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 border="0" cellpadding="0" cellspacing="0" width="574">
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			<td height="26" style="height:26px;width:140px;">Name</td>
			<td style="width:73px;">Company</td>
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			<td style="width:292px;">Comments</td>
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			<td height="26" style="height:26px;width:140px;">Pentobarbital</td>
			<td style="width:73px;">CTS</td>
			<td style="width:69px;">170066</td>
			<td style="width:292px;">Concentration: 60 mg / ml&#160; in physiological saline.</td>
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			<td height="40" style="height:41px;width:140px;">Big scissors&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">14001-16</td>
			<td style="width:292px;">Any large scissors or a guillotine with sufficiently sharp edges can be used for decapitation&#160;</td>
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			<td height="40" style="height:41px;width:140px;">Iris scissors&#160;</td>
			<td style="width:73px;">Prestige medical</td>
			<td style="width:69px;">48,148</td>
			<td style="width:292px;">Any fine tip scissors can be used, provided the scissor blades are not longer than 1.5 - 2 cm</td>
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			<td height="26" style="height:27px;width:140px;">Fine tip forceps&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">11254-20</td>
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			<td height="26" style="height:27px;width:140px;">Scalpel&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">91003-12</td>
			<td style="width:292px;"></td>
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		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Scalpel blade #11</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">10011-00</td>
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			<td height="26" style="height:27px;width:140px;">Small spatula&#160;</td>
			<td style="width:73px;">Fisher&#160;</td>
			<td style="width:69px;">2350</td>
			<td style="width:292px;"></td>
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		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Filter paper</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">Any laboratory brand can be used.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Petri dishes</td>
			<td style="width:73px;">Duroplan</td>
			<td style="width:69px;">Z231509-1</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Glass beakers&#160;</td>
			<td style="width:73px;">SCHOT</td>
			<td style="width:69px;">10022846</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Pasteur pipette&#160;</td>
			<td style="width:73px;">Maple Leaf Brand</td>
			<td style="width:69px;">14672-029</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Super glue&#160;</td>
			<td style="width:73px;">LOCTITE</td>
			<td style="width:69px;">4091361/1</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Slicer</td>
			<td style="width:73px;">Campden</td>
			<td style="width:69px;">7000-smz</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Ceramic slicing blade</td>
			<td style="width:73px;">Campden</td>
			<td style="width:69px;">7550-1-C</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Magnetic heater/stirrer</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For heating up the SPS for the procedure</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Electric kettle</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For heating up water for temperature control</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:56px;width:140px;">Slice recovery chamber + heating unit</td>
			<td style="width:73px;">Warner instruments</td>
			<td style="width:69px;">&#160;BSC-HT +&#160; BSC-BUW</td>
			<td style="width:292px;">Home-built models may also be used.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Thermometer</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">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

25.6K 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

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

TMS의 국가 종속성 효과 : 동기 Phosphene 동작 좀보세요

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

연구

신경과학

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

이미징 페로몬은 마우스 Vomeronasal 급성 조직 슬라이스 준비에 감지

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

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 ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Immunoprecipitation 및 아실-비오틴 거래소 (아베)에 의한 양식 Hippocampal 뉴런의 단백질 Palmitoylation의 감지

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

뇌 슬라이스 바이오 티 닐화 :<em&gt; 전의 VIVO</em성인 뉴런에서 지역 고유의 플라즈마 막 단백질 인신 매매를 측정하는&gt; 접근

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

유도 된 신경 세포로 성숙한 인간 두뇌의 혈관 주위 세포에서 파생 된 세포의 리니지 - 프로그래밍

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

마우스 보습 코 기관의 급성 조직 조각에서 정의 된 세포 집단의 심층 생리 학적 분석

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

급성 Hippocampal 조각에 작은 볼륨 재활용-, 관류-침수 형 챔버 시스템 유지에 시 냅 스가 소성을 기록

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

초파리 애벌레의 열 환경 설정을 결정 하는 온도 분석 결과

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

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 ...