본 연구는 NIH 보조금 (R15DA029863)에 의해 지원되었다, 플로리다 애틀랜틱 대학의 학부 연구 보조금 (M30014) 및 동물 용 의약품의 연구 보조금의 로스 대학. 우리는이 일을 위해 (±) 3,4-methylenedioxymethamphetamine (± MDMA)를 제공하는 국립 약물 남용 연구소 (락빌, MD)에 감사의 말씀을 전합니다.

아래에 설명 된 동물 사용 절차는 동물 용 의약품의 로스 대학에서 기관 동물 관리 및 사용위원회 (IACUC)와 플로리다 애틀랜틱 대학에 의해 승인되었다. 멸균 기술과 장갑이 필요하지만이 프로토콜을 사용하는 동안의 RNase없는 환경이 필요하지 않습니다. 1. 준비 <ol><li> 조직 준비와 절편 <ol><li> 세 그룹으로 무작위로 쥐를 할당 :,, SAL / 접두사 및 / 3,4-methylenedioxymethamphetamine 접두사 신선한 / 식염수 (0.9 % NaCl을 SAL) (MDMA를 표 1에서 자세한 내용을 참조). </li><li> 2 시간 간격으로 SAL 및 MDMA 그룹, 복강, 각각 1 ㎖ / ㎏ SAL 및 10 ㎎ / ㎏ MDMA의 세 가지 용량을 관리합니다. 동물 6 일 동안 치료 살아남을 수 있습니다. </li><li> 7 일에, 5 mg의 조합에 100 ㎎ / ㎏ 케타민을 관리 / 복강 깊은 마취에 대한 쥐 (에 자일 라진을 kg 즉, 각막 깜짝 및 타 손실김정일이) 반사 신경을 꼬집어. </li><li> 신선한 / SAL 그룹의 경우, 신속하게 쥐 목을 벨과 (기준 5에서 세부 사항을 참조) 신선한 뇌 테스트 뇌를 제거합니다. </li><li> SAL / 접두사와 접두사 / MDMA 그룹의 경우, 흉골 약 10 -12 cm 따라 절단을 한 후 흉골의 끝을 노출. </li><li> 지혈제와 흉골의 끝을 잡고 날카로운 가위로 양쪽 측면 다이어프램을 절단 한 후 폐에 갈비와 병렬을 통해 위로 잘라. </li><li> 한 손으로, 작은 집게로 마음의 복부 끝을 잡고. 다른 한편으로, 18 G 주사기 바늘 좌심실 피어스 (빙냉 SAL과 파라 포름 알데히드 및 용기 연동 펌프에 Y 자형 실리콘 호스와 연결되어 바늘 어댑터 측에 유의 참조 기준 9). </li><li> 유량 (~ 5 ml / 분)의 중간 수준에서 SAL 펌프를 켜고 반환 CIRC의 탈출을 할 수 있도록 집게로 우심방에 구멍위험률. 30 분 동안 SAL 관류를 유지한다. </li><li> 중간 수준에서 파라 포름 알데히드 펌프를 켜고 얼음처럼 차가운 4 % 파라 포름 알데히드는 30 분 동안 동물을 관류 할 수 있습니다. </li><li> 관류 악기를 제거하고 동물의 목을 벨. 두개골의 정중선을 통해 절단 후 전방을 확인하고 두개골 캐비티 (10)를 노출하는 두개골 플랩. </li><li> 두개골 캐비티에서 주걱으로 뇌를 제거하고 25 ml를 4 % 파라 포름 알데히드를 포함하는 50 ML의 원심 분리기 튜브에 두뇌를 놓습니다. 24 시간 동안 4 ºC 냉장고에서 파라 포름 알데히드 함유 튜브 내 뇌를 유지합니다. </li><li> 3 일 4 ºC에서 30 % 자당 용액 25 ㎖를 함유하는 50 ㎖ 원심 분리 튜브로 이동 뇌. 마지막으로, 사용까지 -80 ° C에서 비닐 봉지 내 뇌를 저장합니다. </li><li> 내장 미디어 척에 두뇌를 탑재하고 -26 ºC의 온도에서 저온 유지 장치에 뇌가 포함 된 내장 미디어를 동결. 신선한 동결을 잘라n 개의 20 μm의 섹션으로 뇌와 10 μm의 섹션으로 뇌를 파라 포름 알데히드는-접두사. 섹션에 슬라이드를 터치하여 RT 현미경 슬라이드에 섹션을 전송합니다. </li><li> 공기 건조 4 시간 동안 실온에서. 