We thank Souvik Maiti for help in designing the antagomirs. We also acknowledge Rangeetha J. Naik, Rakesh Dey, and Bijay Pattnaik for their help with experimental methods. This work was funded by the Council of Scientific and Industrial Research (BSC0123). HS, MV and RR acknowledge fellowship from the Council of Scientific and Industrial Research, India. MAS acknowledge fellowship from the University Grants Commission, India.

참고 : 동물 과목을 포함한 모든 절차는 기관 동물 윤리 유전체학 연구소의위원회 (IAEC)와 통합적인 생물학, 뉴 델리에 의해 승인 된 (IGIB / AEC / 2,013분의 10). 이 프로토콜은 특히은 miR-29의 뇌와 최저의 Antagomir-29의 표적 전달을 위해 조정됩니다. 1. Antagomir 디자인 전략 <ol><li> miRBase (11)의 성숙한 miRNA의 순서를 검색 ( <a href="http://www.mirbase.org/">http://www.mirbase.org/</a> ). </li><li> miRBase 레코드 &quot;유전자 패밀리&quot;링크를 사용하여 관련 miRNA의 가족 구성원의 서열을 검색 ( <a href="http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000009">http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000009</a> ). </li><li> 순서를 보완 역 : <a href="http://www.bioinformatics.org/sms/rev_comp.html">http://www.bioinformatics.org/sms/rev_comp.html이</a> 할 수있는 편리한 도구를 제공합니다보완 순서를 역. </li><li> 다음 지침에 따라 LNA에 의해 수정 될 수있는 위치를 표시 : <ol><li> LNA의 피리 미딘는 퓨린 (12)보다 더 안정적이기 때문에, LNA의 수정을 위해 3 ~ 4 기지 떨어져 배치 티민 잔류를 선택합니다. </li><li> 이전 단계가 5 개 미만의 수정을 생성하는 경우 3-4 잔류 물에 의해 분리 된 시토신 잔류를 선택합니다. </li><li> 이 시드 서열을 방지하기 때문에, antagomir의 5 &#39;말단에 다섯 수정 우선 순위. </li></ol></li></ol> 2. Antagomir - 신경 펩타이드 복합 준비 주 :이 프로토콜에서 가장 중요한 단계이며 표준화를 필요로한다. 임의의 형광 표지 된 올리고 뉴클레오타이드 (FLO)는 antagomir 9 대신에 사용될 수있는 프로토콜을 표준화. 펩티드 고순도이어야한다 (HPLC 등급은 98 % 순도) 및 .Sequences 펩타이드 antagomir의 전하를 표 1에 나타내었다. <ol><li> MO의 수를 결정할antagomir의 레는 마우스의 antagomir, 독성 및 무게에 몰 요금을 기준으로 주입한다. 펩티드 : 펩티드의 몰수를 계산하는 분사 antagomir 몰 투입 비율에 기초한다. 참고 : 마우스 뇌의 독성 및 효과적인 전달을위한 펩타이드 : antagomir 몰 충전 비율을 표준화. 표준화를위한 펩타이드 : 형광 표지 된 올리고 뉴클레오티드의 다른 몰 충전 비율을 사용합니다. 