Abstract

1. GABAB receptor antagonism abolishes the learning impairments in rats with chronic atypical absence seizures (가바B 수용체 길항작용은 만성 비전형 압상스 발작을 가진 쥐의 학습장애를 없앤다.) ***약물에는 상승작용, 길항작용, 상협작용이 있으며 길항작용은 약물이 서로의 작용을 못하게 하여 효과를 막거나 약하게 하는 것, 상반되는 두 가지 요인이 동시에 작용하여 그 효과를 서로 상쇄시킴.

2. Abstract Chronic atypical absence seizures are a component of the Lennox–Gastaut syndrome, a disorder invariably associated with severe cognitive impairment in children. However, the cause of this intellectual delay remains unclear. The AY9944 model of chronic atypical absence seizures in rats reliably reproduces the electrographic, behavioral, pharmacological and cognitive features of clinical atypical absence. Using this model, we tested the hypothesis that the cognitive impairment associated with this disorder involves a γ-aminobutyric acid B (GABAB) receptor-mediated mechanism. Therefore, we examined the effect of a specific, high affinity GABAB receptor antagonist, CGP35348, on the atypical absence seizures, the working memory deficits, and the altered long-term potentiation that we have observed in the AY9944 model. CGP35348 blocked atypical absence seizures, restored long-term potentiation to normal level, and reversed the cognitive deficit in the AY9944-treated animals. However, dose–response studies showed that lower doses of CGP35348 that failed to influence atypical absence seizure activity, completely reversed the spatial working memory deficit. These data suggest that GABAB receptor-mediated mechanisms are responsible for the cognitive dysfunction in the AY9944 model of chronic atypical absence seizures and further, that their cognitive impairment is independent of the seizure activity. The data raise the possibility that GABAB receptor antagonists may have therapeutic potential for the treatment of cognitive impairment in epilepsy syndromes where atypical absence seizures are a component. Keywords : GABAB receptor; CGP35348; Atypical absence seizure; Cognitive deficit; Radial arm maze

3. Article Outline 1. Introduction 2. Methods 2.1. Drugs 2.2. The AY9944-model 2.3. Surgery, electrocorticography and drug testing 2.4. Radial arm maze task 2.5. Hippocampal slice recording 3. Results 3.1. Minimal attenuation of chronic atypical absence seizures by low dosages of CGP35348 3.2. CGP35348 reversed the hippocampal-dependent spatial memory deficit in the radial arm maze task of AY9944-treated rats 3.3. CGP35348 reversed the impairment of long-term potentiation in AY9944 animals 4. Discussion Acknowledgements References

4. 1. Introduction In spite of the similar pharmacological profile of antiepileptic drugs between typical and atypical absence seizures, atypical absence differs from the more benign condition of typical absence in a number of ways clinically. First, atypical absence seizures are electrographically characterized by slow(i.e. <3Hz) spike-and-wave discharges that emanate from both hippocampal and thalamocortical circuitries while typical absence seizures are constrained within the thalamus and the cortex . Second, children with atypical absence can move in a semi-purposeful manner during seizures, while in typical absence seizures, there is a complete immobility with no purposeful movement. Third, semiologically the slow spike-and-wave discharges of atypical absence appear and disappear gradually, and thus are not time-locked to ictal behavior; whereas the spike-and-wave discharges in typical absence are time-locked to the ictal bahavior with abrupt onsets and offsets. Finally, children with typical absence seizures usually have normal cognition, but those with atypical absence seizures commonly show severe developmental delays . Rats with typical absence showed intact working memory, while AY9944-treated rats with atypical absence manifest spatial working memory deficit in the radial arm maze task and an impaired long-term potentiation, a form of synaptic plasticity that represents the molecular correlate of learning and memory. The seizures in rats with both typical and atypical absence seizure respond to ethosuximide and valproate, are blocked by γ-aminobutyric acid B (GABAB) receptor antagonists, and are intensified by GABAA and GABAB receptor agonists.

