Name: correlating gene-specific dna methylation changes with expression and transcriptional activity of astrocytic kcnj10 (kir4.1)
Uploaded: 2015-09-26T15:00:00.000+00:00
Duration: 11 min 19 s
Description: Watch this Scientific Journal Video about Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1 at JoVE.com

This work was supported by R01NS075062-01A1. FACS sorting performed at UAB Comprehensive Flow Cytometry Core facility (P30 AR048311, P30 A1027767). Dr. Scott Philips from the UAB Neurobiology Core facility and Dr. Susan Nozell from UAB CDIB assisted with technical aspects of the luciferase assay.

모든 동물은 건강 지침의 국립 연구소에 따라 처리되었다. 버밍엄에서 알라바마 대학의 동물 관리 및 사용위원회는 동물의 사용을 승인했다. 1. 전체 뇌 조직에서 성상 (FACS)을 정렬 형광 활성 셀을 사용하여 풍부한 성상 세포 인구에서 RNA와 DNA를 얻기 <ol><li> 1 분 동안 이산화탄소 후 빠르게 목을 벨과 진정 동물. 앨버 커키 등의 알에 설명 된대로 피질을 해부, 26.; 그것은 수막을 제거 할 필요가 없다. 참고 :. S100β에서 eGFP는 (성상 세포 마커) 프로모터를 발현하는 형질 전환 쥐 이타 쿠라 등 (27)에 의해 생성 및 성상 세포의 FACS 정렬을 위해 사용되었다. </li><li> 제조사의 프로토콜에 따라 비 멸균 조건 하에서 파파인 해리 키트를 사용하여 전체 뇌 균질 물을 준비한다. 10 분 동안 물을 욕조에 37 ℃에서 파파인을 열 활성화합니다. 노트 :파파인 솔루션은 제조업체에서 제공 및 L 시스테인 및 EDTA를 (재료의 표 참조)이 포함된다. <ol><li> 절단 또는 튜브는 95 % O 2 반송하도록 50 ㎖ 원뿔형 튜브의 상단에 구멍을 드레 멜 : 5 % CO 2는 폐쇄 원추형 튜브에 공급되는 (도 1a 참조). 해리 미디어 함유 10mm 배양 접시에 해부 피질 배치 (하여 20 mM 글루코스 및 페니실린 / 스트렙토 마이신 (500 U / ml)로 보충 된 95 %의 O를 평형화 MEM을 2 : 5 % CO 2) (1)에 조직을 말하다 깨끗한 면도날을 사용 X 1mm 2 개. </li></ol></li><li> 파파인 함유 용액 50 ㎖ 원뿔형 튜브에 10 ㎖를 사용하여 수동 pipetman 전사 조직. 조직이 이월 해리 미디어의 양을 최소화하기 위해 배출 전에 전송 피펫의 바닥에 정착하도록 허용합니다. 95 % O 2로 평형화 파파인 용액을 보관 : 5 % CO 2 항온 처리 기간 동안 표면을 통하여 가스 교환. 