Name: rodent brain microinjection to study molecular substrates of motivated behavior
Uploaded: 2015-09-16T14:00:00.000+00:00
Duration: 10 min 5 s
Description: Watch this Scientific Journal Video about Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior at JoVE.com

MSB is supported by the Alcohol Beverage Medical Research Foundation, a Center for Translational Research Award (UL1 TR000058), the National Institutes for Alcohol Abuse and Alcoholism (P50 AA022537), and startup funds provided by the Virginia Higher Education Equipment Trust Fund and the VCU School of Medicine.

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None of the authors have competing or conflicting interests.

여기에 제시된 방법은 마이크로 인젝션 캐뉼라 및 동기 동작의 분자 기판을 밝히는데 도움이 될 microinjectors를 제조하는 효율적인 수단이다. 이 방법은 몇 가지 장점을 제공한다. 첫째, 자신의 임플란트와 microinjectors을 제조하여, 새로운 실험 매개 변수를 신속하게 최적화 할 수있다, 즉, 하나가 도착하기를 맞춤 제작 한 부품을 기다릴 필요가 없습니다. 둘째, 정맥의 작은 직경으로 인해, 더 캐뉼라를 동시에 주입 할 수있다. 이것은 생존을 향상시킬 수 있습니다 필요한 수술 시간을 단축하고, 또한 동물 당 여러 개의 임플란트를 할 수 있습니다. 고정비 패러다임 빠르게 단순히 원하는 보강 일정 포함 된 이벤트 전이 파라미터리스트를 적용하여 프로그레시브 비율 패러다임으로 전환 될 수 있기 때문에, 셋째, 조건화 챔버를 제어하는​​데 사용되는 소프트웨어를 용이 프로그레시브 비 스케줄을 수용한다. 되려고광범위하게 유용한, 일반적인 미세 주입 절차는 현재 거의 모든 시약의 미세 주입을위한 광범위하게 적용해야한다고 제시했다. 따라서, 우리는이 기술은 약간의 수정과 향후 유사한 높은 유틸리티로 계속 될 것으로 예상하고있다. 단지 약간의 변수를 변경함으로써, 이러한 접근 방법은 시약의 다양한 개수에 적용될 수있다. 가장 일반적으로 조작 할 것이다 매개 변수는 마이크로 인젝터는 정맥 돌출 길이, 사출의 볼륨 및 주입 속도를 포함한다. 예를 들어, 하나의 주사기는 일반적으로 만성 임플란트 주위 아교 흉터 형성을 방지하기 위해 캐뉼라 팁에서 더 돌출 할 수있다. 또한, 하나의 더 큰 부피를 주입해도된다. 그 사용에 비하여 (10 분의 추가 시간 확산 자주 7 - - 10 분 및 3) 선조체 바이러스 microinjections 들어, 1 μL의 부피는 통상적으로 사용되며,이 볼륨은 전형적으로 장기간에 걸쳐 주입하여약리 시약 D (일반적으로 0.3 - 3 분 플러스 1 - - 2 이상 0.5 μL 3 분의 추가 확산 시간). 사용자는 문헌을 참조 및 / 또는 경험적으로 자신의 요구에 가장 적합한 매개 변수를 결정해야한다. 주사 전에 마이크로 인젝터의 분사 패턴의 1) 캐 뉼러의 길이, 2) 마이크로 인젝터 길이, 3)의 품질, 및 4) 시스템 무결성에 관계없이,이 절차의 성공은 4 변수 결정적으로 의존한다. 미세 주입 위치가 마이크로 인젝터는 정맥에서 돌출 깊이에 의존하기 때문에 모두 캐 뉼러 (단계 1.2.8) 및 마이크로 인젝터 길이 (포스트 굽힘, 단계 2.2.1)를 모두 정확하게 모든 주제 사이에 알려진 균일 한 것을,이 필수적이다 . 