5. The GABAB receptor agonists have been shown both clinically, and in animal models, to exacerbate absence seizures and to impair cognition. we hypothesized that treatment with a GABAB receptor antagonist would reverse the cognitive deficit associated with AY9944-induced seizures. Our goals were (1) to address the question whether there was a cause–effect relationship between atypical absence seizure activity and cognitive impairment; and (2) to investigate the mechanisms of cognitive impairment using the AY9944 rat model of chronic atypical absence.

6. 2. Methods 2.1. Drugs AY9944 (trans-1,4-bis-cyclohexane dihydrochloride) was a gift from Wyeth-Ayerst (Philadelphia, PA). Ethosuximide (2-Ethyl-2-methylsuccinimide) was purchased from Sigma (Toronto, Canada). CGP35348, 3-Aminopropyl(diethoxymethyl)phosphinic acid, was a gift from Novartis (Basel, Switzerland). ***Ethosuximide : 뇌에서 비정상적인 전기활동의 증식을 줄인다. Absence seizure에 일차 선택 약. <부작용> 만성투에서 오심, 구토가 일어나며 혼미, 기면, 졸림, 불안, 집중력저하가 관찰, 민감한 사람에게선 두드러기가 일어날 수 있으며 백혈구 저하증, 재생불량성 빈혈 등이 일어남. 2.2. The AY9944-model Animals were maintained in a controlled environment with a 12-h light/12-h dark cycle (lights on at 6:00h) with food and water ad libitum. Experimental subjects received AY9944 at 7.5mg/kg s.c, while age-matched littermate control subjects were given an equivalent volume of 0.9% saline. AY9944 or saline was administered every six days from postnatal days 2 to 20 to all the pups. On postnatal day 21, male rats were randomly separated into two animals per cage and used for the following studies; all female animals were excluded to avoid the possible confound of their estrous cycle on slow spike-and-wave discharges and spatial performance.

7. 2.3. Surgery, electrocorticography and drug testing Chronic epidural electrodes were surgically implanted at postnatal day 22 for electrocorticographic recording to quantify the total slow spike-and-wave discharge duration of developmentally AY9944-induced atypical absence seizures. This age in rat is roughly analogous to 5–10 years in human. Surgery and then Following four days of recovery, electrocorticograms were recorded individually from freely moving rats in Plexiglas chambers between the hours of 9:30 and 14:30. All animals received saline, ethosuximide and all four doses of CGP35348. A total of twelve AY-treated animals were tested.

8. 2-4. Radial Arm Maze This task is used primarily to measure spatial learning and memory.  Some versions of the task can be used to examine concurrently both working and reference memory. This task is sensitive to hippocampal function.  The task is designed to mimic natural foraging behaviors..

9. All animals were handled regularly prior to the commencement of the radial maze task on postnatal day 24. Data were graphed as mean ± S.E.M. Differences among the groups related to the number of trials required to reach criterion, number of perfect entries, working memory errors and reference memory errors were compared using Analysis of Variance (ANOVA) followed by the Student–Newman–Keuls (SNK) post hoc test, with P < 0.05 considered as statistically significant. For the radial maze task, there were a total of 6 AY9944 and 6 control groups. The AY9944 groups were treated with: Saline (1 ml/kg; n = 9), ethosuximide (100 mg/kg; n = 7), CGP35348 (12.5 mg/kg; n = 9), CGP35348 (25 mg/kg; n = 9), CGP35348 (50 mg/kg; n = 9), or CGP35348 (100 mg/kg; n = 8). The control groups were also treated with: saline (1 ml/kg; n = 10), ethosuximide (100 mg/kg; n = 7), CGP35348 (12.5 mg/kg; n = 7), CGP35348 (25 mg/kg; n = 7), CGP35348 (50 mg/kg; n = 7), or CGP35348 (100 mg/kg; n = 7).