하지 거품 파파인 soluti를 수행에. 37 ° C의 물을 욕조에 20 분 동안 조직을 품어. </li><li> 파파인 솔루션에 다음과 배양, 씹다 조직 느린 속도로 10 ml의 전송 피펫으로 10 배. 실온에서 5 분 1,000 XG에 원심 분리기 흐린 세포 현탁액. <ol><li> 표면을 통한 가스 교환 (제조사에 의해 제공된)의 DNase / 억제제 알부민 용액과 평형의 DNase / 억제제 알부민 용액 3 ml의 세포 펠렛을 다시 일시. 제조업체의 지침에 따라 상업 불연속 밀도 기울기를 준비합니다. </li></ol></li><li> 6 분 동안 1,000 XG 불연속 밀도 구배 스핀. 피펫을 사용하여 바닥 펠렛을 흡입에 의해 튜브의 바닥에서 해리 세포를 분리. </li><li> 0.02 % 소 혈청 알부민 및 1 ㎎ / ㎖의 DNase 또는 배양 배지 HEPES 완충 바람직 DPBS로 2-3 ml 중에 해리 된 세포를 다시 일시 중지. FACS (그림 2A) 전에 40 μm의 필터를 통해 전달합니다. 정렬 될 때까지 얼음 세포를 유지합니다. <ol><li> 외과 (28)를 수행합니다. &lt;/ 리&gt; <li> 4 ℃에서 5 분 2,000 XG에 1.5 ㎖의 원심 분리기 튜브에서 펠렛 세포. 주 : 펠릿의 성상은 즉시 사용 또는 DNA 추출까지 -80 ° C에 보관 될 수있다. </li></ol></li><li> RNA와 DNA를 선호하는 분리 방법 (29) (30)를 사용하여 압축을 풉니 다. 분광 광도계와 bioanalyzer (31)를 통해 RNA 및 DNA 농도와 품질을 평가합니다. 주의 : 후속 단계에서 단지 고품질 RNA 및 DNA를 활용하여 각각 280분의 260 = 2.0~2.2 및 1.8-1.9를. Bioanalyzer 분석은 RNA 분해 또는 DNA 파편에 대해 평가하는 것이 필수적이다. RNA 또는 DNA는 즉시 사용하거나 또는 -20 ° C, 각각 후속 연구를위한 C °에서 -80 저장할 수 있습니다. </li></ol> 2. 메틸화에 민감한 고해상도 용융 분석 (MS-르마)를 사용하여 유전자의 DNA 메틸화 상태를 평가 <ol><li> 유전자에 어떤의 CpG 섬을 식별하는 것이 바람직 온라인 메틸화 매핑 소프트웨어에 관심의 유전자 서열을 입력관심 (32). <ol><li> 중아 대해 디자인 된 프라이머는 DNA 서열 (33)를 변환하여 제조사의 프로토콜에 따라 바람직한 DNA 중합 효소를 이용하여 증폭한다. 45 분 동안 100 V에서 1 % 아가 로스 DNA 젤을 실행하여 증폭 산물의 크기를 확인합니다. -20 ° C에서 20 μM의 재고 농도 스토어 프라이머. </li></ol></li><li> 중아는 같은 동물 종 (34)로부터 0~100%에 이르기까지 각각의 샘플과 메틸화 된 DNA 표준 DNA의 500 ~ 1,000 NG를 변환합니다. 용출 시료 20 NG / μL의 농도를 제공한다. 분광 광도계 (31)를 통해 중아 변환 된 DNA의 농도를 확인합니다. </li><li> 표 1 및 2에 따른 5 μM 농도에서 바람직한 DNA 폴리머 라제 및 프라이머를 사용하여 MS-HRM 증폭 용 셋업 20 μL 반응. 