사용까지 -80 ° C에서 꽉 밀봉 된 봉투에 섹션 슬라이드를 저장합니다. </li></ol></li><li> 탈수 <ol><li> -80 ° C의 냉동고에서 슬라이드를 타고 1 ~ 3의 시험에 섹션을 할당합니다 HT2A, ppiB (양성 대조군) 또는 dapB (음성 대조군); 마크와 공 펜 라벨 섹션 (연필을 사용하지 마십시오). 5 분 동안 실온에서 100 % 알코올에 슬라이드를 담근다. 흄 후드에서 5 분 동안 슬라이드를 건조. </li></ol></li><li> 전처리 <ol><li> 피펫 슬라이드 부분에 전처리-1 시약 20 μL (알칼리 포스 파타 아제의 불 활성화). 수분 트레이 선반 레일에 슬라이드를 배치 (트레이 반응 solutio로부터 증발을 방지하기위한 높은 습도를 유지하는 덮개로 덮여 있음에 유의N). </li><li> 조심스럽게 10 분 동안 저속으로 수평 통에 습기 트레이를 흔들. 2 분 동안 두 번 증류수 (DDH 2 O)로 두 번 슬라이드를 씻으십시오. (파라 포름 알데히드 고정에 의한 가교 결합의 파괴) -2- 전처리 시약을 150 ㎖을 함유하는 200 ml의 비이커에 슬라이드를 담근다. 100 ºC (RNA 구조 (11)의 열 - 유도 복원)에서 5 분의 총 슬라이드를 끓인다. </li><li> 2 분 동안 DDH 2 O로 두 번 슬라이드를 씻으십시오. 5 분 동안 100 % 알코올에 슬라이드를 놓습니다. 흄 후드에서 5 분 동안 슬라이드를 건조. 조직 섹션에서 선택된 영역 주위에 원을 그릴 소수성 펜을 사용하여 (예를 들어, 그림 2에 설명 된대로 시상과 시상 하부를 포함한 비 대뇌 피질의 구조). </li><li> 피펫 전처리-3 시약 선택한 각 영역 (프로브 침투의 증가)의 10 μL, 그리고 뚜껑 수분 트레이 커버. 하이브리드 오븐에서 트레이를 배치수평 교반기 장착 40 ºC 오븐 온도를 설정; 부드럽게 30 분 동안 트레이를 흔들. 2 분 동안 DDH 2 O로 두 번 슬라이드를 씻으십시오. </li></ol></li></ol> 2. 하이브리드 및 증폭 <ol><li> 피펫 선택된 영역에 각각 htr2a, ppiB 및 dapB 프로브 시약 10 μL,,. 시약의 증발을 방지하기 위해 뚜껑 수분 트레이 커버. 부드럽게 저속으로 설정 수평 진탕 기에서 흔들어. 2 시간 동안 오븐에서 40 ºC에서 슬라이드를 배양한다. </li><li> 2 분 동안 세척 버퍼로 두 번 슬라이드를 씻으십시오. 피펫 선택한 각 지역의 증폭-1 시약 10 μL, 흔들어 30 분간 40 ºC에서 슬라이드를 품어. </li><li> 2 분 동안 세척 버퍼로 두 번 슬라이드를 씻으십시오. 피펫 선택한 각 지역의 증폭-2 시약 10 μL, 흔들어 15 분 40 ºC에서 슬라이드를 품어. </li><li> 두 번 W 슬라이드를 씻으2 분 동안 세척 버퍼 번째. 피펫 선택한 각 지역의 증폭-3 시약 10 μL, 흔들어 30 분간 40 ºC에서 슬라이드를 품어. </li><li> 2 분 동안 세척 버퍼로 두 번 슬라이드를 씻으십시오. 피펫 선택한 각 지역의 증폭-4 시약 10 μL, 흔들어 15 분 40 ºC에서 슬라이드를 품어. </li><li> 2 분 동안 세척 버퍼로 두 번 슬라이드를 씻으십시오. 피펫은 각각의 선택된 영역 상에 증폭 -5- 시약 10 μL, 진탕​​하고, 30 분간 RT에서 슬라이드를 배양한다. </li><li> 2 분 동안 세척 버퍼로 두 번 슬라이드를 씻으십시오. 피펫 선택한 각 지역의 증폭-6 시약 10 μL, 흔들 실온에서 15 분 동안 슬라이드를 품어. 2 분 동안 세척 버퍼로 두 번 슬라이드를 씻으십시오. </li></ol> 3. 신호 감지 <ol><li> 각 선택 영역에 레드 시약의 피펫 10 μL은 (시약 레드-B와 레드의 혼합물이 1:60 비율로 내가 사용되어야합니다mmediately). 실온에서 수평 통에 습기 트레이에 슬라이드를 삽입하고 10 분 동안 저속으로 부드럽게 흔들어. </li><li> 10 분 동안 (DDH 2 O)로 두 번 슬라이드를 씻으십시오. 