FONCICYT 000000000091984,, Jeansson의 기초, Söderström Königska sjukhemmet, 마사 Lundqvist 및 시거드 루시 엘사이 작품은 스웨덴 의학 연구 협의회 (K2009 - 75SX - 21028-01 - 3, K2008 - 61X - 20700-01 - 3)에 의해 추진하는 사업 Goljes 민, 그리고 의약 SLS - 95151의 스웨덴어 학회. 교수 진 D. 블록, UCLA는 기꺼이 원고에 대한 귀중한 의견을 인정받고 있습니다. 우리는 비디오 현미경 제공을위한 HCN 채널 차단에 대한 박사 마이클 Andäng 및 박사 헬레나 Johard 감사하고, 교수 압델 엘 - Manira. 윤리적인 고려 사항 : 동물의 모든 실험은 Karolinska Institutet와 &quot;스톡홀름의 Norra Djurförsöksetiska Nämnd&quot;에 의해 명시된 지침과 규정에 따라 수행되었다. 모든 동물 실험은 모든 가능한 응력이나 동물에 불편을 최소화하기 위해 의도로 수행됩니다.

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루시페라제 리포터 기술과 장점과 단점 낮의를 공부하기 위해서는 여러 시간 포인트 (샘플링 주파수에 따라 일반적으로 2-4 시간의 낮은 시간 해상도를 제공)에서 조직 샘플을 필요로 원위치 하이브리드화과 서양 얼룩 이러한 RT - PCR로 전직 생체내 방법, 달리 유전자와 단백질 표현의 변화, 루시페라제 리포터 기술은 높은 해상도 (10-10 분) 같은 준비에 많은 일 동안 circadian oscillations의 연구를 수 있습니다. 리듬의 위상 및 기간에 대한 효과 그러므로, 사용하는 동물의 수를 최소화하고 상세한 연구는 일반적으로 낮은 시간 해상도로 기존의 샘플링 기법을 사용 불가능한지, 가능합니다. 리듬의 상대 진폭 측정이 가능 있지만 그러나, 그것은 기자의 기술은 양적하지 않으며 따라서 베꼈는데 유전자 또는 단백질 번역의 양을 측정하는 데 사용할 수 없다는 것을 강조해야합니다. 조직 녹음 organotypic SCN 문화는 성인 뇌의 장기 (주) 약리 조작을 허용하기 시작하고 즉시 culturing, 직접 생체내 빛을 유도 위상 교대 등, 생체내 스트레스의 분자 효과를 공부에 대한 큰 장점 후 분석할 수 손상 성숙한 신경 네트워크와 루시페라제 리포터 기술을 포함하는 조직은 여러 주 동안 안정적인 레코딩이 가능합니다. 따라서 조직은 건강한 문화를 얻기 위해서는 신생아 또는 출생 후의 초기 필요하지 않으며, 급성 만성 슬라이스 약리 치료와는 대조적으로 수행할 수 있습니다. epigenetic 변경은 연구해야하는 경우없이 염색체에 DNA의 결합으로 이어질 할 셀 문화에 플라스미드 - 기자 transfections, 대조적으로, 유전자 변형 루시페라제 - 동물 모델 (뿐만 아니라 lenti 바이러스 transfections)이 유리합니다. 그것은이 상황에서도 발광 이미징은 녹음도 circadian 리듬을 공부 전공 실험실로 활용 단일 SCN의 뉴런 (~ 60-10 μm의)와 다른 세포 유형에 있으므로, 매우 민감한 CCD 카메라 11 요즘 가능하다는 것을 언급해야합니다. 마지막으로, 여러 circadian 조사는 일반적으로 시계 유전자 / 단백질 oscillations 16-19를 공부하기 위해서는 이러한 그린 형광 단백질과 같은 다른 기자를 사용하여 분자 표현의 모니터링을 사용합니다. 슬라이스 두께의 측면 여기에서 권장하는 SCN 슬라이스 (200-300 μm의)의 두께가 감소하거나 원할 경우 증가 수 있습니다. 