We would like to acknowledge the significant contribution Dr. Shiwei Huang (Australian National University) in validating the method. Furthermore, we would like to thank Ms. Kasia Pietrajtis for helpful comments regarding Golgi cells and Mr. Vitaly Lerner for the cortex experimental data. This work was supported by PITN-GA-2009-238686 (CEREBNET), FP7-ICT (REALNET), ELSC and ISF.

이 프로토콜에 설명 된 모든 실험 절차는 히브리 대학의 동물 관리 및 사용위원회에 의해 승인되었습니다. 1. 슬라이스에 대한 솔루션 및 도구 준비 <ol><li> 표 1에 기재된 이온을 함유하는 표준 생리 용액 (SPS)의 1 L를 준비한다. <ol><li> 탈 이온수 (0.055 μS / cm의 전도도) 1,000 ml로 채워진 저장소 유리 병에 미리 10 배의 최종 농도에서의 염을 함유하는 스톡 용액을 준비한다. 소금 (염화나트륨, KCl을, KH 2 PO 4, 황산)을 추가하고 완전히 용해 될 때까지 저어. 4 ℃에서 원액을 저장합니다. </li><li> 실험 당일 최종 SPS 용액을 확인. 자기 교반 막대를 이용하여 글루코스 (3.6 g)과의 NaHCO3 (2.18 g)을 용해, 정제수 700 ml의 스톡 용액 100 ㎖를 첨가하고, 그 다음 1 L. 정제수 위로 추가 </li><li> soluti를 평형에서 ~ 20 분 동안 95 % O 2 / 5 % CO 2 가스 처리를하여. 이 후, 1 M CaCl2를 용액 2 ㎖를 추가합니다. </li></ol></li><li> 다음과 같은 도구 (그림 1)를 조립합니다. <ol><li> 두개골을 여는 큰 가위 잘린 다른 도구, 작은 수술 가위를 준비, 뇌를 노출 뇌의 원하는 부분을 해부를 위해 블레이드와 메스 두개골을 리프팅을위한 좋은 팁 집게는 슬라이스를 조작하기위한 작은 주걱한다 해부하는 뇌의 부분. </li><li> 또한, 뇌에서 초과 액체를 제거하기 위해 필터 종이의 작은 조각을 준비, 뇌에 대 한 두 개의 작은 페트리 접시에 뇌를 붙이는 포함 따뜻하게 정제 물과 SPS, 및 시아 노 아크릴 레이트 접착제에 대해 두 개의 유리 500 ml의 비커에 해부한다 분할 단계. </li><li> 분할 및 복구 차에 슬라이스 화장실에서 조각을 이동하는 입이 큰 유리 파스퇴르 피펫 동안 조각을 처리하기위한 작은 브러시를 준비합니다mber. 따뜻한 슬라이스 화장실에서 박테리아의 성장을 방지 할 수있는 도구를 소독. 또한, 깨끗한 실험실 장갑을 사용합니다. </li></ol></li><li> 깁과 에드워즈 (15)에 의해 기술 등의 침수 슬라이스 복구 챔버를 준비합니다. 지속적으로 95 %, O2 / 5 % CO 2로의 SPS를 가스와 36 ° C로 유지한다. 가스 처리에 슬라이스가 손상 될 수 욕조에 갇혀 작은 기포가 발생하지 않는 것을 확인하십시오. </li><li> 따뜻한 ~ 자석 교반기 또는 수조에 히터 접시에 36 ° C의 SPS 300 ml의. 히터 플레이트가 사용되는 경우, 이온 침전 될 수 과열로부터 용액을 방지하기 위해주의를 기울여야. 경우는, 대신에 이전의 냉각 따뜻한 신선한 SPS 발생합니다. </li><li> 전기 주전자를 사용하여 끓여 정제수 ~ 500 ml의를 가져오고, 비커, 슬라이싱 온도보다 약간 따뜻한 물을 얻기 위해 차가운​​ 물로 300 mL로 결합한다. 이 잘린 동안 정제 물을 데워 사용하고 remai를 사용나중에 분할 목욕의 생리적 온도를 유지하기위한 삶은 물 닝. </li><li> 복강 내에 펜토 바르 비탈 0.1 ㎖ (60 ㎎ / ㎖)을 주입하여 마우스를 마취시키다. 몇 분 후 동물이 감각의 부족을 확인하기 위해 강력한 발가락 또는 꼬리 핀치에 응답하지 않습니다 있는지 확인하십시오. </li></ol> 2. 뇌를 해부 <ol><li> 두개골의 뒷면 근처 큰 가위로 목을 절단하여 마우스를 빠르게 목을 벨, 그리고 머리가 초과 혈액을 씻어 따뜻하게 정제수로 비커에 빠질 수 있습니다. </li><li> 아마도 작은 가위로 목을 더 제거, 목의 근육과 피부 아래 두개골에서 난원 매그넘 노출. </li><li> 명확 두개골의 개구를 안내하기 위해 두개골 봉합을 볼 수 있도록 두개골 위에 피부를 당겨. </li><li> 난원 매그넘을 통해 작은 가위의 하단 끝을 삽입하고 즉시 기수를 돌려 열린 두개골을 잘라측면. 