이것은 쉽게 용이라도 그 최종 재 측정에 필요한 길이 아니다 구현 거부에 의해 제어 될 수있다. 이 가이드 뉼러 바로 아래에 발생하는 경우 또한, 주입 위치 만 예측 될 수있다. 따라서, 어떤 마이크로 인젝터를 미상 그테스트에 긴, 미세 스트림을 뿌리지 S (단계 2.4.6)을 거부해야합니다. 품질 주입은 또한 종래 주입 시스템의 무결성에 관한 것이다. 여러 관광 명소 랩 닦아에서 관찰 된 인젝터에서 모든 물을 분배 후 (전 시약과 충전에) 경우, 누수가 (단계 2.4.8에 대한 참고) 해결 될 필요가있다. PE20 튜브 내의 물에서 약물을 분리 버블 (단계 2.4.9)가없는 한, 하나의 기포 (마이크로 인젝터 시약을 충전 한 후) 인 경우, 또한, 후 분사가 부분적으로 막혀있다. 이 방해하거나 예방 주사를 전환 할 수 중 하나. 이 너무 쉽게 해결 될 수있다 (참고 단계 2.4.8에서). 하나는 세 가지 대안이있는 동물이 고정대에있는 동안 microinject하고자합니다. 첫째, 하나는 정위 매니퓰레이터 의해 단단히 유지되고 또한 PE20 튜브에 연결을 허용하도록 충분히 연장 될 수 있도록 마이크로 인젝터 칼라의 길이를 증가시킬 수있다. 둘째,에전자는 일시적으로 정맥을 이식하고 여기에 제시된 표준 마이크로 인젝터를 사용할 수 있습니다. 셋째, 하나 그린과 광택 유리 피펫 사용할 수 있습니다. (16, 17) 여기에 제시된 방법의 중요한 한계는 최상의 절차에 익숙 잘 처리 래트에서 수행된다는 점이다. 동일한 조사자 2 개월 이상 매일 랫트를 취급하기 때문에 결과 절에 설명 된 데이터에 사용되는 래트 특별한 처리 절차가 필요 없다. 이것은 최소 2 주 동안 외과 임플란트의 매일 관찰 및 조작을 포함했다. 그러나, 래트 급격히 응력에 의해 영향을받을 수 프리 펄스 억제 분석 이전에 사용되는 다양한 기술에 의해 순응된다. 이러한 특수 습관화 기술들이 앞서 상세히 좋게되어있다. (43)이 절차 이외에, 그것은 쥐 단축 microinjectors가 뒤 사용되는 마이크로 인젝션 절차에 순응하는 것이 바람직하다반지 &#39;가짜&#39;주사. 이러한 허위 주사 동안, 마이크로 인젝터가 조직 손상을 제한하기 위하여 조직으로 돌출하지 않는 것이 중요하다. 즉, 마이크로 인젝터가 더 이상 14mm 이상 절곡되어서는 안된다. 따라서, 본 기술의 구현에 필요한 최적의 철저한 습관화는 제한으로 간주 될 수있다. 여러 행동 패러다임 동기를 측정하기 위해 존재하지만, 일반적으로는 비 진보적 피사체 보강제를 수득 발휘 기꺼이 노력을 정량화하는데 사용된다. 누진 비율 패러다임은 종종 최종 완료된 비율 레버 프레스의 최대 수로서 정의되는 브레이크 포인트로 알려진 측정 값을 만들어 낸다.. 즉, 최대 그 보강재를 생성 응답 21 프로그레시브 비가 크기 보강제 민감하다. 예를 들어, 높은 코카인 (또는 수 크로스) 투여 량은 더 높은 브레이크 코카인 및 하부 (또는 수 크로스) 복용 breakp 낮은 수율을 생산OINT. (21, 22) 따라서, 브레이크 포인트는 동기 부여 및 / 또는 보강 효과에 대한 일상적으로 사용하는 프록시입니다. 21,23-26 중단 점의 의도는 동물이 응답하지 않을 때 결정하기 때문에, 진보적 인 비율 패러다임의 중요한 매개 변수입니다 세션 길이. 세션 길이가 유한 한 브레이크 포인트는 거짓 값에 캡을 넣을 수 있으며, 이는 비정상적자가 투여 또는 증가 포스트 보강 일시의 속도를 감소 사전 치료에 의해 악화 될 수있다. .이 혼란은 접근 방법의 수에 의해 극복 될 수있다. 예를 들어, 세션 동물은 평균 간 투여 간격의 일부 복수로 응답을 원천 징수 한 경우 종료 (44)이 방법의 더 일반적으로 적용 변형이 응답 한 번 세션을 종료하는 것입니다 주제에 걸쳐 일정하게 유지된다 일부 경험적으로 결정된 값으로 원천 징수하고. 우리는 단계 4.4.9.11에이 방법을 적용 할 수있는 방법을 제공하고 있습니다.