10. 2.5. Hippocampal slice recording Hippocampal slice recordings were carried out exactly as detailed in Chan et al. (2004). Groups of 3–5 months old AY9944 and control rats were sacrificed and the brains were rapidly removed. Hippocampal slices were cut cold at 400 μm in thickness and constantly bathed in artificial cerebrospinal fluid (aCSF); aCSF contained (in mM) 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1.6 MgSO4, 2 CaCl2, 26 NaHCO3 and 10 d-glucose, equilibrated with 95% O2–5% CO2. The magnitude of long-term potentiation was averaged from 35–45 min after high frequency stimulation and compared with Student's t-tests for statistical differences at P < 0.05 between AY9944 and control subjects. At least two slices were tested from each animal, and eight animals from each group were used in this procedure.

11. 3. Results 3.1. Minimal attenuation of chronic atypical absence seizures by low dosages of CGP35348 The effects of saline, 100 mg/kg ethosuximide and 12.5–100 mg/kg CGP35348 on atypical absence seizures were determined using a randomized crossover design in pre-pubertal AY9944-treated animals (n = 12). All AY9944 animals exhibited similar discharge duration prior to injection with saline, ethosuximide and CGP35348. Following the injection, all agents significantly reduced discharge duration from baseline during the first 20 min (P < 0.05, RM-ANOVA using post hoc Holm–Sidak method). At subsequent periods, however, only 100 mg/kg of either ethosuximide or CGP35348 significantly attenuated slow spike-and-wave discharges until al least 120 min post-injection (P < 0.05, RM-ANOVA using post hoc Holm–Sidak method). For CGP35348, dosages < 100 mg/kg (i.e., 12.5, 25, and 50 mg/kg) did not statistically change from baseline seizure duration during the period of 20–180 min after injection.

12. Fig. 1. Chronic atypical absence seizures were attenuated with 100 mg/kg of CGP35348 and ethosuximide. Seizure durations were quantified from electrocorticographic recording of twelve AY9944 animals during 1 h prior to and 20 or 60 min epochs after saline, CGP35348 or ethosuximide injection. All animals were tested in a random order with all the dosages and/or drugs. *denotes statistical difference from pre-injected baseline duration with P < 0.05 using RM-ANOVA followed by the post hoc Holm–Sidak method.

13. 3.2. CGP35348 reversed the hippocampal-dependent spatial memory deficit in the radial arm maze task of AY9944-treated rats The cognitive performance of all groups is presented in Fig. 2. This was evaluated by the number of training trials required to learn the task (Fig. 2A), the number of working (Fig. 2B) and reference (Fig. 2C) memory errors in attaining the criterion, and the number of perfect entries (Fig. 2D). drug-naïve (saline-treated) AY9944 animals performed more poorly than all controls animals (i.e., non-AY9944-treated). The former required a significantly greater number of trials to reach criterion (P < 0.05, ANOVA using SNK post hoc; Fig. 2A), committed a higher number of working memory errors (P < 0.05, ANOVA using SNK post hoc; Fig. 2B), and scored a fewer number of perfect entries during acquisition (P < 0.05, ANOVA using SNK post hoc; Fig. 2D).

15. Fig. 2. All dosages of CGP35348 treatment alleviated the impaired performance of AY9944-treated animals during the acquisition of radial arm maze task. Both AY9944 (AY) and control (CTL) animals were administered with saline (Sal), 100 mg/kg ethosuximide (ESM) or 12.5–100 mg/kg CGP35348 (CGP) at least 20 min prior to the maze testing. The number of animals in each group is specified in the Method section. Their performance was compared with ANOVA followed by the post hoc test of SNK and *indicates statistical significance at P < 0.05 from saline-treated AY9944 animals. A) The number of training trials required to reach the criterion of obtaining ≥ 3 baits consecutively with zero working memory error during a trial. B) The number of average working memory error committed in reaching the criterion. C) The number of average reference memory error committed in reaching the criterion. D) The total number of perfect entries scored during acquisition. In summary, the impaired spatial hippocampal-dependent memory function in the radial maze task of AY9944 rats can be completely corrected by all dosages of CGP35348, but not by ethosuximide.