중으로, FACS의 DNA 메틸화 및 표준을 포함하여 모든 샘플을 실행할. </li><li> 분석 소프트웨어에 따라, 미리 설정하고 시작 매개 변수를 중지 포스트용융 곡선의 천이 주위. 세트 미리 용융 개시 및 둘 사이의 차이가 0.​​2 정도로 미리 용융 정지 파라미터 - 0.5 ° C. 후 용융 시작 및 사후 용융 유사 중지를 설정합니다. 각 샘플에 대한 피크 온도 차이 데이터의 압축을 풉니 다. </li><li> 메틸화 백분율 기준 (Y 값)과 대응하는 평균 피크 온도차 (x 값)을 사용하여 선형 회귀 방정식 (도 3A-B, 표 3-4)을 생성한다. 미지 시료 (35)의 메틸화 상태를 추정하기 위해 선형 회귀 방정식을 사용합니다. </li></ol> 3. 루시퍼 라제 분석의 사용을 통해 하이퍼 메틸화 발기인 활동 평가 <ol><li> 표적 유전자 (2.1 단계)의의 CpG 섬을 식별합니다. 이 PCR의 CpG 섬 luc2-37 플라스미드를 제조 luc2 반딧불 루시퍼 라제 리포터 유전자의 상류에 관심 36 클론의 영역을 증폭. </li><li> 제한을 통해의 CpG 섬 - luc2 플라스미드의 30 μg의 선형화효소 소화 38. 제한 다이제스트에 대한 사이트를 확인하고 원하는 커터 소프트웨어 (38)를 사용하여 두 번 상처를 피할 수 있습니다. 제조 업체의 프로토콜에 따라 소화를 다음과 같은 적절한 온도와 시간에 효소를 열 - 불 활성화. DNA의 최소 손실은 선형화 단계에서 발생합니다. </li><li> 30 ° C에서의 CpG 메틸화 (M.Sssl) O / N을 사용하거나 다음과 같은 조정을 제외하고 처리되지 않은 다음과 같은 제조 업체 프로토콜을 남겨 메틸화 선형화 플라스미드. </li><li> 50 μL 반응을 수행합니다. </li><li> 선형화 플라스미드 700 NG 메틸화에의 CpG 메틸화의 5 단위를 사용합니다. </li><li> 13 ~ 19 시간 동안 반응 O / N을 실행합니다. </li><li> 의 CpG 메틸화 반응에 따라, 시판되는 실리카 - 겔 막의 제조 업체의 프로토콜에 따라 키트를 정리 표준을 사용하여 DNA 정리를 수행합니다. DNA의 상당한 손실은 메틸화 된 CpG 반응 30-60 %의 감소에 따라 발생한다. </li><li> HPA II 제한 다이제스트를 통해 플라스미드의 메틸화를 확인합니다. </lI&gt; <li> 메틸화 또는 비 메틸화 된 DNA 1 μg의를 가지고 제한은 37 ℃에서 1 시간 동안 HPA II 다이제스트. 각 μg의 DNA를 위해 HPA II의 5 ~ 10 단위를 사용합니다. HPA II 소화에 따라, 선호, 시판되는 실리카 - 겔 막 청소 키트를 사용하여 DNA 정리를 수행합니다. 시각화를위한 45 분 (그림 4B) 100 V에서 TAE 나 TBE 버퍼에 1 % 아가 로스 DNA 젤에 모두의 CpG 메틸화 및 비 메틸화 된 플라스미드를 실행합니다. </li><li> 적절한 제한 효소와 더블 소화에 따라 모두 메틸화 및 비 메틸화 플라스미드는 전체 길이 루크 2 (벡터)과의 CpG 섬 (삽입) 38 조각을 분리합니다. 제조 업체의 프로토콜에 따라 소화 다음 적절한 온도와 열을 비활성화 효소에서 더블 소화 O / N을 수행합니다. DNA 농도의 손실을 최소화 이중 제한 다이제스트 동안 발생합니다. </li><li> O를 분리 할 수​​ 있도록 1 시간 동안 100 V에서 1 %의 DNA 아가 로스 젤을 두 번 소화 플라스미드를 실행F 벡터 삽입 (그림 4C). 