조직 문화의 몇 일 후에 막에 나가 flattens하지만 그러나, 그것은 조직 7 생존을 보존하기 위해 처음 컷 단면 500 μm의 두께를 초과하지 않는 것이 좋습니다. 100 μm의보다 얇은 슬라이스가 처리하기 어려운 기계 및 실질적인 이유입니다. SCN (예를 들어 &#39;껍질&#39;, 그리고 지느러미 대 복부 지역 대의 &quot;핵심&quot;) 이내에 다른 지역은 다른 단계와 함께 진동 SCN 조직 이질 때문에 슬라이스의 두께가 루시페라제 출력 신호의 위상에 영향을 미칠 수, 이후 20 differentially이 단계 이후에 다시 동기화 21 교대. 더 많은 조직이 광자의 큰 숫자를 방출 이후 슬라이스의 두께는 광자 개수의 기준에 영향을 미칩니다. 분자 oscillations의 진폭이 방식으로 간접적으로 explant의 크기에 의해 영향을받을 수 있습니다. 이러한 이유로, 제어 및 치료 문화는 항상 같은 크기, 두께가 같은 위상과 같은 진폭과 진동하기 위하여 SCN의 동일한 영역을 포함해야합니다. 두 일방적 핵은 반면에, 서로와 한 유사한 amplitudes와 단계에 진동 vibratome 컷 (차례로 소뇌와 두 반구 사이의 절단 분리는 직각입니다에 의존하는 직각 수평이 아니므로 테이블 표면). SCN의 culturing을 수행하는 조사는 SCN의 문화가 서로 단계에서 진동하지 않는 경험을 할 수도 있습니다. 연습과 깔끔히 기술의 표준화, 조각은 항상 SCN의 동일한 rostro - 꼬리 수준 버렸어요 즉, 결과를 향상됩니다. 중요 단계 <ol><li> 산소 공급이 중요합니다. 두뇌는 산소 22이 많이 필요하기 때문에, 깔끔히 절차 빨리해야합니다 (3.1-3.10 가까이수록 그것은 조직을 건강하게 유지하기 위해서입니다 ~ 5~6분하는 것입니다.) </li><li> SCN은 온도 변화와 매체 교환을 구분합니다. SCN은 온도 민감하고 큰 온도 변화가 단계 맥박 조정기를 이동하고 실험 유물 23가 발생할 수 있습니다. 중간이나 솔루션은 문화에 며칠 후에 약물을 적용하는 예를 들어, 지속적인 실험, 매체 또는 솔루션을 도중에 교환 또는 추가하는 경우n은 사전 예열 36-37로 ° C (챔버와 동일한 온도)뿐만 아니라 전에해야합니다. 중간 교류를 포함 약리 실험에서, 항상 사전에 시설 세포 배양 매체와 함께 작업하는 것이 중요합니다. 또한 / 신선한와 함께 씻어, 무조건 매체는 잠재적으로 분자 SCN 리듬 24뿐만 아니라 다른 세포 유형 문화 25 단계 이동하실 수 있습니다. </li><li> 중간 구성. 정확한 산도와 osmolality 문화 조직을하는 것이 중요합니다. 공기 버퍼링 매체 산도 6.8-8.2의 범위에서 최적의 버퍼 용량을 가지고 있으며, 세포 호흡의 결과로 발생할 수있는 산도의 변화를 버퍼링에 적합합니다 HEPES 많은 양의가 포함되어 있습니다. HEPES는 반면에, 같은 문화 매체 소량에 포함되어있는 나트륨 중탄산염,에 비해 온도 변화에 더 민감합니다. 나트륨 중탄산염는 CO 2 분위기 때문에 적절한 낮은 PH (5.1-7.1) 범위 26에서 큰 버퍼링 용량이 있습니다. 그러나, 공기 버퍼링 매체 (DMEM 2902)에서 나트륨 중탄산염의 금액을 증가 상당히 osmolality를 증가 동시에 매체, 아미노산, 비타민 및 이온의 잘못된 구성에 차례 결과 인치를 diluting없이 추가할 수 없습니다 매체를 준비 조언 : <ol><li> 매체를 혼합하면 정확하고주의하십시오. </li><li> 오직 신선한 또는 갓 해동 주식 솔루션을 사용합니다. </li><li> 매우 높은 osmolality의 경우 그것은 diluting 가치가되지 않습니다. 이상 5~6% - 희석 매체는 대부분 작동하지 않습니다. 