우리는 Antagomir-29 및 제어 마우스의 4microgram / g의 체중에서 독성이 두 사실을 발견했습니다. Antagomir-29 및 제어 2microgram / g 체중으로 사용되었지만,이 유효 농도는 각각의 마이크로 RNA에 따라 다를 가능성이있다. </li><li> 1 단계에서 계산 된 목적하는 최종 농도로 멸균 10 % D 글루코스 원액으로부터 미세 원심 분리 튜브에 펩티드 antagomir 희석. </li><li> 적당한 텍싱 속도 소용돌이에 펩타이드 용액으로 미세 원심 튜브를 유지한다. </li><li> 1/3 번째 추가이 와류 믹서에서 충분히 혼합하면서 펩티드 용액 튜브에 ntagomir 용액을 천천히, 적가. </li><li> 다음으로 1 분 동안 와동에 용액의 혼합을 계속 한 다음, 혼합물을 1 분 동안 방치한다. </li><li> 같은 미세 원심 분리 튜브에 펩타이드 솔루션 나머지 antagomir 솔루션을 혼합하는 단계 4와 5를 두 번 더 반복합니다. 느린 첨가는 형질 효과적이다 모노 - 분산 복합체의 형성에 중요하다. 빠른 추가는 단지 그들의 강수량의 집합으로 이어질 수 있습니다. </li><li> 텍싱없이 RT에서 30 분 동안 인큐베이션 복합체. 배양이 기간 동안 주사가 수행 될 실험실 쥐를 순응. </li></ol> Antagomir - 신경 펩타이드 콤플렉스 3. 꼬리 정맥 주입 <ol><li> 동물을 억제하기 위해, 적절한 크기의 제한 수단 또는 decapicone에 마우스를 놓습니다. </li><li> 면봉 홍보를 사용하여 꼬리의 표면을 청소전자 적신 따뜻한 물 (약 40 ℃)에서. 이는 혈관 확장을 촉진하고 혈관의 가시성을 증가시킬 것이다. </li><li> 15 ~ 20 ° 각도로 - (1.0 CC 0.5) 인슐린 주사기 작은 볼륨 꼬리에 접근. 주사기에있는 공기를 도입하지 않도록주의하십시오. 꼬리의 말단부에 시작한다. 단지를 주입. 바늘을 제거하고 출혈을 멈추게하기 위해 주사 부위 (약 5 ~ 10 초)에 직접 살균 면봉을 적용합니다. </li><li> 농축과 옥수수 속대와 티슈 페이퍼로, 3-5의 그룹에서 개별적으로 환기 케이지에 마우스를 교체합니다. 같은 케이지에서 uninjected 및 주입 쥐를 보관하지 마십시오. </li></ol> 4. 행동 분석 실험 주 : 주사 후 회복을 허용하기 위해 주입 및 행동 분석 사이에서 3-4 시간의 시간 간격을 유지. 조용한 방에 마우스를 가져 오십시오. 적응의이 기간 동안 동물을 방해하지 마십시오. <ol><li> 마우스를 순응새로운 방 30 분 후 다음 행동 분석을 시작합니다. 각각의 행동 분석 사이에 10 분의 간격을 유지합니다. </li><li> 뒷다리 물림쇠 : 참고 :이 테스트는 운동 기능 장애 및 신경 손상을 나타냅니다. <ol><li> 분명 배경에 꼬리 근처에 기반을 잡고 부드럽게 마우스를 올립니다. </li><li> 10 ~ 15 초 동안 사지의 위치를​​ 확인합니다. 실조 마우스가 시간의 50 % 이상 13 현탁 복부쪽으로 한쪽 또는 양쪽 뒷다리 경향 후퇴하면서 야생형 마우스 splays 멀리 복부에서 뒷다리. </li><li> 케이지에 마우스를 반환 </li></ol></li><li> 난간에 시험 : <ol><li> 꼬리를 잡고 부드럽게 마우스를 올려 케이지의 선반에 보관하십시오. </li><li> 케이지의 모서리에 구체적으로 마우스를 관찰합니다. 야생형 마우스는 두려움과 균형을 잃지 않고 쉽게 모서리를 전달할 수 있습니다. 케이지 선반을 따라 코너에 걷는 동안 운동 실조증 마우스는 균형, 동결을 잃거나 흔들립니다. </li></ol></li><lI&gt; 마우스 발 인쇄 분석 : 참고 :이 분석은 좁은 통로의 바닥에, 준비 흡수 시트를 유지 (~ 70 센티미터 길이 ~ 5 cm 전체 ~ 5 cm의 높은 벽.) <ol><li> 한 손으로 부드럽게 마우스를 잡고 브러시에 의해 뒷다리에 잉크를 적용합니다. </li><li> 좁은 활주로 바닥에 흡수 시트를 놓습니다. </li><li> 마우스를 걷거나 다른 한 끝에서 활주로에 직선으로 흡수 시트를 통해 실행할 수 있습니다. </li><li> 신선한 흡수 시트를 사용하여 각 마우스로 과정을 두 번 더 반복합니다. </li><li> 전방 이동에 두 개의 연속 단계들 사이의 거리를 측정한다. 동물은 단지 각각 시작과 실행을 마무리된다 처음과 마지막 몇 발자국를 포함하지 마십시오. </li></ol></li></ol>

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The authors have nothing to disclose.