16. 3.3. CGP35348 reversed the impairment of long-term potentiation in AY9944 animals The effect of 100 μM CGP35348 on in vitro long-term potentiation was examined in the hippocampal slices from adult AY9944 (n = 8) and control (n = 8) animals. For comparison purposes, we have included in Fig. 3A the attenuated long-term potentiation of AY9944 (142.68 ± 6.13%) versus the control (180.03 ± 5.75) animals in the absence of CGP35348, which has been previously published in Chan et al. (2004). Using the same electrophysiologic paradigm, 100 μM CGP35348 rescued the impaired neuroplasticity in AY9944 animals and allowed them to obtain similar long-term potentiation as the control animals.

19. 전기적 시냅스 두 세포 사이에 connexin이라는 단백질이 통로를 만들어 (이 통로를 gap junction 이라고 함.) 시냅스 전 세포의 전기적 신호가 바로 시냅스 후 세포에 전달 됨. 화학적 시냅스에 비하여 빠르게 신호를 전달 할 수가 있으며, 심장 세포 등에 분포. 화학적 시냅스 보통 신경 세포에서 말하는 시냅스. 신경세포체에서 만들어진 활동전압이 축삭을 따라서 말단으로 전달되면, 축삭 말단의 전압 의존성 칼슘 이온 통로가 열리고, 상대적으로 칼슘 이온 농도가 높은 세포 외부로부터 칼슘이 세포 내부로 유입. 신경 세포 축삭 말단의 세포질 내 칼슘 이온 농도가 높아지고, 그러면 이것을 신호로 신경 전달 물질을 담고 있던 vesicle(작은 주머니, 소포체)이 세포막에 융합되며, 신경 전달 물질이 신경 연접부(synapse)로 방출됨. 이 때 방출되는 신경 전달 물질의 종류는 세포 마다 아주 다양한데, 대표적으로 흥분성 시냅스의 경우 glutamate, 억제성 시냅스의 경우 GABA가 exocytosis에 의해 방출됨. 이러한 신경 전달 물질이 시냅스 후 세포의 수용체(receptor)에 작용하게 됨. 시냅스 후 세포의 수용체도 몇 가지 종류가 있는데, 수용체 자체가 이온통로일 경우, 신경 전달 물질에 의해 이온 통로가 열리고, 이온이 세포 내로 또는 방출되어, 시냅스 후 세포에서 또 다시 활동 전압을 일으키거나 억제 함. 경우에 따라 수용체가 효소이거나 효소 활동을 매개하는 단백질인 경우도 있음.

22. 압상스"(absence) 발작이란 ? 과거에 소발작이라고 불리던 것으로 전신발작. 대개 5 - 15초 동안 의식만 살짝 잃음. 자세히 보지 않으면 옆에서도 모르고지나가는 경우가 많으며 대개 말을 하다가 또는 어떤 행 동을 하다가 잠깐 멈추게 됨. 잠깐 멍한 상태로 표출되며 눈을 깜박거리거나 눈동자가 살짝 위로올라가기도 함. 발작이 끝나면 곧 정상으로 돌아옴. 대개 어린이들에서 주로 발생하는데 적시에 진단을 받지 못하게 되면, 학교에서 정신집중을 못한다고 선생님께 혼나는 경우도 있는데 때때로 학습의 장애를 초래할 수도 있음. 대개 청소년기에 들어가면 없어지나 간혹 다른 형태의 발작으로 진행하는경우도 있으며, 필요시 적절한 항경련제의 투입으로 이를 막을 수도 있음.