Rodents and humans respond in remarkably similar ways to behaviorally relevant stimuli1-3. This suggests that rodents are appropriate subjects for elucidating the molecular substrates of behavior and complex psychiatric conditions4. Understanding the molecular substrates of complex behavioral processes, such as motivation, frequently requires brain microinjection. Both the brain microinjection technique and a primary motivation assay will be presented here. Rats will be used as subjects, but these procedures can readily be adapted to well-handled mice. Included herein are procedures for the manufacture of the required cannulae, obturators (dummy cannulae or stylets), and microinjectors. The method presented is significantly more flexible and more cost-efficient than prefabricated implements. This flexibility will prove valuable when optimizing conditions. Importantly, because the microinjection procedure can be used to test a myriad of hypotheses; the techniques presented here should be broadly applicable. For example, receptor ligands can be microinjected to understand neurochemistry3,5,6; cell-permeable peptides and small-molecules can be microinjected to understand intracellular signaling pathways7-10; toxins, ion channel blockers, or antagonist cocktails can be microinjected to understand circuitry1,11,12.
While the generic protocol presented here can be readily adapted by the user for their particular needs, the procedure is particularly well suited for behavioral assays since microinjection occurs in awake rodents that are only under mild hand restraint. No anesthesia or special restraints are required. This is possible because the brain itself lacks pain sensation. However, if anesthesia is not used, microinjection must occur through cannulae that were previously stereotaxically implanted. This is because nociceptors are present on the scalp, meninges,13 which are the membranes surrounding the brain, and the periosteum,14 which is the membrane covering the skull. It should be noted that microinjection under anesthesia is sometimes desirable. One example is when the virus is being injected, and one may wish to inject virus directly through either stainless steal needles15 or glass pipettes because this can reduce tissue damage and improve transduction efficiency.16,17 The microinjectors described below can be modified for this purpose and suggestions on how to do this can be found in the Discussion. Because other JoVE articles have demonstrated stereotaxic brain cannula implantation,18-20 these procedures will not be covered here.
We present these microinjection procedures together with an assay that quantifies motivation. Several rodent models of motivated behavior are currently in use, such as the runway box and barrier scaling. Here, we describe how to use an operant progressive ratio schedule of reinforcement to quantify motivation where operant responding is being maintained by a reinforcer. Responding on the progressive ratio is responsive to reinforcer magnitude.21,22 Accordingly, this assay is routinely used as a proxy for motivation and/or reinforcing efficacy. 21,23-30 Because several excellent reviews have covered this topic in detail,21,24 we will focus mainly on practical concerns.