Here we demonstrate a widely accessible methodology to study the effects of miRNA modulation. Currently, most attempts at in vivo characterization of miRNA functions involve the creation of knockout mice or a transgenic that expresses a miRNA sponge. Most miRNAs, even the cell type specific ones are expressed in more than one organ. For instance, miRNAs initially thought to be specific to the hematopoietic system are also expressed in the brain, due to the presence of microglia. Thus even a cell type specifi...

마이크로 RNA에 의한 유전자 발현과 질병에의 참여에 대한 직접적인 증거의 규제에서의 보편적 인 역할에 새로운 치료 대상으로 등장했다. 약물이 1, 2를 대상으로 miRNA가 적극적으로 자신의 잠재력에 대한 탐구되고있다. 더욱이, miRNA의 발현의 변화가 여러 질환 3의 miRNA 발현의 인공 섭동에 의한 이러한 변화의 시뮬레이션과 관련된 질병의 발현에 관여하는 세포 경로를 연구하기 위해 사용될 수있다. miRNA의 표적 약물의 조직 특정 배달은 현재의 miRNA 기반 신약 개발의 주요 과제입니다. Antagomirs 및 miRNA의 모방은 miRNA의 레벨 4-6을 교란하기위한 유망 에이전트입니다. 그러나, 특이성 및 효능을 향상 특징들은 miRNA의 발현 생체 섭동에 사용될 수 있기 전에 antagomirs의 디자인에 혼입 될 수있다. 마이크로 RNA는 특히 관련이<html> 현재 불치의 퇴행성 신경과 신경 발달 질​​환 대상으로. 혈액 - 뇌 장벽은 뇌에 antagomirs의 전달에 제한을 포즈. 정위 주사 널리 7 뇌의 특정 위치로 분자를 전달하는 설치류 모델에서 사용된다. 그것은 기술, 계측 및 시간에 광범위한 투자를 필요로한다. 정위 주사는 적어도 경미한 부상의 원인, 수술을 포함하고 로컬 배달로 제한됩니다, 침략이다. 표적 뉴런의 설정에 셀 관통 펩티드의 사용은 트랜스 혈관 경로를 통해 전달 될 수 있기 때문에 이러한 제한에 대응하지만, 혈액 뇌 장벽을 위반할 수있다. 광견병 바이러스 당 단백질 (RVG) 유래의 펩타이드 등, 이전 쥐 8 일본 뇌염 바이러스에 대한 siRNA를 전달하기 위해 사용되었다. 우리는 antagomir 배달 펩타이드를 사용하여,의 miRNAs 효과적으로 할 수 마우스 뇌 9 무너 뜨렸다 수 있다는 것을 발견했다. ontent &quot;&gt;의 miRNA의 두 번째 주요 과제는 노크 다운의 miRNAs의 작은 크기와 밀접한 관련이 시퀀스 동종의 존재에서 발생한다. 우리는 세 밀접하게 관련 동종 구성-은 miR-29 MMU 가족의 예를 취할 미르-29A 핵산 (LNA들)이 그들은 심지어 열 안정성을 향상시키고 또 다른 장점을 제공 잠금, b와 c. Antagomirs는 일반적으로 안정성을 높이고 뉴 클레아의 공격에 저항하는 렌더링하는 백본을 따라 수정됩니다. 이상과 그 이후 저하를 대상으로지도 입체 장애 (10). 모든 백본을 따라 수정을 소개하는 것은 효과가 있지만 비용이 많이들 수 있습니다. 우리는 이전의 최적의 수를 넘어 수정이 더 효과를 강화하지 않을 수 있음을 보았다. antagomir의 디자인은 따라서 antagomir의 최적의 수정을 포함한다. 복잡한 antagomir 비공유 노나 아르기닌 확장에 neurotropic 펩타이드, 충전 헵타와 함께 사용된다. D-아르기닌그들은 단백질 분해 효소에 의해 분해에 민감하지 않은으로 높은 안정성을 부여하기 때문에 잔류 물이 사용된다. 노나 아르기닌까지 뻗어 헵타은 세포 유형 특이성을 부여하지 않지만, 효율적인 세포 침투제의 역할. 공유 노나 아르기닌 링커 neurotropic, 세포 투과 펩티드가 생성되었던 RVG 펩티드 연결함으로써. 펩티드의 양으로 하전 된 잔기가 복합체를 형성하기 위해, 음으로 하전 된 핵산 백본과 상호 작용. 이러한 착물은 효과적으로 배양 세포 내로 생체 내에서 조직 내로 DNA 또는 RNA를 형질 감염 할 수있다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Vortex</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Restrainer or Decapicone</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow runway</td>
			<td></td>
			<td></td>
			<td>~70-cm-long, ~5-cm-wide with ~5-cm-high walls.</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescently labelled oligonucleotides (siGLO)</td>
			<td>GE Healthcare Dharmacon INC</td>
			<td>D0016300120</td>
			<td></td>
		</tr>
		<tr>
			<td>10% sterile D-glucose</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antagomir-29</td>
			<td>Exiqon</td>
			<td>custom synthesis</td>
			<td></td>
		</tr>
		<tr>
			<td>Antagomir-control</td>
			<td>Exiqon</td>
			<td>custom synthesis</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuropeptide RVG</td>
			<td>G.L.Biochem (Shanghai) Ltd.</td>
			<td>custom synthesis</td>
			<td>&#62;98% purity</td>
		</tr>
		<tr>
			<td>Neuropeptide RVM</td>
			<td>G.L.Biochem (Shanghai) Ltd.</td>
			<td>custom synthesis</td>
			<td>&#62;98% purity</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Warm water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbent sheets</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ink</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antiseptic</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple alternative to stereotactic injection for brain specific knockdown of mirna