15 분 동안 오븐에서 60 ºC에서 슬라이드를 건조. 5 분 흄 후드에서 크실렌의 슬라이드를 놓습니다. 피펫 (20) 선택한 각 지역에 크실렌 기반 설치 미디어의 μL 및 커버 슬립 커버. </li></ol> 4. 이미지 캡처 <ol><li> 4 × 20 × 대물 렌즈의 배율 선택 영역 (예, 시상 하부는, 1과 3도)의 디지털 현미경 사진을 가져 가라. 이미지 파일을 저장합니다. </li></ol> 5. 이미지의 입자 분석 <ol><li> 드래그하여 주 ImageJ에 창에 이미지 파일을 놓습니다. 메뉴 표시 줄로 이동하여 &#39;이미지&#39;를 선택, 다음 드롭 다운 메뉴에서 &#39;형식&#39;을 선택하고 &#39;8 비트&#39;를 선택합니다. </li><li> 선택한 다음 &#39;의 Thr를 선택,&#39;조정 &#39;eshold은 &#39;대화 상자를 엽니 다. 원치 않는 배경이 제거되도록 대화 상자에서 낮은 바 값을 조정합니다. 다음 두 번째 대화 상자를 엽니 다 &#39;입자 분석&#39;을 선택, 메뉴 표시 줄로 이동하여 &#39;분석&#39;을 선택합니다. </li><li> (10)에서 입자 크기를 설정, 다음 &#39;쇼 개요&#39;를 선택하고 &#39;결과 표시&#39;와 &#39;요약한다&#39;상자를 선택합니다. 마지막으로, 대화 상자에 입자 데이터를 볼 수있는 &#39;확인&#39;을 클릭합니다. </li></ol> 6. 스프레드 시트 계산 <ol><li> 엑셀 시트에 데이터를 입력하고 기준으로 SAL 그룹에 입자의 수​​를 평균. 각 섹션의 백분율 레벨을 계산하고 그에 따라 그래프 (도 4)를 만든다. </li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+5-HT2A+receptor+mRNA+expression+following+withdrawal+from+a+chronic+escalating+dose+regimen+of+D-amphetamine.">Differential regulation of 5-HT2A receptor mRNA expression following withdrawal from a chronic escalating dose regimen of D-amphetamine.</a> Brain Re. 1390, 10-20 (2011).</li><li>Mocci, G., Jimenez-Sanchez, L., Adell, A., Cortes, R., Artigas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+5-HT2A+receptors+in+prefrontal+cortex+pyramidal+neurons+projecting+to+nucleus+accumbens.+Potential+relevance+for+atypical+antipsychotic+action.">Expression of 5-HT2A receptors in prefrontal cortex pyramidal neurons projecting to nucleus accumbens. Potential relevance for atypical antipsychotic action.</a> Neuropharmacolog. 79, 49-58 (2014).</li><li>Reneman, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+and+chronic+effects+of+MDMA+('Ecstasy')+on+cortical+5-HT2A+receptors+in+rat+and+human.">The acute and chronic effects of MDMA ('Ecstasy') on cortical 5-HT2A receptors in rat and human.</a> 26, 387-396 (2002).</li></ol>