23. * 레녹스 가스토 증후군(Lennox Gastaut Syndrome)을 흔히 영문 약자로 LGS라 한다. LGS는 약물로 조절하기 매우 힘든 유형의 간질로서 일반적으로 1-8세 사이의 어린 나이에 시작되며, 그 특성은 여러 유형의 발작이 나타나고 발달 장애를 동반한다. 간질 아동의 3-11% 정도가 LGS로 진단되고 있는 것으로 추산되고 있다. 평균적으로 만 3세에 LGS가 시작된다. * 작동기억(working memory) : 우리가 무엇을 생각하거나, 계산하거나, 말하거나 하는 그때마다 사용하는 기억을 말한다(이러한 활동을 통틀어 '인지활동'이라한다).

24. LTP(Long-Term Potentiation) 현상은 1973년 영국의 생리학자인 Timoth Bliss와 노르웨이의 Terje Lomo가 최초로 발견한 신경의 현상, 두뇌의 해마 부분(해마 상융기)에서 일어나는 학습과 기억을 설명하는 신경가소성(plasticity)의 기제를 말한다.

25. 만성 비전형 압상스 발작은 어린이들에게 있는 심한 인지장애와 관련된 장애인 레녹스-가스토 증후군의 구성 요소이다. 그러나 이 지적지체의 원인은 불분명하게 남아있다. 쥐에 있는 만성 비전형 압상스 발작의 AY9944 모형은 임상의 비전형 압상스의 전기기록, 행동, 약리학과 인식 특징을 확실히 재생한다. 이 모형을 사용하여, 우리는 이 장애와 관련된 인지손상이 가바B 수용체에 중재 된 메커니즘과 밀접한 관계가 있다는 가설을 시험했다. 그러므로 우리는 AY9944 모형에서 관찰한 특정하고 높은 친화력 가바B 수용체 길항제와 CGP35348과 비전형 압상스 발작에서의 작동기억장애 및 대체된 LTD(장기 깅억 강화 현상)의 효력을 시험했다. CGP35348은 AY9944 처리한 동물에 있는 비전형 압상스 발작을 막고, 장기약효증가를 정상수준까지 복구하고, 인지손상을 반전시켰다. 그러나 복용량 반응연구는 CGP35348의 낮은 복용량은 불규칙한 압상스 발작 행동에 영향을 끼치는 공간의 작동기억장애를 완전히 반전시키는 데는 실패했다는 것을 보여주었다. 이 자료는 가바B 수용체에 중재 된 메커니즘은 만성 비전형 압상스 발작의 AY9944 모형에서 인지기능장애에 책임이 있고, 더 나아가서 인지손상은 발작 행동과 관계가 없다는 것을 암시한다. 이 자료는 가바B 수용체 길항제가 비전형 압상스 발작의 구성 요소인 간질 증후군의 인지손상을 치료할 수 있는 가능성을 높인다.

26. <GABA의 효능> 1.뇌중의 GABA 함량이 감소되면 알콜성 뇌질환을 유발할 가능성이 높고, 또한 간질환자 및 간 경화증 환자의 경우 정상인 보다 GABA함량이 낮은 것으로 보고되고 있다. 2. 식물에서는 병원성 방어기작에 중요한 역할을 함 (Zimmerli et al., 2000). 3. 일반적으로 숙취해소, 불안감 해소, 고혈압강하, 인슐린 효과의 증대, 식욕 감퇴 및 우울증 등에 효과가 있음 4. 뇌세포 대사 기능을 활발하게 함으로서 중풍 치매 예방, 정신집중력 강화, 기억력 증진, 불면 등에 효과를 인정받고 있음 - 수험생 관련 기능성 식품 시장에 적용이 용이함 5. 간기능 활성과 알코올 대사 촉진 기능 - 숙취제거 음료에 이용되기도 함 6. 기타 비만해소 작용과 신장 기능 촉진 작용이 있음 7. HGH (human growth hormone)분비 자극- 청소년 성장 및 발육 촉진