부드럽게 눈과 frontoparietal 봉합 뒤에 위치로 정수리 뼈까지의 측면 가장자리를 따라 잘라; 그 다음, (도 2a에 녹색 점선 참조) 두개골의 중심을 향해 회전과 두개골의 다른쪽에 정중선을 가로 질러. </li><li> 좋은 팁 집게를 사용하여 (그림 2A 노란색 점선 화살표 참조) 뇌를 노출 비스듬히 frontoparietal 모서리에서 두개골을 들어 올려 비스듬히 당겨합니다. 두개골 들어 올릴 때 뇌가 장소에 머물 수 있도록 두개골, 머리의 뼈 또는 피부에 부착되지 않았는지 확인합니다. 뇌가 두개골로 움직이는 경우, 두개골과 뇌 사이의 결합 조직을 제거하는 작은 가위를 사용한다. </li><li> 뇌가 노출되면, 뇌가 건조하는 것을 허용하지 않는다. 두개골과 따뜻하게 가득 페트리 접시의 뇌를 유지하면서 따라서, 다음 단계를 수행, SPS를 가스실. </li><li> 전자에 사용되는 뇌의 일부를 분리메스와 주걱을 이용하여 뇌의 나머지 xperiment. 소뇌를 사용하는 경우, (그림 2B-D에 붉은 라인을 따라) 중뇌와 뇌교를 통해 하나의 컷에 의해 전뇌에서 분리하고 신선한 포함하는 작은 페트리 접시로 이동, SPS를 가스실. </li><li> 수평면에서 소뇌를 슬라이스 할 때 무대를 절단에 뇌를 붙이는 직선과 넓은 기반을 형성하는 뇌간을 제거합니다. 전뇌에서 절단 된면이 아래로 향하도록 간단히 필터 종이의 젖은 부분에 소뇌를 배치합니다. <ol><li> (그림 2B에서 파란색 선으로 표시됨)의 기본을 형성하는 메스와 뇌간을 차단하고, SPS에 소뇌를 반환합니다. 절단의 다른면이 필요할 때, 그에 따라 소뇌 블록 (그림 2C &amp; D에 파란색 선)을 트림. </li></ol></li></ol> 3. 뇌 조각화 <ol><li> 가 D가되는 것을 확인하는 의해 절단 단계 준비공예, 무대의 중앙에 superglue의 작은 방울을 적용하기 전에. </li><li> 올바른 쪽이 위로 주걱을 사용하여 SPS에서 뇌 블록을 들어 올리고 필터 종이의 작은 조각을 사용하여 뇌에서 초과 액체를 제거합니다. 그런 다음, 하나의 움직임, 무대에서 접착제의 드롭에 주걱의 두뇌를 밀어 넣습니다. </li><li> 건조으로부터 뇌 조직의 측면에 닫 실행 한 접착제를 방지하기 위해, 파스퇴르 피펫 SPS의 몇 방울을 적용하고 슬라이싱 챔버에서 절단 단을 배치. 관심 영역 (예 : 소뇌 피질) 블레이드를 향하도록 뇌 블록을 맞추고, 따라서 분리되기 전에, 기계적 압력의 최소량을 실시한다. </li><li> 끊임없이 절단 실에서 SPS 가스 처리뿐만 아니라 34-37 ° C에서의 온도를 유지하면서 제조업체의 지시에 따라 각 조각을 잘라. 통근자를 충전함으로써,이 범위에서 절단 실의 온도 유지가온 정제수 온도가 너무 낮게 떨어질 때마다 물 교체와 슬라이서의 알 챔버. 참고 : 분할 매개 변수를 실험적으로 검토되고있는 조직과 신경 세포의 각 유형을 찾을 수 있어야합니다. 세라믹 블레이드와 소뇌 골지 세포와 캠든 SMZ 700 슬라이서의 경우 0.75 mm의 진동 진폭 65 Hz에서 0.05 mm / sec의 속도의 발전을 사용; 소뇌 핵 신경 세포 높은 진폭과 주파수 (1mm, 90 Hz에서, 각각)와 느린 진행 속도 (0.01-0.02 mm / 초)을 권장합니다. 골지과 소뇌 핵 신경 세포 모두를 들어, 슬라이스 두께 300 μm의를해야한다. </li><li> 슬라이싱 동안, 조각 자체에 접어 허용하지 않습니다. 부드럽게 바람직뿐만 아니라 실험에 관심이있는 지역에서 조각을 터치, 부드러운 브러시로를 지원함으로써이 문제를 방지합니다. vibratome 블레이드를 만지지 브러시를 방지하기 위해주의; 특히 세라믹 블레이드 경우에도 광 접촉블레이드가 손상 될 수 있습니다. </li><li> 입이 큰, 화재 광택 유리 파스퇴르 피펫을 사용하여 챔버를 유지 / 복구에 분할 실에서 각 조각을 이동합니다. </li><li> 슬라이스 실험을 시작하기 전에 최소한 1 시간 동안 챔버 휴식하자. 이 손상되거나 죽어가는 조직과 세포의 분해를 허용함으로써 청소기 슬라이스 표면에 발생합니다. 처음 시간 동안 34-36 ° C의 범위의 챔버 내에 SPS의 온도 유지; 슬라이스는 슬라이스가 손상되거나이 떠 할 수 기포에 감동되지 않도록. </li></ol> 4. 실험 <ol><li> 1 시간 후, 복구 챔버 온도를 실온 (17 ~ 25 ° C에서)까지 냉각 할 수 있습니다. NOTE :이 슬라이스는 박테리아의 성장뿐만 아니라 세포 대사를 느리게하여 사용할 수있는 기간을 연장시킬 수있다. 이 기간은 뇌 영역 및 세포의 종류에 따라 달라집니다. 예를 들어, 골지체 특정 유형의 세포 조각이 건강한 것으로 판명 될 수있다8 시간 다른 유형 반면 절단 후 만 6 시간 지속. </li><li> 1 시간이 슬라이스에서 전달 된 후, 다양한 전기 생리학 실험에 대한 슬라이스를 사용합니다. </li></ol>