<table><tbody><tr><td>Cannula Tubing</td><td>Amazon Supply/ Small Parts</td><td>HTXX-26T-60</td><td>26 G, Hypotube S/S 316-TW 26GA</td></tr><tr><td>Obturator</td><td>Amazon Supply/ Small Parts</td><td>GWXX-0080-30-05</td><td>33 gauge, Wire S/S 316LVM 0.008 IN</td></tr><tr><td>Microinjector Wire</td><td>MicroGroup</td><td>33RW 304</td><td>33 gauge</td></tr><tr><td>Super Glue</td><td>Loctite</td><td>3924AC</td><td>Liquid, Non-gel, can be autoclaved</td></tr><tr><td>Microinjector Plastic Tubing</td><td>Becton Dickson</td><td>427406</td><td>PE20</td></tr><tr><td>Medium Weight Hemostats</td><td>World Precision Instruments</td><td>501241-G</td><td></td></tr><tr><td>Ruler</td><td>Fisher</td><td>09-016</td><td>150 mm</td></tr><tr><td>#7 Forceps</td><td>Stoelting</td><td>52100-77</td><td>Dumont, Dumostar</td></tr><tr><td>Rotary Tool</td><td>Dremmel</td><td>285</td><td>Two-speeds</td></tr><tr><td>Cut-off Disc</td><td>McMaster Carr</td><td>3602</td><td>15/16&quot; x 0.025&quot;</td></tr><tr><td>Microinjection Pump</td><td>Harvard Apparatus</td><td>PhD 2000</td><td></td></tr><tr><td>1 ul Glass Syringe</td><td>Hamilton</td><td>7001KH</td><td>Needle Style: 25s/2.75&quot;/3</td></tr><tr><td>Cotton Tipped Applicator</td><td>Fisher</td><td>23-400-101</td><td></td></tr><tr><td>Lab Wipes</td><td>Kimwipes</td><td>34133</td><td></td></tr><tr><td>Operant Software</td><td>Coulbourn</td><td>Graphic State</td><td></td></tr><tr><td>Operant Chambers</td><td>Coulborun</td><td>Habitest</td><td></td></tr></tbody></table>

rodent brain microinjection to study molecular substrates of motivated behavior

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as appropriate models, partly because the response to behaviorally relevant stimuli and the circuitry parsing stimulus-action outcomes is astonishingly similar between humans and rodents. In studying molecular substrates of complex behaviors, the microinjection of reagents that modify, augment, or silence specific systems is an invaluable technique. However, it is crucial that the microinjection site is precisely targeted in order to aid interpretation of the results. We present a method for the manufacture of surgical implements and microinjection needles that enables accurate microinjection and unlimited customizability with minimal cost. Importantly, this technique can be successfully completed in awake rodents if conducted in conjunction with other JoVE articles that covered requisite surgical procedures. Additionally, there are many behavioral paradigms that are well suited for measuring motivation. The progressive ratio is a commonly used method that quantifies the efficacy of a reinforcer to maintain responding despite an (often exponentially) increasing work requirement. This assay is sensitive to reinforcer magnitude and pharmacological manipulations, which allows reinforcing efficacy and/ or motivation to be determined. We also present a straightforward approach to program operant software to accommodate a progressive ratio reinforcement schedule.