저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

One of the major concerns of in situ hybridization test is to choose appropriate techniques used for RNA preservation because of RNase enzymes. It is well known that these enzymes are widely present in the cells and environment which can affect the results. However, enzyme activity can be quickly distinguished by placing the tissue in the dry ice/alcohol solution5,12. Although quick preservation is critical for hybridization using a riboprobe13, little is known about experimental conditions...

Serotonin (5-hydroxytryptamine; 5-HT) syndrome is an acute neurologic disorder caused by 5-HT-promoting drugs such as antidepressants1, while also occurring in situations of MDMA use for recreational purposes2. Molecular mechanisms responsible for mood swings, learning and memory deficits that occur in association with the acute syndrome are not well understood3,4. In situ hybridization is a powerful research tool allowing the detection and quantification of specific mRNAs expressed potentially at a single-cell level. The conventional way to perform in situ hybridization is to utilize a radioactive-labeled riboprobe that specifically hybridizes the gene of interest. However, a major drawback is that the method requires complicated and time-consuming probe preparation and hybridization steps, as well as access to fresh frozen tissues maintained in an RNase-free environment5,6. 
Oligonucleotide probes have been recently developed to hybridize shorter RNA fragments than those required for riboprobes7. Furthermore, the probes produce a low background signal without sacrificing specificity8. This newly-developed probe technology can be used in situ hybridization on paraformaldehyde-prefixed brain tissues commonly available in immunocytochemical laboratories.
Here, we describe a protocol for in situ hybridization using oligonucleotide probes on paraformaldehyde-prefixed rat brain and compare findings with those noted in a fresh frozen brain5,6. This protocol is used to test the hypothesis that MDMA substance abuse causes changes in 5-HT2A receptor gene htr2a mRNA in the brain. We began the procedure with MDMA treatment followed by paraformaldehyde tissue perfusion of the animal, in situ hybridization of the thr2a probe, and data analysis. Note that dapB (the bacterial gene coding for dihydrodipicolinate reductase) is used as a negative control, and ppiB (housekeeping gene coding for peptidylprolyl isomerase B) as a positive control.

<table><tbody><tr><td>RNAscope Negative Control Probe-DapB</td><td>Advanced cell diagnostic, INC</td><td>310098</td><td></td></tr><tr><td>RNAscope Pretreat 4</td><td>Advanced cell diagnostic, INC</td><td>320046</td><td></td></tr><tr><td>RNAscope 2.0 HD Reagent Kit - Red</td><td>Advanced cell diagnostic, INC</td><td>310036</td><td></td></tr><tr><td>RNAscope Probe - Rn-Ppib</td><td>Advanced cell diagnostic, INC</td><td>313921</td><td></td></tr><tr><td>Rat Htr2a</td><td>Advanced cell diagnostic, INC</td><td>300031</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM 1850</td><td></td></tr><tr><td>Horizontal shaker</td><td>VWR</td><td>88032-088</td><td></td></tr><tr><td>Hybridization oven</td><td>Thermo Fisher Scientific</td><td>222000</td><td></td></tr><tr><td>Superfrost Plus microscope slide</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Hydrophobic pen</td><td>Vector</td><td>H-4000</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>Provis AX70</td><td></td></tr></tbody></table>

rapid in situ hybridization using oligonucleotide probes on paraformaldehyde-prefixed brain of rats with serotonin syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

올리고 뉴클레오티드 RNA 프로브 (&lt;25 NT)를 사용하여, 하이브리드는 파라 포름 알데히드 - 접두사 및 냉동 뇌에서 준비 시상 하부 세포에서 빨간 점으로 검출 할 수있다. htr2a mRNA의 분자 (실선 화살표로 표시됨) 일부 셀에 존재하는, 그러나 기타 (개방 화살표). 우리는 파라 포름 알데히드 접두어 및 냉동 조직 (도 1) 사이에 차이가없는 것을 관찰했다. 성공적인 하이브리드는 육?...