주의. 보호 자외선 얼굴 가리개를 착용, 밴드를 시각화 탁상 블랙 라이트에 DNA 젤을 배치합니다. 깨끗한 수술 블레이드를 사용하여 크기, 소비세 메틸화 및 비 메틸화 삽입 및 비 메틸화 된 벡터 기준으로합니다. </li><li> 포화 페놀, pH가 6.6을 사용하여 젤 추출 DNA. 간단히, 벡터 또는 삽입을 포함하는 DNA 젤 무게. DNA 젤 0.1 g 당 페놀의 100 μl를 사용합니다. 유리 다운스 균질 기에서 페놀을 사용하여 DNA 겔 균질화. </li><li> 클로로포름 (페놀 량의 1/5 사용)을 추가하고 20 초 동안 샘플을 흔들. 2 ~ 3 분 동안 실온에서 샘플을 품어. 4 ° C에서 최대 속도 (16.1 X 1,000 XG)에서 15 분 동안 원심 분리기. </li><li> 수용액을 제거하고, 3 M 아세트산 나트륨 및 에탄올 2.5 배 부피의 0.1X 볼륨. -80 ° C에서 1 시간 동안 샘플을 품어. 인큐베이션 후, 에탄올을 제거하고 바람직한 완충액 30 μL에 DNA를 다시 일시. DNA의 상당한 손실이 삽입 및 벡터의 다음 분리를 발생, 30 % ~ 50 % 싸다SS. </li><li> 재 결찰을 메틸화 및 비 메틸화 된 인서트 T4 DNA 리가 제 (38)를 이용하여 벡터를 비 메틸화. 결찰 반응에 삽입하는 벡터의 4 비율 : 1을 사용합니다. 제조업체의 지침에 따라 설치 반응. 반응 50 μL의 총 부피를 사용하여 C ° 또는 얼음 양동이 위에 뚜껑 얼음에 -20 O / N을 품어. </li><li> 결찰 후, 선호, 시중에서 판매하는 실리카겔 막 청소 키트를 사용하여 DNA 정리를 수행합니다. DNA의 상당한 손실은 연결 반응, DNA의 대략 30 % ~ 50 %의 손실에 따라 발생한다. 45 분 동안 100 V에서 1 % 아가 로스 DNA 젤에 실행하여 재 결찰을 확인하고 재 결찰 플라스미드 (그림 4D)의 농도를 평가합니다. </li><li> 제조사의 프로토콜에 따라 상업적 형질 전환 시약을 이용하여 세포 내로 D54 메틸화 또는 비 메틸화 된 플라스미드를 형질 감염. 잘 0.14 × 10 6 세포 / 12 웰 판에 씨앗 D54 셀. </li><li> 24 시간 후, 형질 세포 위스콘신(1) 비 methyated의 CpG-luc2 플라스미드 + 레 닐라 또는 다른 제어 루시 페라 벡터 또는 (2) 메틸화 된 CpG-luc2 플라스미드 + 레 닐라 또는 다른 제어 루시 페라 벡터 중 하나의 일 동일 농도. </li><li> 세포가 듀얼 루시 페라 제 분석을 수행하기 전에 24 시간 동안 형질을 허용합니다. 제조업체의 지침에 따라 분석을 수행합니다. 루미를 사용하여 측정을 수행합니다. 세중에서 잘 각을 읽어보십시오. </li><li> 레 닐라하는 반딧불 루시 페라 제 활동의 비율 또는 기타 제어 루시퍼 라제 활성을 계산합니다. 비 메틸화를 제어 반딧불 루시페라아제 활성 : 메틸화 반딧불 정상화 제어 루시퍼 라제 활성을 비 메틸화 된 루시퍼 라제 활성에 의해 메틸화 된 루시퍼 라제 활성을 나누어 </li></ol>