너무 낮으면 osmolality를 증가 D - 포도당을 사용합니다. </li></ol></li><li> 단계는 준비 시간으로 인해 교대 슬라이스 준비의 시간은 중요합니다. 슬라이스 절차가 ZT 12월 6일부터 12일까지 사이에 수행되는 경우에는 단계에 제로 또는 최소한의 효과를 얻을 수 있습니다. 모든 슬라이스 해부은 준비 사이의 유사 콘텐츠를 위상으로 인한 오류를 최소화하기 위해 낮의 또는 circadian 사이클의 동일한 단계에서 수행해야합니다. </li></ol> 가능한 수정 <ol><li> 깔끔히 시작하기 전에 산소 (95% O 2, 5 % CO 2)의 포화 버퍼; 급성 SCN의 전기 생리학은 깔끔히는 링어 (ACSF 인공 세리 브로 척수)으로 수행되어야한다. 또한, 산소는 전기 시냅스 활동이 산소 공급 22에 직접 의존으로 전체 깔끔히 절차를 수행하는 동안 버퍼에 지속적으로 추가되어야합니다. </li><li> 수평 및 화살 조각도 성공적으로 준비하고 SCN의 교양 수 있습니다. 저자는 코로나 컷과 비교 경험에 따라 낮은 진폭과 circadian oscillations를 제공 수평 조각에 루시페라제 기록과 경험을했습니다. </li><li> SCN은 약 1mm 길이 rostro - caudally이며 깔끔히 여기에서 설명한 기법은 중앙 지역의 SCN 부분을 취득으로 타겟팅됩니다. 그러나, 하나 이상의 SCN 섹션은 SCN의 핵보다 주동이의 비교 꼬리 수준 circadian 유전자 / 단백질 표현의 조사를 허용, 마우스 및 쥐 두뇌에서 구할 수 있습니다. </li><li> 심지어 어린 동물, 출생 후의 새끼 또는 배아에서 슬라이스 문화가도 준비하실 수 있습니다. 아주 어린 새끼의 두뇌와 두개골이 아주 부드럽고 구분 유의하시기 바랍니다. 또한, 시신경이 얇고 완벽하게 개발되지 않습니다. SCN 영역이 손상되지 않도록 새끼의 머리를 해부 때주의를 가져가라. 그들이 너무 큰 있기 때문에 예를 들어, 마이크로 rongeur 도구는 마우스 새끼의 두개골을 열어 사용할 수 없습니다. 좋은 가위는 어린 새끼에있는 부드러운 조직을 절단 정도입니다. </li><li> 여기에 설명된 커버 유리 기반 문화 기술은 또한 하나의 SCN의 뉴런에서 CCD / EM - CCD 카메라를 사용하여 이미지 발광에도 사용할 수 있습니다. </li><li> 기타 중추 신경계의 지역 및 유전자 변형 PER2에서 말초 장기의 조직 : 루크 동물 쉽게 PER2로, 분자 oscillations 측면에서 얻은 교양 및 분석할 수 : 루크 표현은 다른 조직의 숫자도 많은 일 동안 진동을 계속 및 세포 유형 10. 이러한 주변 조직의 대부분은 인스턴스 간, 생존을위한 멤브레인 필요하지 않지만 대신에 직접적으로 매체 11 목욕 양식입니다. 인해 sectioning로 변화되는 단계는, culturing와 매체 교환이 SCN과 동일한 원칙을 따르지 않을 수 있습니다 주시기 바랍니다. </li></ol> 의미 유전자 변형 마우스 및 쥐 종자 (mPER2 : 륙, mPer1 - 륙 8, 10, 27)에서 루시페라제 효소 활동은 단백질이나 유전자 발현의 리듬을 반영하고 bioluminescence 기록에 의해 평가하실 수 있습니다. 루시페라제 생성된 bioluminescence는 약한 신호를 제공하지만 배경 발광이 방법이 유리하고, 제로에 가까운 것입니다. 또한, 루시페라제 분자가 불안정하고 급속하게 저하되기 때문에 PH가 없습니다장기적인 흥분성의 조명 28 동안 나타날 수 있습니다 ototoxicity. 함께 촬영이 속성은 circadian 연구 루시페라제 리포터 기술이 매우 유리하게 오랫동안 실험 수 있습니다.