Watch this Scientific Journal Video about Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome at JoVE.com

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

빠른<em&gt; 현장에서</em세로토닌 증후군 쥐의 파라 포름 알데히드 - 접두사 뇌에 올리고 뉴클레오티드 프로브를 사용하여&gt; 하이브리드

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

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Neuroscience

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

8.8K 조회수.  Ross University School of Veterinary Medicine. This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

비디오: Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

더블 형광 현장에서</em신선한 두뇌 섹션> 하이브 리다이 제이션

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

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신경과학

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

PTSD의, 신경 Hematologic, 그리고 행동 상관 관계를 누설하는 행위에 대해서는 동물 모델에서 생체

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

의학

DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is characteristic of disease states like major depressive disorder (MDD) and Alzheimer's disease. Traditional techniques to investigate synapse numbers include genetic expression of fluorescent markers (e.g., green fluorescent protein (GFP)), dyes that fill a neuron (e.g., carbocyanine dye, DiI), and immunofluorescent detection of spine markers (e.g., postsynaptic density 95 (PSD95)). A major caveat to these proxy techniques is that they only identify postsynaptic changes. Yet, a synapse is a connection between a presynaptic terminal and a postsynaptic spine. The gold standard for measuring synapse formation/elimination requires time-consuming electron microscopy or array tomography techniques. These techniques require specialized training and costly equipment. Further, only a limited number of neurons can be assessed and are used to represent changes to an entire brain region. DetectSyn is a rapid fluorescent technique that identifies changes to synapse formation or elimination due to a disease state or drug activity. DetectSyn utilizes a rapid proximity ligation assay to detect juxtaposed pre- and postsynaptic proteins and standard fluorescent microscopy, a technique readily available to most laboratories. Fluorescent detection of the resulting puncta allows for quick and unbiased analysis of experiments. DetectSyn provides more representative results than electron microscopy because larger areas can be analyzed than a limited number of fluorescent neurons. Moreover, DetectSyn works for in vitro cultured neurons and fixed tissue slices. Finally, a method is provided to analyze the data collected from this technique. Overall, DetectSyn offers a procedure for detecting relative changes in synapse density across treatments or disease states and is more accessible than traditional techniques.

DetectSyn is an unbiased, rapid fluorescent assay that measures changes in relative synapse (pre- and postsynaptic engagement) number across treatments or disease states. This technique utilizes a proximity ligation technique that can be used both in cultured neurons and fixed tissue.

DetectSyn: Synapse 밀도의 변화를 감지하는 빠르고 편향되지 않은 형광 방법

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is ...

생화학

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

고해상도를 사용하여 뇌 섹션에서 Axonally 지역화 된 mRNA를 검출<em&gt; 현장에서</em&gt; 하이브리드

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

더블 형광 결합<em&gt; 현장에서</em&gt; 마우스 뇌 섹션에서 세 가지 유전자의 발현의 검출을위한 Immunolabelling와 하이브리드

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

탐지 기술 현장에서 혁신적인 RNA에 의해 설치류 핵 Accumbens에서 도파민 D1-알파 수용 체의 식별

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

근 적외선 형광 및 고 분해능 주사를 사용 하 여 설치류 뇌의 단백질 발현 측정

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Section(신선 냉동 또는 고정 마우스 뇌 절편에서 Multiplex Fluorescence In Situ Hybridization과 형광 면역조직화학의 결합)

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

In Situ Hybridization Using Oligonucleotide Probes to Study RNA Expression in Rat Brain Sections

Source: Shokry, I. M., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/53165">Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome.</a>&#160;J. Vis. Exp.&#160;(2015).
This video demonstrates the detection of serotonin receptor RNA synthesis using oligonucleotide probes in rat brain tissue affected by serotonin syndrome. The protocol includes tissue pretreatment, probe hybridization, and enzymatic detection to visualize and quantify serotonin receptor RNA expression under a microscope.

Source: Shokry, I. M., et al. Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin ...

JoVE Encyclopedia of Experiments

Neuropathology

Neurodegenerative Diseases

 Performing Double Fluorescence In Situ Hybridization to Detect Gene Expression in Mouse Brain Sections

<a href='https://appjove.unicartagenaproxy.elogim.com/v/53976/combining-double-fluorescence-situ-hybridization-with-immunolabelling'>Source: Jolly, S., et.al. Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections. J. Vis. Exp. (2016).</a>This video demonstrates the use of double fluorescence in situ hybridization to visualize the expression of two RNA targets in mouse brain sections. The protocol involves sequential detection of RNA using probes labeled with fluorescein isothiocyanate and digoxigenin, followed by enzymatic signal amplification to enhance sensitivity. The resulting fluorescence enables co-localization analysis of the target RNAs within the tissue.

Source: Jolly, S., et.al. Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse ...

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