여기에서 우리는 상기 절차를 얻을 수있다 결과의 예를 도시한다. 표시된 첫 번째는 바이러스 벡터 (그림 3)에 의해 형질 전환 된 전형적인 지역이다. 일반적으로 3 ~ 1mm의 형질 볼륨 선조체 조직을 얻을 수있다. 형질 영역의 부피 카발리의 방법을 사용하여 직렬 섹션에 걸쳐 정량화 될 수있다 (32)이 중요한 것은, 형질 볼륨은 형질되는 조직의 형태와 같은 많은 요인에 의존한다.; 바이러스 혈청 형; 프로모터; 속도 및 주입 부피; 주입 된 입자의 수​​; 인젝 산도; 및 만니톨과 같은 삼투압 시약, 여부, 사용되었다. 33, 34 일반적으로, 우리는 10 분에 걸쳐 10 ~ 12 입자 / ㎖, 1 μL /면을 microinject 및 obturators 교체 7 분 추가 이전 할 수 있습니다. 또한, pH가 테이트 8. 다음 주위 통상, 우리는 동기가 어느 유전자의 발현에 의해 조작 될 수 있음을 보여준다 (도 4)또는 약리 시약 미세 주입 (그림 5). 그림 4에서 우리는 독점적으로 디자이너 마약 (DREADDs)에 의해 활성화 디자이너 수용체를 표명했다. DREADD 수용체 코딩 영역은 내부 리보솜 항목 사이트 및 mCitrine 카세트으로 이어졌습니다. mCitrine는 형질 감염된 세포의 편리를 육안으로 확인 할 수 있습니다. DREADD는 이종삼 G- 단백질 Gαq에 커플 링시켰다. 성상 세포, 28,35 및 DREADD 자체를 자극 할 수 Gαq 결합 DREADD의 활성화는 클로자핀-N-산화물 (CNO, 3 ㎎ / ㎏, IP)의 전신 투여에 의해 활성화 될 수있다. 14,22,36 쥐에 훈련을받은 레버는 3 레버 프레스 60 연속 일 동안 매일 1 시간 세션 동안 마실 1 기회를 얻었다 에탄올 보강에 대한 반응한다. 다음, 쥐 금욕을 강요하고, 핵 핵심 성상 세포를 중격 의지에 Gαq 결합 DREADDs 표현했다. 삼주의 절제, 자기 ADMI에 의욕 후<html> nister 에탄올 브레이크 포인트에 의해 측정 하였다. 핵 21,27-30 활성화 CNO 전신 투여를 통해, 절제는 차량에 비해 후 에탄올자가 투여를 재개 쥐의 의욕을 감소 코어 성상 중격 의지. 중요한 것은, CNO 대신 DREADD의 녹색 형광 단백질을 발현시켰다 동등 훈련 코호트에 영향을 미치지 않았다. <img alt="그림 3" src="/files/ftp_upload/53018/53018fig3.jpg" /> 마이크로 인젝션 된 바이러스에 의해 형질도 3 대표 복측 선조체 지역.이 이미지는 핵의 영역은 위의 방법에 따라 형질 감염시켰다 아스트로 중격 의지를 나타낸다. 데이터는 저작권 소유자의 허가를 불 등. 13에서 재 인쇄되어 있습니다. 그 세부 사항은 첨부 된 간행물 및 보충 자료에서 발견 될 수있다.&quot;빈&gt;이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오. <img alt="그림 4" src="/files/ftp_upload/53018/53018fig4.jpg" /> 자기 관리] 에탄올에 쥐 그림 4. 동기 부여는 핵 핵심 성상 세포를 중격 의지에. 표현을 통해-바이러스가 표현 할 일주를 허용 한 후 바이러스가 미세 주입하고, 동기는 브레이크 포인트를 통해 측정 한 형질 전환 유전자를 활성화하여 감소 하였다. 유전자는 전용 디자이너 마약 (DREADDs)에 의해 디자이너 수용체 활성화 된 표현. 일원 분산 분석 (F (2,30) = 3.29, P = 0.04)은 Scheffe 사후에 의해 밝혀 다음에 그 클로자핀-N-산화물의 전신 투여에 의한 DREADD의 활성화 (CNO, 3 ㎎ / ㎏, IP ) 크게 대신 DR의) CNO는 녹색 형광 단백질 (GFP를 표현했다 동등하게 교육을받은 집단에 영향을 미치지 아니합니다 차량에 비해 절제 후자가 관리] 에탄올에 쥐의 의욕을 감소EADD. 데이터는 저작권 소유자의 허가를 불 등. 13에서 재 인쇄되어 있습니다. 그 세부 사항은 첨부 된 간행물 및 보충 자료에서 발견 될 수있다. <img alt="그림 5" src="/files/ftp_upload/53018/53018fig5.jpg" /> 그림 간극 결합 차단제 5. 미세 주입은 절제 후 에탄올을-관리 자체에 쥐의 의욕을 증가 두 간극 결합 차단제는 에탄올 관리 자체에 쥐 (브레이크 포인트를 통해 측정) 동기 부여에 미치는 영향을 평가 하였다 :. 메플로퀸 및 18 α - 글리시 레틴 산 (18-α)를. 두 일원 분산 분석은 핵으로 갭 접합 차단제의 미세 주사가 핵심이 절제 후 에탄올을-관리 자체에 쥐의 의욕을 증가 중격 의지 것으로 나타났다 (F (3,40) = 5.56, P = 0.003). 데이터는 저작권 소유자의 허가를 불 등. 13에서 재 인쇄되어 있습니다. 자세한 사항은이 책에서 찾을 수 있으며첨부 된 보충 자료.