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+Inhibitory+Input+to+the+Inferior+Olive+Decreases+Electrical+Coupling+and+Blocks+Subthreshold+Oscillations.">Cerebellar Inhibitory Input to the Inferior Olive Decreases Electrical Coupling and Blocks Subthreshold Oscillations.</a> Neuron. 81 (6), 1389-1400 (2014).</li><li>Bourne, J. N., Kirov, S. A., Sorra, K. E., Harris, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Warmer+preparation+of+hippocampal+slices+prevents+synapse+proliferation+that+might+obscure+LTP-related+structural+plasticity.">Warmer preparation of hippocampal slices prevents synapse proliferation that might obscure LTP-related structural plasticity.</a> Neuropharmacology. 52, 55-59 (2007).</li><li>Kirov, S. A., Sorra, K. E., Harris, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slices+have+more+synapses+than+perfusion-fixed+hippocampus+from+both+young+and+mature+rats.">Slices have more synapses than perfusion-fixed hippocampus from both young and mature rats.</a> J. Neurosci. 19, 2876-2886 (1999).</li><li>Kirov, S. A., Petrak, L. J., Fiala, J. C., Harris, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+spines+disappear+with+chilling+but+proliferate+excessively+upon+rewarming+of+mature+hippocampus.">Dendritic spines disappear with chilling but proliferate excessively upon rewarming of mature hippocampus.</a> Neuroscience. 127, 69-80 (2004).</li><li>Tanaka, Y., Tanaka, Y., Furuta, T., Yanagawa, Y., Kaneko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+cutting+solutions+on+the+viability+of+GABAergic+interneurons+in+cerebral+cortical+slices+of+adult+mice.">The effects of cutting solutions on the viability of GABAergic interneurons in cerebral cortical slices of adult mice.</a> J. Neurosci. Methods. 171, 181-125 (2008).</li></ol>

The authors declare that they have no competing financial interests.