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

여기에 제시된 절차를 이용하여 형광 표지 된 올리고 뉴클레오티드 50microgram (FLO)와 전하 ~ 1시 15분 몰 비율의 850microgram RVG 펩타이드 복합체 (FLO : 펩티드)을 준비하고, 꼬리 정맥을 통해 한 번만 주입 하였다. 비 neurotropic 광견병 바이러스 매트릭스 (RVM) 펩티드 및 FLO의 복합체는 대인 대조군으로 사용 하였다. 다음날, 마우스 뇌 및 간을 분리 하였다 및 단일 세포 현탁액을 제조 하였다. 세포는 녹색?...

Watch this Scientific Journal Video about A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA at JoVE.com

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

miRNA의 뇌의 특정 최저 대한 정위 주입하는 간단한 대안

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

a-simple-alternative-to-stereotactic-injection-for-brain-specific

Research

JoVE Journal

Neuroscience

12.6K 조회수.  CSIR-Institute of Genomics and Integrative Biology, New Delhi, India. MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

비디오: A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

베르 미 첼리와 Capellini 처리 시험 : 쥐 및 생쥐의 손재주가 앞발 기능의 단순 양적 측정

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

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신경과학

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

비 심실 플라스미드 주입 및 Electroporation에 의해 출생 후의 쥐 뇌에 유전자 전달

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

성인 쥐의 특정 두뇌 영역의 Excitotoxic 병변에 대한 Stereotaxic 외과

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).
The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

Optogenetic 및 동결 골절 복제 Immunolabeling는 마우스 편도체에서 글루타메이트 수용체의 입력 특정 배열을 검사하기 위해 결합

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

망막 하 공간에 액세스하기위한 대안의 검증 사출 방법<i&gt;를 통해</i&gt;를 Transcleral 후방 접근

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

사용 보상 추구 행동의 변화를 측정하는 간단한 방법<em&gt; 노랑 초파리</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

외상 성 부상에 의해 영향을 하는 뇌 영역의 정위 적 아틀라스 유도 레이저 캡처 기정

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

성인 인간 피부 섬유 아 세포에서 유도 된 신경의 고수익 문화의 간단한 세대

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

연 접 기능 그대로 신경 회로 내에 유전자 최저 효과 특성 인공 microRNAs Optogenetics 결합

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Neurodegeneration Ex Vivo 쥐 두뇌 분할 영역을 사용 하 여 기본 이벤트를 공부 하는 양자 택일 접근

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...