Watch this Scientific Journal Video about Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior at JoVE.com

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented.

쥐 뇌 미세 주입은 분자 기판 동기를 행동을 연구하기 위해

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as ...

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JoVE Journal

Behavior

14.3K 조회수.  Virginia Commonwealth University School of Medicine. Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented. 

비디오: Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

의 정위 주입 MicroRNA의 발현 마우스 해마 CA1 영역으로 렌티 바이러스 및 행동 결과의 평가

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

연구

행동분석학

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes of interest allows for enhanced temporally- and regionally-specific control of gene deletion, compared to existing methods. While conditional deletion can also be achieved by mating mice that express Cre recombinase under the control of specific gene promoters with mice carrying a floxed gene, stereotaxic microinjection allows for targeting of discrete brain areas at experimenter-determined time points of interest. In the context of cocaine conditioned place preference, and other cocaine behavioral paradigms such as self-administration or psychomotor sensitization that can involve withdrawal, extinction and/or reinstatement phases, this technique is particularly useful in exploring the unique contribution of target genes to these distinct phases of behavioral models of cocaine-induced plasticity. Specifically, this technique allows for selective ablation of target genes during discrete phases of a behavior to test their contribution to the behavior across time. Ultimately, this understanding allows for more targeted therapeutics that are best able to address the most potent risk factors that present themselves during each phase of addictive behavior.

This article describes how to microinject viral vectors into mouse brain and then test in a conditioned place preference paradigm that includes an acquisition, extinction and reinstatement phase.

크레 재조합이 코카인 컨디셔닝 장소 환경에있는 표적 유전자의 역할을 연구하는 표현 바이러스 성 벡터의 정위 미세 주입

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes ...

Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific genes in encoding experience is one of the main objectives of molecular neuroscience. Furthermore, the functional association of defined genes with specific behaviors has implications for understanding the basis of neuropsychiatric disorders. Induction of robust transcription programs has been observed in the brains of mice following various behavioral manipulations. While some genetic elements are utilized recurrently following different behavioral manipulations and in different brain nuclei, transcriptional programs are overall unique to the inducing stimuli and the structure in which they are studied1,2.
In this publication, a protocol is described for robust and comprehensive transcriptional profiling from brain nuclei of mice in response to behavioral manipulation. The protocol is demonstrated in the context of analysis of gene expression dynamics in the nucleus accumbens following acute cocaine experience. Subsequent to a defined in vivo experience, the target neural tissue is dissected; followed by RNA purification, reverse transcription and utilization of microfluidic arrays for comprehensive qPCR analysis of multiple target genes. This protocol is geared towards comprehensive analysis (addressing 50-500 genes) of limiting quantities of starting material, such as small brain samples or even single cells.
The protocol is most advantageous for parallel analysis of multiple samples (e.g. single cells, dynamic analysis following pharmaceutical, viral or behavioral perturbations). However, the protocol could also serve for the characterization and quality assurance of samples prior to whole-genome studies by microarrays or RNAseq, as well as validation of data obtained from whole-genome studies.

This manuscript describes a protocol that applies comprehensive profiling for analysis of transcriptional programs induced in specific brain nuclei of rodents following behavioral paradigms. Herein, this approach is illustrated in the context of profiling genes induced in the nucleus accumbens (NAc) of mice following acute cocaine exposure, utilizing microfluidic qPCR arrays.