우리는 생리 대신 빙냉 온도 마우스로부터 급성 뇌 조각을 제조하는 방법을 보여준다. 또한, 추운 상태로 제조와 비교시 따뜻한 조건에서 얻어진 조각의 품질이 우수하다는 11 도시 슬라이서 블레이드 최소한 수직 진동을 가지고 제공되었다. 생리적 온도에서 슬라이스는 단일 셀의 수차뿐만 아니라 네트워크 동작에 명시 할 수있다 대사 과정 17 ~ 19의 변화에 ...

제시된 방법의 목적은, 특히 성인 또는 이전 동물을 사용하는 경우, 시험 관내 전기 생리 학적 실험을 위해 고품질 급성 뇌 조각을 얻을 수있다. 두 우아한 문장 Skrede과 Westgaard 1에 의해 설명 된 바와 같이 급성 뇌 분할 방법은, 현대 신경 과학 연구의 기초 중 하나가되었습니다 전 세계적으로 수많은 변형에 사용된다. 슬라이스 품질 슬라이스 당 뉴런 생활의 수에 반영되는 시간의 기간 동안 세포는 조직의 무결성뿐만 아니라 자신의 전기 생리 학적 및 형태 학적 특성을 유지한다. 또한, 안정된 기록 용 최대 기간은 조각의 품질에 의존한다. 따라서, 수십 따라, 원래 슬라이싱 방법은 상기 절단 조성물의 복잡한 변형에 의해 종종 2-10을 절단 후 슬라이스 복구를 강화하기 위해 개별 연구 그룹에 의해 개발 된 오뿐만 아니라 냉각 생리 솔루션과 동물의 내 심장 미리 관류 (예 : 아스 코르 빈산, 티오 우레아 또는 H 2 O 2를 추가하는 등) R 복구 솔루션. 최근 11를 표시 한 바와 같이, 슬라이스 동안 생리적 온도는 신경 세포의 건강에 냉각보다 더 도움이 될 것 같다; 성인 (2~8개월) 설치류와 함께 작업 할 때 개선이 가장 눈에 띄는입니다. 극적인 온도 변화를 방지 인해 이러한 가소성 (13)와 이온 채널 반응 속도 13, 14 등의 세포에서 온도에 따라 프로세스에 아티팩트를 방지 할 수 있습니다. 이러한 변화는 막 전압 및 세포 내 칼슘 신호, 스파이크 임계 값에 영향을 미치는, 모양 스파이크 수 있습니다. 여기서 제시된 &quot;핫&quot;급성 슬라이스 제조 방법은, 임의의 뇌 영역에서 고품질의 급성 뇌 조각을 획득 소뇌 피질 및 해마, 뇌간 16 핵을 포함하는 일반적인 절차 &lt;/ SUP&gt;뿐만 아니라 후각 망울로, 모두 쥐와 생쥐. 특히, 생리적 온도 슬라이싱 절차는 절단 날이 거의 완벽하게 수평 진동 및 구조적 결함이없는 것을 필요로한다. 정밀 이전 슬라이서 모델 달성하지 않을 수 있습니다; 저온 대사 수차의 비용해도, 기계적 손상 조직 저항력 만들 것 같은 경우에, 우리는 동결 - 냉각 조건 슬라이스 준비를 수행 권장.