The authors have no disclosures.

This protocol describes the isolation of an enriched population of astrocytes via FACS as well as a variety of techniques that allow for both correlative and causative studies between DNA methylation and gene expression. These techniques, used in isolation or in combination, are particularly useful for laboratories that work with tissue of high cellular heterogeneity or are interested in the DNA methylation status of a particular gene or gene region versus global DNA methylation changes. One relatively unique challenge i...

Epigenetics is the study of chemical modifications that can affect the transcriptional activity of the genome. Essentially, without a change in the DNA sequence, epigenetic modifications such as DNA methylation, histone acetylation, and histone methylation are sufficient to reversibly alter patterns of gene expression 1. DNA methylation, a potent regulator of gene expression, is the most well characterized epigenetic modification. DNA methylation is the covalent attachment of methyl groups on the C5 position of a cytosine, typically the cytosine of a cytosine-guanine dinucleotide, also known as a CpG site. Areas that contain a high density of CpG sites are known as CpG islands (CGIs). CGIs are frequently associated with transcriptional start sites (TSS) and gene promoters 1-3. Thus, while changes in DNA methylation at CGIs are not always concomitant with changes in cellular expression or function, changes in DNA methylation at CGIs can exert powerful regulation on transcriptional activity 2. 
Historically, DNA methylation was observed to be essential in embryogenesis, imprinting, and development, with little changes in the levels of DNA methylation occurring in post-mitotic cells (with the exception of alterations in cancer-related genes) 4,5. However, the field of neuroepigenetics has highlighted an important non-developmental role for DNA methylation. Specifically, cognitive epigenetics has redefined DNA methylation as a highly plastic mechanism integral in mediating both the transcriptional activation and repression of genes essential for the process of learning and memory 6. Apart from cognitive epigenetics, studies modeling ischemic injury and neuropathic pain characterize DNA methylation as a labile mechanism that responds rapidly to a variety of CNS insults 7-9. In regards to astrocytes, several lines of evidence suggest DNA methylation plays an important role in astrogliogenesis. Fan et al., found that conditional KO of DNMT1 in neural progenitor cells (NPCs) resulted in precocious development of astrocytes concordant with a global state of hypomethylation 10. Additionally, Perisic et al., concluded differential levels of DNA methylation of the GLT-1 promoter mediated differential levels of expression of the glutamate transporter in the cortex and cerebellum, emphasizing a role in DNA methylation in establishing brain-region specific patterns of astrocytic gene expression 11. Overall, numerous studies underscore the dynamic and labile nature of DNA methylation in the CNS as environment, drugs, and injury have all been shown to change DNA methylation and often, gene expression 4,9. Together, these neuroepigenetic studies point to DNA methylation as a feasible therapeutic target with the potential to mitigate a variety of CNS pathologies.
As the field of epigenetics expands its understanding of the role of DNA methylation in neurodevelopment and disease, the challenge of moving DNA methylation towards a therapeutic target is performing not only correlative, but causative studies that define specific gene targets and sites. Additionally, surveying changes in DNA methylation specific to brain region and cell type remains an ongoing and time worthy challenge unique to the field of neuroepigenetics. This protocol utilizes a variety of techniques including fluorescence-activated cell sorting (FACS) of astrocytes, methylation-sensitive high resolution melt analysis (MS-HRM), and a methylation luciferase assay to investigate the DNA methylation status of KCNJ10, a gene that encodes for Kir4.1. Kir4.1 is a glial specific potassium channel that demonstrates both brain region and cell specific patterns of expression in the CNS 12-16. Kir4.1 expression increases moving from rostral to caudal CNS regions, with the highest expression occurring in the spinal cord 15. Although the channel is expressed in ependymal cells, oligodendrocytes and their precursor cells, Kir4.1 is predominantly expressed in astrocytes and thought to be essential for maintaining homeostatic levels of potassium as well as supporting glutamate uptake by setting the astrocytic resting membrane potential at a hyperpolarized -80mV 12,16-19. Importantly, the expression of Kir4.1 is non-static both during development and following multiple forms of CNS injury 20-25. We wished to examine the epigenetic regulation of this channel, specifically in astrocytes during development. The techniques utilized offer gene-specific and targeted CpG site analyses that provide causal evidence for a role of DNA methylation in regulating KCNJ10 gene expression. These techniques can be applied to other genes.