행동 경험에 따라 뇌 샘플에서 전사 역학의 종합 분석

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific ...

Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or knockdown gene expression in a relatively focused region without significant damage to the brain tissue. After recovery, the injected mouse can be tested on various behavioral tasks such as the Open Field Test (OFT) and the Forced Swim Test (FST). The OFT is designed to assess locomotion and the anxious phenotype in mice by measuring the amount of time that a mouse spends in the center of a novel open field. A more anxious mouse will spend significantly less time in the center of the novel field compared to controls. The FST assesses the anti-depressive phenotype by quantifying the amount of time that mice spend immobile when placed into a bucket of water. A mouse with an anti-depressive phenotype will spend significantly less time immobile compared to control animals. The goal of this protocol is to use the stereotactic injection of a lentivirus in conjunction with behavioral tests to assess how genetic factors modulate animal behaviors.

Stereotactic injection of lentiviruses expressing cDNAs or shRNAs can modulate gene expression in specific brain areas of mice. Here, we present a protocol to combine stereotactic injections with behavioral tasks, such as the Open Field Test (OFT) and the Forced Swim Test (FST).

정위 사출 기술을 적용하는 것은 동물의 행동에 유전 적 영향을 연구하기

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or ...

Survivable Stereotaxic Surgery in Rodents 

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different systemic challenges (pharmacological or physiological) to the CNS. For example, one can directly measure how the animal's midbrain dopamine projections respond to dopamine-releasing drugs like d-amphetamine or natural stimuli like food. In this video, we show you how to implant guide cannulas targeting specific sites in the rat brain, how to insert and implant a microdialysis probe and how to use high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to measure extracellular levels of oxidizable neurotransmitters and metabolites. Local precise introduction of drugs through the microdialysis probe allows for refined work on site specificity in a compound s mechanism of action. This technique has excellent anatomical and chemical resolution but only modest time resolution as microdialysis samples are usually processed every 20-30 minutes to ensure detectable neurotransmitter levels. Complementary ex vivo tools (i.e., slice and cell culture electrophysiology) can assist with monitoring real-time neurotransmission.

Survivable Stereotaxic Surgery in Rodents

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

설치류에 살아서 Stereotaxic 외과

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

생물학

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

동시 전기 생리 녹화 및 설치류의 뇌에서 억제 에이전트의 마이크로 주사

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

신경과학

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

다운스트림 분석을 위한 냉동 설치류 뇌 영역 모음

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

Adaptable Angled Stereotactic Approach for Versatile Neuroscience Techniques

Stereotactic surgery is an essential tool in the modern neuroscience lab. However, the ability to precisely and accurately target difficult-to-reach brain regions still presents a challenge, particularly when targeting brain structures along the midline. These challenges include avoiding of the superior sagittal sinus and third ventricle and the ability to consistently target selective and discrete brain nuclei. In addition, more advanced neuroscience techniques (e.g., optogenetics, fiber photometry, and two-photon imaging) rely on targeted implantation of significant hardware to the brain, and spatial limitations are a common hindrance. Presented here is a modifiable protocol for stereotactic targeting of rodent brain structures using an angled coronal approach. It can be adapted to 1) mouse or rat models, 2) various neuroscience techniques, and 3) multiple brain regions. As a representative example, it includes the calculation of stereotactic coordinates for targeting of the mouse hypothalamic ventromedial nucleus (VMN) for an optogenetic inhibition experiment. This procedure begins with the bilateral microinjection of an adeno-associated virus (AAV) encoding a light-sensitive chloride channel (SwiChR++) to a Cre-dependent mouse model, followed by the angled bilateral implantation of fiberoptic cannulae. Using this approach, findings show that activation of a subset of VMN neurons is required for intact glucose counterregulatory responses to insulin-induced hypoglycemia.

