open field test in an angelman syndrome mouse model

行動試験を用いたアンジェルマン症候群のマウスモデルの検証

Angelman syndrome (AS) is a rare neurodevelopmental disease. The most common genetic origin of AS is a large deletion of the 15q11-q13 region of the maternally-derived chromosome, which is found in nearly 74% of patients1. Deletion of this region causes the loss of UBE3A, the main causative gene of AS that encodes an E3 ubiquitin ligase. The paternal allele of the UBE3A gene in neurons is silenced in a process known as imprinting. As a consequence, paternal imprinting of the gene allows only maternal expression in the central nervous system (CNS)2. Therefore, UBE3A gene deletion from the maternally-derived chromosome leads to the development of AS symptoms. In humans, AS manifests at around 6 months of age, with developmental retardation that persists throughout all developmental stages and results in severe debilitating symptoms in affected individuals3,4. The core symptoms of the disorder include the deficit of fine and gross motor skills, including jerky ataxic gait, serious speech impairment, and intellectual disability. Approximately 80% of AS patients also suffer from sleep disturbances and epilepsy. To date, the only available treatment are symptomatic drugs, which reduce epileptic seizures and improve sleep quality1. Therefore, the development of robust animal models with reproducible behavioral phenotypes alongside refined phenotyping analysis will be essential to elucidate the pathophysiological mechanisms of the disorder and discover effective medications and treatments.
The complexity of the human disorder affecting the CNS demands model organisms to possess a comparable genome, physiology, and behavior. Mice are popular as a model organism due to their short reproductive cycle, small size, and relative ease of DNA modification. In 1984, Paul Willner proposed three basic disease model validation criteria: the construct, face, and predictive validity, which are used to determine the model&#39;s value5. Simply, construct validity reflects the biological mechanisms responsible for the disorder development, face validity recapitulates its symptoms, and predictive validity describes the model response to therapeutic drugs.
To adhere to the above principles, we have chosen the most common genetic etiology, a large deletion of the maternal 15q11.2-13q locus including the UBE3A gene, to create AS model mice. We used the CRISPR/Cas9 technique to delete a 76,225 bp long region spanning the entire UBE3A gene, encompassing both the coding and non-coding elements of the gene, in mice from a C57BL/6N background6. We then bred the animals to obtain UBE3A+/&#8722; heterozygous mice. For face validation of the model, we used animals from crosses of UBE3A+/&#8722; females and wild-type males to gain UBE3A+/- progeny (strain named C57BL/6NCrl-UBE3A&#60;em1(IMPC)Ccpcz&#62;/Ph and later assigned as UBE3AmGenedel/+) and control littermates. We tested their fine and gross motor skills, emotionality, and affect to recapitulate core AS symptoms. In a previous article, we also evaluated the animals&#39; cognitive functions, as AS patients also suffer from intellectual disability6. However, we found no cognitive impairments in UBE3AmGenedel/+ mice, perhaps due to the young age of the animals at the time of testing7. Later examination of the older animals, around 18 weeks old, revealed a deficit in behavioral flexibility during reversal learning in the place preference paradigm. However, the complexity of the employed equipment for this analysis requires a separate methodological module and it is not included here.
The behavioral tests presented here belong to the common phenotyping tools in genetic research, thanks to their high predictive value and sufficient construct validity8,9,10. We used these tests to validate a mouse model of AS by recapitulating core symptoms of the human disease in a reproducible, age-independent manner. The emotionality of the animal was evaluated in the elevated plus maze and open field tests. Both of these tests are based on the approach-avoidance conflict, where animals explore a new environment in search of food, shelter, or mating opportunities while simultaneously avoiding anxiogenic compartments11. Additionally, the open field test is used to test a mouse&#39;s locomotor activity8. The tail suspension test is widely used in depression research to screen for new antidepressant drugs or depressive-like phenotypes in mouse knockout models12. This test evaluates the despair that animals develop over time in an inescapable situation. Motor learning and detailed gait characteristics were determined on the rotarod and in DigiGait, respectively. Animal endurance on the accelerating rod characterizes its balance and movement coordination skills, while detailed analysis of a mouse&#39;s step patterns is a sensitive evaluation of neuromuscular impairments connected to many neurogenerative movement disorders13,14,15. The nestlet shredding test is part of the standard methodology for detecting impulsive behavior in rodents, and since it utilizes natural rodent building behavior, it indicates the animal&#39;s well-being16,17.
The size of the experimental groups was a result of a compromise to meet the 3R rule demands and efficient usage of colony breeding performance. However, to obtain statistical power, the groups had no fewer than 10 individuals, due to the establishment of a sufficient amount of breeding pairs. Unfortunately, breeding performance did not always result in a sufficient number of animals.