<table><tbody><tr><td>Papain Dissociation System</td><td>Worthington Biochemical Corporation</td><td>LK003150</td><td></td></tr><tr><td>AllPrep DNA/RNA Mini Kit</td><td>Qiagen</td><td>80204</td><td></td></tr><tr><td>Methyl Primer</td><td>Applied Biosystems</td><td>online</td><td>software to localize CpG Islands</td></tr><tr><td>EZ DNA methylation Kit</td><td>Zymo Research</td><td>D5001</td><td></td></tr><tr><td>Rat Premixed Calibration Standard</td><td>EpiGenDx</td><td>80-8060R-Premix</td><td></td></tr><tr><td>CpG Methylase (M.Sssl)</td><td>Zymo Research</td><td>E2010</td><td></td></tr><tr><td>QIAquick Gel Extraction&amp;nbsp;</td><td>QIagen</td><td>28704</td><td>Used for gel extraction and DNA cleanup</td></tr><tr><td>Restriction enzymes</td><td>New England BioLabs</td><td></td><td></td></tr><tr><td>NEB cutter</td><td>New England BioLabs</td><td>online</td><td>verify restriction digest sites</td></tr><tr><td>Dual Luciferase Reporter Assay System</td><td>Promega</td><td>E1910</td><td></td></tr><tr><td>Luc2 vector, pGL4.10</td><td>Promega</td><td>E6651</td><td></td></tr><tr><td>renilla vector, pGL4.74</td><td>Promega</td><td>E2241</td><td></td></tr><tr><td>TD-20/20 Luminometer</td><td>Turner Designs</td><td></td><td></td></tr><tr><td>Lipofectamine LTX and Plus Reagent</td><td>Life Technologies</td><td>A12621</td><td></td></tr><tr><td>Phenol, saturated pH 6.6/6.9</td><td>Fisher Scientific</td><td>BP 17501-100</td><td></td></tr><tr><td>Nanodrop 2000/2000c Spectrophtometer</td><td>ThermoScientific</td><td></td><td></td></tr><tr><td>MeltDoctor Master Mix</td><td>Life Technologies</td><td>4415440</td><td></td></tr><tr><td>High Resolution Melt (HRM) Software v2.0</td><td>Life Technologies</td><td>4397808</td><td></td></tr><tr><td>AB SDS software v2.3</td><td>Life Technologies</td><td>online</td><td></td></tr><tr><td>AB High Resolution Melting Getting Started Guide&amp;nbsp;</td><td>Life Technologies</td><td>online</td><td></td></tr><tr><td>AB 7900HT Fast Real-Time System</td><td>Life Technologies</td><td></td><td></td></tr></tbody></table>

correlating gene-specific dna methylation changes with expression and transcriptional activity of astrocytic kcnj10 (kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

성상 세포의 풍부한 인구는 eGFP는-S100β 형질 전환 동물 (27)의 FACS 정렬을 통해 인수되었다. 때문에 P0-P40 세 세포와 출생 후 하루 50 (P50)보다 오래된 동물에서 분리 된 분자 분자, 동물의 품질을 감소 그러한 실험에 최적이다. 대뇌 피질 조직을 다음 실험에 사용 하였다. 2-6 동물의 피질 함께 모았다. FACS는 UAB 종합 유동 세포 계측법 핵심 시설에서 수행되었다. 정렬은 벡톤 디킨슨 (Becton Dickins...

Watch this Scientific Journal Video about Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1 at JoVE.com

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

유전자 특이 발현과 DNA 메틸화 변화와 성상 세포의 전사 활동의 상관 관계<em&gt; KCNJ10</em&gt; (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

correlating-gene-specific-dna-methylation-changes-with-expression

Research

JoVE Journal

Biology

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

7.9K 조회수.  University of Alabama at Birmingham. DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression. 

비디오: Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

뇌의 작은, 해부학적인 몸의 구조 정의 분야의 DNA Methylation과 유전자 발현의 최적화 분석

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

연구

신경과학

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

급성 Hippocampal 슬라이스에 Synaptically-evoked Astroglial와 Neuronal 응답의 듀얼 Electrophysiological 레코딩

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

마이크로 유체 문화 플랫폼에 Glia 상호 작용 (MCP) 기반 Neuronal 축삭과 Glia 공동 문화 시스템에 신경의 영상 분석

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the &#34;open&#34;, transcriptionally active chromatin in developing and postmitotic mouse retina.

The objective of this report is to describe the protocol for robust immunohistochemical detection of epigenetic markers, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing and postmitotic mouse retina.

5-Methylcytosine 개발에 5-Hydroxymethylcytosine의 Immunohistochemical 검출 및 Postmitotic 마우스 망막

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a ...