<table><tbody><tr><td>Pentobarbital</td><td>CTS</td><td>170066</td><td>Concentration: 60 mg/ml in physiological saline.</td></tr><tr><td>Big scissors&amp;nbsp;</td><td>FST</td><td>14001-16</td><td>Any large scissors or a guillotine with sufficiently sharp edges can be used for decapitation&amp;nbsp;</td></tr><tr><td>Iris scissors&amp;nbsp;</td><td>Prestige medical</td><td>48,148</td><td>Any fine tip scissors can be used, provided the scissor blades are not longer than 1.5&amp;#8211;2 cm</td></tr><tr><td>Fine tip forceps&amp;nbsp;</td><td>FST</td><td>11254-20</td><td></td></tr><tr><td>Scalpel&amp;nbsp;</td><td>FST</td><td>91003-12</td><td></td></tr><tr><td>Scalpel blade #11</td><td>FST</td><td>10011-00</td><td></td></tr><tr><td>Small spatula&amp;nbsp;</td><td>Fisher&amp;nbsp;</td><td>2350</td><td></td></tr><tr><td>Filter paper</td><td></td><td></td><td>Any laboratory brand can be used.</td></tr><tr><td>Petri dishes</td><td>Duroplan</td><td>Z231509-1</td><td></td></tr><tr><td>Glass beakers&amp;nbsp;</td><td>SCHOT</td><td>10022846</td><td></td></tr><tr><td>Pasteur pipette&amp;nbsp;</td><td>Maple Leaf Brand</td><td>14672-029</td><td></td></tr><tr><td>Super glue&amp;nbsp;</td><td>LOCTITE</td><td>4091361/1</td><td></td></tr><tr><td>Slicer</td><td>Campden</td><td>7000-smz</td><td></td></tr><tr><td>Ceramic slicing blade</td><td>Campden</td><td>7550-1-C</td><td></td></tr><tr><td>Magnetic heater/stirrer</td><td></td><td></td><td>For heating up the SPS for the procedure</td></tr><tr><td>Electric kettle</td><td></td><td></td><td>For heating up water for temperature control</td></tr><tr><td>Slice recovery chamber + heating unit</td><td>Warner instruments</td><td>&amp;nbsp;BSC-HT +&amp;nbsp; BSC-BUW</td><td>Home-built models may also be used.</td></tr><tr><td>Thermometer</td><td></td><td></td><td>For monitoring SPS temperature during dissection and slicing</td></tr></tbody></table>

slice it hot: acute adult brain slicing in physiological temperature

여기에서 우리는 급성 뇌 조각의 준비를위한 프로토콜을 제시한다. 이 절차는 크게 결과의 품질을 결정하는 전기 생리 패치 클램프 실험을위한 중요한 요소이다. 이 절차를 절단하는 동안 냉각 단계를 생략하는 성숙한 동물에서 매우 유수의 뇌 구조를 다루는 특히, 건강한 슬라이스와 세포를 얻는 데 도움이되도록 표시되었습니다. 승온 만에 추측 할 수 신경 건강을 지원함으로써 정확한 메커니즘은, 그것이 그 추론하기 위하여 스탠드에도 가능한 한 슬라이싱이 수행되는 온도는 온도 관련 아티팩트를 방지하는 생리 학적 조건에 근접한다. 이 방법의 또 다른 중요한 장점은 절차의 단순성 때문에 짧은 준비 시간이다. 증명 방법에서는 성인 마우스는 사용되지만 동일한 과정 어린 마우스뿐만 아니라 래트에 적용 할 수있다. 또한, 다음 패치 카스티A 실험 수평 소뇌 슬라이스 수행되지만 동일한 절차가 다른 평면뿐만 아니라 뇌의 다른 후방 영역에서 사용될 수있다.

기술 된 방법으로 제조 슬라이스 다양한 전기 생리 및 optogenetic 실험에 사용될 수있다. 도 3a 및도 3c에서, 우리는 각각의 차동 간섭 (DIC) 하에서 광학 보아 수평 소뇌 슬라이스 관상 대뇌 피질 슬라이스의 대표적인 예를 나타낸다. 소뇌 슬라이스에서, 소뇌 뉴런 여러 종류의 용이, 그들의 위치 및 셀 체형에 의해 인식 될 수있는 전기 생리 녹화 대상으로 허용한다. 그림 (b)와 3D에서...

Watch this Scientific Journal Video about Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature at JoVE.com

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

슬라이스 뜨거운 것이 : 생리적 온도에서 급성 성인 뇌 슬라이스

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

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Neuroscience

26.2K 조회수.  Hebrew University of Jerusalem. In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

비디오: Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

최적화 된 N를 사용 하 여 급성 뇌 조각의 준비-메 틸-D-glucamine 보호 복구 방법

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

연구

신경과학

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

흑색질 도파민 뉴런의 Somatodendritic 도메인에서 세포 내 패치 클램프 녹음

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

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, ...

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called &#34;directional motility&#34;, toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice.

Microglial 프로세스 매력 ATP 또는 급성 뇌 조각에 세로토닌의 2 광자 영상

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

성인 인간의 뇌에서 단기 자유 부동 슬라이스 문화

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.

성인과 노화 마우스에서 해마 조각에서 저산소증 최소화

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high ...