著者スポットライト:アンジェルマン症候群マウスモデルを検証するための再現性の高い一連の行動試験 

アンジェルマン症候群モデルマウスの行動特性

Watch this Scientific Journal Video about Open Field Test in an Angelman Syndrome Mouse Model at JoVE.com

Open Field Test in an Angelman Syndrome Mouse Model  

アンジェルマン症候群マウスモデルにおけるオープンフィールド試験

4つのオープンフィールド(OF)テストボックスをカメラの下のテストプラットフォームに配置します。壁のポテンショメーターを使用して、調整中にセンサーが各ボックスの中央に配置されたルクスメーターを使用して、各OFテストの中央で光強度を200ルクスに調整します。ビューアソフトウェアアイコンをダブルクリックしてソフトウェアを開き、設定タブの左上にあるアイコンをクリックしてEFテスト用の設定をロードします。実験タブの対応するフィールドに動物情報を入力します。ゾーンの位置がOFテストボックスと一致することを確認し、必要に応じて調整します。ビジュアルコントロールとコンピューターマウスを使用して、仮想アウトラインの中心ゾーンと周辺ゾーンが対応するOFテストゾーンと一致していることを確認します。取得タブの左上にある矢印アイコンにマウスカーソルを置きます。ホームケージから4匹の動物を取り出し、各テストボックスの隅に置きます。コンピューターのマウスを左クリックして実験を停止します。OF試験では、母体のヘテロ接合型Ube3a変異動物は、より短い横断距離、より低い平均速度、およびより長い休息時間によって反映されるように、有意に低活性であった。しかし、不安様行動に関連するパラメータはいずれもUbe3a変異体では変化しなかった。

open-field-test-in-an-angelman-syndrome-mouse-model

Research

JoVE Journal

Behavior

Open Field Test in an Angelman Syndrome Mouse Model

570 ビュー. 4つのオープンフィールド(OF)テストボックスをカメラの下のテストプラットフォームに配置します。壁のポテンショメーターを使用して、調整中にセンサーが各ボックスの中央に配置されたルクスメーターを使用して、各OFテストの中央で光強度を200ルクスに調整します。ビューアソフトウェアアイコンをダブルクリックしてソフトウェアを開き、設定タブの左上にある?...

ビデオ: Open Field Test in an Angelman Syndrome Mouse Model  

Behavioral Characterization of an Angelman Syndrome Mouse Model

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of AS. We use the rotarod learning paradigm, detailed gait analysis, and nest building test to detect and characterize animal motor impairments. We test animal emotionality in the open field and elevated plus maze tests, as well as the affect in the tail suspension test. When AS mice are tested in the open field test, the results should be interpreted with care, since motor dysfunctions influence mouse behavior in the maze and alter activity scores.
The reproducibility and effectiveness of the presented behavioral tests has already been validated in several independent Uba3a mouse lines with different knockout variants, establishing this set of tests as an excellent validation tool in AS research. Models with the relevant construct and face validity will warrant further investigations to elucidate the pathophysiology of the disease and grant the development of causal treatments.

This manuscript presents a set of highly reproducible behavioral tests to validate an Angelman syndrome mouse model.

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of ...

行動学

Elevated Plus Maze Test in an Angelman Syndrome Mouse Model

アンジェルマン症候群マウスモデルにおける上昇プラス迷路テスト

Tail Suspension Test in an Angelman Syndrome Mouse Model

アンジェルマン症候群マウスモデルにおける尾懸垂試験

Gait Analysis in an Angelman Syndrome Mouse Model

アンジェルマン症候群マウスモデルにおける歩行解析

Rotarod Test in an Angelman Syndrome Mouse Model

アンジェルマン症候群マウスモデルにおけるロータロッド試験

Nestlet Shredding: Nest Building Test in an Angelman Syndrome Mouse Model

ネストレットシュレッダー:アンジェルマン症候群マウスモデルにおける巣作り試験