold determination. In contrast, none of the wild type mice were ever observed to have additional seizures after threshold testing. Thus, while the lack of NPY did not markedly alter the efficacy of drugs in raising seizure threshold, the possibility cannot be ruled out that a lack of NPY may confer drug resistance in the sense that antiepileptic drugs may be less effective in blocking secondary, or subsequent, seizures in the absence of NPY. Further studies recording EEG and behavioral seizures over longer periods of time after an electrically-induced seizure are required in order to more clearly define the potential role of NPY in suppressing such secondary seizures. Kainate and pilocarpine produce similar limbic-type seizures, although through different mechanisms. Kainate acts as a direct acting agonist at kainate receptors to produce limbic seizures (e.g., Sperk, 1993), while pilocarpine acts as a direct acting agonist at muscarinic cholinergic receptors to produce limbic seizures (e.g., Cavalheiro, 1995; Turski et al., 1989). In the present studies, the NPY knockout mice were significantly more susceptible to kainate- and pilocarpine-induced seizures than were the wild type mice both in terms of the lower dose required to produce seizures and the shorter latency to seizures, although it should be kept in mind that dose and latency were not entirely independent measures in the present studies since injections were administered

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every 20 min until seizures occurred. More knockout than wild type mice died after receiving kainate, even though the knockout mice received a lower average total dose of kainate. The present findings with kainate are generally consistent with those of Baraban et al. (1997) who reported that, while doses of kainate required to produce limbic seizures in NPY knockout mice as well as latencies to seizure onset were not different between knockout and wild type littermates, seizures in knockout mice progressed uncontrollably and with much higher mortality than in wild type mice. On the other hand, we did not observe a higher mortality rate in the NPY knockout mice after pilocarpine-induced seizures; rather, a higher mortality rate was observed in the wild type mice that had received the higher total dose of pilocarpine. Thus, within the 4-h observation period in the present studies, pilocarpine-induced limbic seizures did not progress uncontrollably in the same manner as kainate-induced seizures. However, since these doses of pilocarpine were lethal in most of the wild type mice, it would be of interest to evaluate the effects of lower doses of pilocarpine over longer time periods to determine if, at doses that were not lethal in NPY wild type mice, seizures progressed to a greater extent in NPY knockout mice. Thus, a lack of NPY had a similar influence on the seizure threshold for these two chemoconvulsants, but appeared to differentially alter their morbidity. The reasons for the greater mortality produced by kainate compared with pilocarpine are presently unclear. Both kainateand pilocarpine-induced seizures appear to produce similar long-term changes in expression of NPY and its receptors (e.g., Vezzani et al., 1994; Röder et al., 1996; Lurton and Cavalheiro, 1997; Borges et al., 2003), although acute changes in, for example, release of NPY after kainate and pilocarpine treatment have not been described. Further studies would be of interest to more clearly delineate potential changes in NPY release and expression produced by kainate and pilocarpine, as well as other chemoconvulsants with different mechanisms of action as well. In the present studies, we used colony bred 129S6/SvEv mice as wild type controls for knockout mice maintained by homozygous (−/−) matings. The present approach is consistent with the recommendations for wild type controls for lines maintained by homozygous matings published by The Jackson Labora-

tory (http://jaxmice.jax.org/library/faq/controls.html). Tschopp et al. (2002) have previously used the same wildtype controls. However, it is possible that the genetic background differed between the colony bred wild type and knockouts due to, for example, spontaneous mutations arising after separation of the stocks or random allele fixation during derivation (see e.g., Silva et al., 2002). One approach to ensuring that the phenotypic differences observed in the present studies are due to the presence or absence of the NPY gene is to use littemate controls from heterozygous matings and to carry out the breeding for several generations until the line is congenic, an approach that could be used in future studies. However, even this approach does not guarantee that the wild type and knockout mice differ only in the presence and absence of the gene product of interest as there is considerable genetic material in the regions flanking the mutant gene which can co-segregate with the mutant gene, but is not present with the wild type gene, that could have an impact on the phenotype of the mutant (e.g., Silva et al., 2002). Another approach is to study the mutation of interest in two or more genetic backgrounds to determine if the same phenotype is observed in each background, and determine if there are interactions between genetic background and the presence or absence of the gene product of interest. It would be of importance to address these issues in future studies. In summary, taken together with previous studies, the present results reinforce the important role of NPY in altering seizure thresholds and seizure severity, and provide further support for the hypothesis that agonists at one or more NPY receptors might provide a novel approach to the treatment of epilepsy. References Baraban, S.C., Hollopeter, G., Erickson, J.C., Schwartzkroin, P.A., Palmiter, R.D., 1997. Knock-out mice reveal a critical antiepileptic role for neuropeptide Y. J. Neurosci. 17, 8927– 8936. Bijak, M., 2000. Neuropeptide Y reduces epileptiform discharges and excitatory synaptic transmission in rat frontal cortex in vitro. Neuroscience 96, 487–494. Borges, K., Gearing, M., McDermott, D.L., Smith, A.B., Almonte, A.G., Wainer, B.H., Dingledine, R., 2003. Neuronal and glial pathological changes during epileptogenesis in the mouse pilocarpine model. Exp. Neurol. 182, 21–34.

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