발생학

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

신경 발사 환율의 조작에 의해 성상 세포 수용체의 가소성을 유도

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

이미징 세포 내 칼슘<sup&gt; 2 +</sup&gt; 유전자 인코딩 된 칼슘 표시기를 사용하여 성인 마우스의 선조체 성상의 신호

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively in the neuron and is essential for the proper functioning of Cl- homeostasis and consequently functional GABAergic inhibition. Failure in proper regulation of KCC2 is deleterious and has been associated with the prevalence of several neurological diseases, including epilepsy. There has been considerable progress with regard to understanding the mechanisms involved in the regulation of KCC2, accredited to the development of techniques that enable researchers to study its functions and activities; either via direct (assessing kinase regulatory sites phosphorylation) or indirect (observing and monitoring GABA activity) investigations. Here, the protocol highlights how to investigate KCC2 phosphorylation at kinase regulatory sites - Thr906 and Thr1007- using western blotting technique. There are other classic methods used to directly measure KCC2 activity, such as rubidium ion and thallium ion uptake assay. Further techniques such as patch-clamp-electrophysiology are used to measure GABA activity; hence, indirectly reflecting activated and/or inactivated KCC2 as informed by the assessment of intracellular chloride ion homeostasis. A few of these additional techniques will be briefly discussed in this manuscript.

The present protocol highlights the application of western blotting technique to study the functions and activities of neuronal K-Cl co-transporter KCC2. The protocol describes the investigation of KCC2 phosphorylation at kinase regulatory sites Thr906/1007 via western blotting. Also, additional methods to confirm KCC2 activity are briefly highlighted in this text.

웨스턴 블로팅을 이용한 뉴런 K-Cl 공동수송체 KCC2의 기능 및 활성 연구

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively ...

생화학

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

쥐 뇌 조직 검사 정렬 형광 활성 셀을 사용하여 셀 타입 별 유전자 발현

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Analyzing the Size, Shape, and Directionality of Networks of Coupled Astrocytes

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network level. Astrocytes are strongly connected to each other through gap junctions and coupling through these junctions is dynamic and highly regulated. An emerging concept is that astrocytic functions are specialized and adapted to the functions of the neuronal circuit with which they are associated. Therefore, methods to measure various parameters of astrocytic networks are needed to better describe the rules governing their communication and coupling and to further understand their functions.
Here, using the image analysis software (e.g., ImageJFIJI), we describe a method to analyze confocal images of astrocytic networks revealed by dye-coupling. These methods allow for 1) an automated and unbiased detection of labeled cells, 2) calculation of the size of the network, 3) computation of the preferential orientation of dye spread within the network, and 4) repositioning of the network within the area of interest.
This analysis can be used to characterize astrocytic networks of a particular area, compare networks of different areas associated to different functions, or compare networks obtained under different conditions that have different effects on coupling. These observations may lead to important functional considerations. For instance, we analyze the astrocytic networks of a trigeminal nucleus, where we have previously shown that astrocytic coupling is essential for the ability of neurons to switch their firing patterns from tonic to rhythmic bursting1. By measuring the size, confinement, and preferential orientation of astrocytic networks in this nucleus, we can build hypotheses about functional domains that they circumscribe. Several studies suggest that several other brain areas, including the barrel cortex, lateral superior olive, olfactory glomeruli, and sensory nuclei in the thalamus and visual cortex, to name a few, may benefit from a similar analysis.

Here we present a protocol to assess the organization of astrocytic networks. The described method minimizes bias to provide descriptive measures of these networks such as cell count, size, area, and position within a nucleus. Anisotropy is assessed with a vectorial analysis.

크기, 모양, 및 결합 된 이다의 네트워크의 방향 분석

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network ...

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.

In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes

Astrocytes tile the cerebral cortex uniformly, making the analysis of their complex morphology challenging at the cellular level. The protocol provided here uses multicolor labeling based on in utero electroporation to single out cortical astrocytes and analyze their volume and morphology with a user-friendly image analysis pipeline.

피질 마우스 성상세포를 개별화하기 위해 다각형 게놈 통합 색상(MAGIC) 마커의 자궁 전기포게에서

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at ...

유전자 특이 발현과 DNA 메틸화 변화와 성상 세포의 전사 활동의 상관 관계

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