Described here is a stereotactic procedure that can target challenging and difficult-to-reach brain regions (due to spatial limitations) using an angled coronal approach. This protocol is adaptable to both mouse and rat models and can be applied to diverse neuroscientific applications, including cannula implantation and microinjections of viral constructs.

다목적 신경 과학 기술을 위한 적응가능한 각진 입체 적 접근 법

Stereotactic surgery is an essential tool in the modern neuroscience lab. However, the ability to precisely and accurately target difficult-to-reach brain ...

Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and deafferentation of neuronal groups, which are both invasive methods that permanently impair the integrity of the target area. These methods are accompanied by collateral effects that can affect the analysis of acute and temporal regulatory mechanisms. The stereotaxic implantation of guide cannulas aimed at specific brain regions, followed by a recovery period, allows researchers to microinject different drugs after the disappearance of the undesired effects of the surgery. Tetrodotoxin has been used to determine the roles of several brain areas in diverse physiological processes because it transiently inhibits the sodium-dependent action potentials, thus blocking all neural activity in the target region. This protocol combines this method with strategies for the assessment of the estrous cycle and ovulation to reveal the role of discrete brain regions in the regulation of ovulation at particular times of any given stage of the estrous cycle. Awake and unrestrained rats (Rattus norvegicus) were used to avoid the blocking effects that anesthetics and stress hormones exert on ovulation. This protocol can be easily adapted to other species, brain targets and pharmacological agents to study different physiological processes. Future improvements to this method include the design of a microinjection system using glass capillaries of small diameter instead of guide cannulas. This will reduce the amount of tissue damaged during the implantation and decrease the spread of the infused drugs outside the target area.

This protocol describes the construction of a low-cost microinjection system, its stereotaxic implantation into deep-brain structures and the procedure for timed microinjections of tetrodotoxin in awake and unrestrained rats. The goal is to reveal the participation of hypothalamic structures in the regulation of ovulation by inhibiting their neural activity.

테트로독신 미세 주입에 의해 뒤집을 수 있는 불활성화를 통해 배란의 규칙에 있는 쥐 두뇌의 이산 영역의 역할을 해명

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and ...

Stereotaxic Intracranial Delivery of Chemicals, Proteins or Viral Vectors to Study Parkinson's Disease

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in the substantia nigra. Affected brain areas display intraneuronal fibrillar inclusions consisting mainly of alpha-synuclein (asyn) proteins. No animal model thus far has recapitulated all characteristics of this disease. Here, we describe the use of stereotaxic injection to deliver chemicals, proteins, or viral vectors intracranially in order to mimic various aspects of PD. These methods are well-established and widely used throughout the PD field. Stereotaxic injections are incredibly flexible; they can be adjusted in concentration, age of animal used for injection, brain area targeted and in animal species used. Combinations of substances allow for rapid variations to assess treatments or alter severity of the pathology or behavioral deficits. By injecting toxins into the brain, we can mimic inflammation and/or a severe loss of dopaminergic neurons resulting in substantial motor phenotypes. Viral vectors can be used to transduce cells to mimic genetic or mechanistic aspects. Preformed fibrillar asyn injections best recapitulate the progressive phenotype over an extended period of time. Once these methods are established, it can be economical to generate a new model compared to creating a new transgenic line. However, this method is labor intensive as it requires 30 minutes to four hours per animal depending on the model used. Each animal will have a slightly different targeting and therefore create a diverse cohort which on one hand can be challenging to interpret results from; on the other hand, help mimic a more realistic diversity found in patients. Mistargeted animals can be identified using behavioral or imaging readouts, or only after being sacrificed leading to smallercohort size after the study has already been concluded. Overall, this method is a rudimentary but effective way to assess a diverse set of PD aspects.

We describe how to successfully inject solutions into specific brain areas of rodents using a stereotaxic frame. This survival surgery is a well-established method used to mimic various aspects of Parkinson's disease.

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in ...