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of cellular and synaptic properties. Nevertheless, there is an ever increasing need to perform correlated analyses between different experimental modes in addition to slice electrophysiology, for example: immunohistochemistry, molecular biology, in vivo imaging or electrophysiological recording; to answer evermore complex questions of brain function. However, making meaningful conclusions from these various experimental approaches is not straightforward, as even within relatively well described brain structures, a high degree of sub-regional variation of cellular function exists. Nowhere is this better exemplified than in the CA1 of the hippocampus, which has well-defined dorso-ventral properties, based on cellular and molecular properties. Nevertheless, many published studies examine protein expression patterns or behaviorally correlated in vivo activity in the dorsal extent of the hippocampus; and explain findings mechanistically with cellular electrophysiology from the ventro-medial region. This is further confounded by the fact that many acute slice electrophysiological experiments are performed in juvenile animals, when other experimental modes are performed in more mature animals. To address these issues, this method incorporates transcardial perfusion of mature (&#62;60 day old rodents) with artificial cerebrospinal fluid followed by preparation of modified coronal slices including the septal pole of the dorsal hippocampus to record from CA1 pyramidal cells. This process leads to the generation of healthy acute slices of dorsal hippocampus allowing for slice-based cellular electrophysiological interrogation matched to other measures.

The purpose of this protocol is to describe a method to produce slices of the dorsal hippocampus for electrophysiological examination. This procedure employs perfusion with chilled ACSF prior to slice preparation with a near-coronal slicing angle which allows for preservation of healthy principal neurons.

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of ...

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

급성 마우스 뇌 슬라이스 자발적인 해 마 네트워크 활동을 조사

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...

Assessing Changes in Synaptic Plasticity Using an Awake Closed-Head Injury Model of Mild Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a prevalent health issue in North America. There is increasing pressure to utilize ecologically valid models of closed-head mTBI in the preclinical setting to increase translatability to the clinical population. The awake closed-headed injury (ACHI) model uses a modified controlled cortical impactor to deliver closed-headed injury, inducing clinically relevant behavioral deficits without the need for a craniotomy or the use of an anesthetic.
This technique does not normally induce fatalities, skull fractures, or brain bleeds, and is more consistent with being a mild injury. Indeed, the mild nature of the ACHI procedure makes it ideal for studies investigating repetitive mTBI (r-mTBI). Growing evidence indicates that r-mTBI can result in a cumulative injury that produces behavioral symptoms, neuropathological changes, and neurodegeneration. r-mTBI is common in youths playing sports, and these injuries occur during a period of robust synaptic reorganization and myelination, making the younger population particularly vulnerable to the long-term influences of r-mTBI.
Further, r-mTBI occurs in cases of intimate partner violence, a condition for which there are few objective screening measures. In these experiments, synaptic function was assessed in the hippocampus in juvenile rats that had experienced r-mTBI using the ACHI model. Following the injuries, a tissue slicer was utilized to make hippocampal slices to evaluate bidirectional synaptic plasticity in the hippocampus at either 1 or 7 days following the r-mTBI. Overall, the ACHI model provides researchers with an ecologically valid model to study changes in synaptic plasticity following mTBI and r-mTBI.

Here, it is demonstrated how an awake closed-head injury model can be used for examining the effects of repeated mild traumatic brain injury (r-mTBI) on synaptic plasticity in the hippocampus. The model replicates important features of r-mTBI in patients and is used in conjunction with in vitro electrophysiology.

경미한 외상성 뇌 손상의 깨어 있는 폐쇄 두부 손상 모델을 사용하여 시냅스 가소성의 변화 평가

Mild traumatic brain injuries (mTBIs) are a prevalent health issue in North America. There is increasing pressure to utilize ecologically valid models of ...

Preparation of Brain Sections Using a NMDG-Based Protective Recovery Method

Source: Ting, J. T. et al. <a href="https://app.jove.com/v/53825">Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method.</a> J. Vis. Exp. (2018).
This video describes a protocol to obtain brain slices using an N-Methyl-D-glucamine (NMDG)-based protective recovery method. A dissected brain is incubated in an aCSF with NMDG and HEPES to minimize neuronal cell death and damage during the sectioning. The section is allowed to recover in the aCSF to restore the cellular activities

NMDG 기반 보호 회복 방법을 사용한 뇌 절편 준비

1. Brain Dissection and Slicing

	Decapitate the animal. Use a scalpel to open the skin on the head and expose the skull cap.
	Use fine super-cut scissors ...