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>B27 supplement 50x</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>Cover glasses</td>
<td>&nbsp;</td>
<td>Menzel-Gl&auml;ser</td>
<td>40#1</td>
<td>&#8776; 40 mm</td>
</tr>
<tr>
<td>Culture membranes</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>PICMORG50</td>
<td>0.4 &mu;m</td>
</tr>
<tr>
<td>DMEM low glucose w/o phenol red</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D2902</td>
<td>Powder for 1L</td>
</tr>
<tr>
<td>Filter paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>1003 055</td>
<td>&#8776; 55 mm</td>
</tr>
<tr>
<td>Filtration set</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>431097</td>
<td>Pore size 0.22 &mu;m
Polyethersulfone</td>
</tr>
<tr>
<td>Forceps </td>
<td>&nbsp;</td>
<td>Allgaier Instrumente</td>
<td>0203-7-PS</td>
<td>Dumant #7</td>
</tr>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>15630-056</td>
<td>100 ml</td>
</tr>
<tr>
<td>HBSS 10x</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco </td>
<td>14065-049</td>
<td>500 ml</td>
</tr>
<tr>
<td>NaOCH3 7.5%</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>25080-060</td>
<td>50 ml</td>
</tr>
<tr>
<td>Luciferin </td>
<td>&nbsp;</td>
<td>Promega</td>
<td>E1602</td>
<td>Beetle luciferin, potassium salt</td>
</tr>
<tr>
<td>Micro rongeur tool</td>
<td>&nbsp;</td>
<td>Allgaier Instrumente</td>
<td>332-097-140</td>
<td>&nbsp;</td>
<tr>
<td>PenStrep 10,000 U/ml</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>15140-122</td>
<td>100 ml</td>
</tr>
<tr>
<td>Petri dishes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430165</td>
<td>&#8776; 35 mm</td>
</tr>
<tr>
<td>PMT</td>
<td>&nbsp;</td>
<td>Hamamatsu</td>
<td>H9319-11MOD</td>
<td>&nbsp;</td>
<tr>
<td>Disposable scalpels</td>
<td>&nbsp;</td>
<td>Paragon</td>
<td>P503</td>
<td>Size 11</td>
</tr>
<tr>
<td>Vacuum filter</td>
<td>&nbsp;</td>
<td>Fisher </td>
<td>09-761-5</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum grease</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>&nbsp;</td>
<td>50 g</td>
</tr>
<tr>
<td>Vibratome</td>
<td>&nbsp;</td>
<td>Campden Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

slice preparation, organotypic tissue culturing and luciferase recording of clock gene activity in the suprachiasmatic nucleus

생리와 행동의 일상 리듬을 조정 중앙 circadian (~ 24 시간) 시계 앞쪽에 시상 하부에있는 suprachiasmatic 핵 (SCN)에 있습니다. 시계는 직접 망막과 시신경을 통해 빛이 의해 동기화됩니다. Circadian oscillations는 시대 (당) 유전자를 포함하는 소위 &quot;시계 유전자&quot;와 그들의 단백질 제품의 여러 부정적인 피드백 루프를 상호 작용에 의해 생성됩니다. 코어 클럭은 멤브레인 탈분극, 칼슘 및 캠프 1에 따라 달라집니다. SCN은 시계 유전자 발현, 신진 대사 활동과 자발적인 전기적 활동에 매일 oscillations을 보여줍니다. 현저하게,이 내생 순환 활동 SCN 2-4 성인 조직 슬라이스에 지속. 이러한 방법으로 생물 학적 시계는 쉽게 맥박 조정기 함수의 분자, electrophysiological 및 신진 대사 조사를 허용, 체외에서 공부하실 수 있습니다. SCN 바로 광학 chiasm 5 위에있는 작은, 잘 정의된 양자 구조입니다. 쥐에서는 각 핵에서 ~ 8.000 뉴런을 포함하여 약 947 μm의 (길이, rostrocaudal 축) × 424 μm의 (폭) × 390 μm의 (높이) 6 규격이 있습니다. 그것은 SCN이 확인할 수 있습니다 두뇌의 특정 수준에서 뇌 슬라이스를 잘라하는 데 필요한 SCN을 해부하다합니다. 여기서는 마우스 및 쥐 머리가 비슷합니다 SCN의 해부와 깔끔히 절차를 설명합니다. 또한, 우리는 어떻게 막 7, 야마자키 외 의해 SCN 조직 문화의 개발 기술에 organotypically 해부하는 조직 문화를 보여줍니다. 8. 마지막으로, 우리는 유전자 변형 조직은 시계 유전자 / 동적 루시페라제 리포터 기술, 원래 Geusz 외 의해 circadian 측정에 사용된 방법입니다. 9를 사용 단백질의 표현을 측정하는 데 사용할 수 방법을 보여줍니다. 우리는 여기서 유전자 변형에서 SCN의 조직을 사용하는 PERIOD2 인 노크 : 유 외 제작한 루시페라제 쥐 10.. 마우스는 시대의 융합 단백질 (당) 2 반딧불의 효소 루시페라제가 포함되어 있습니다. 루시페라제은 luciferin의 산화를 catalyzes 때 PER2가 루시페라제 즉, luciferin을위한 기판의 존재로 번역하면 PER2 표현식은 bioluminescence으로 모니터링할 수 있습니다. 방출 광자의 개수는 긍정적으로 생산 PER2 단백질의 금액에 상호 및 bioluminescence 리듬은 생체내 10 PER2 단백질 리듬을 일치합니다. 이런 방식으로 PER2 표현의 주기적 변화는 지속적으로 며칠 동안 실시간으로 모니터링할 수 있습니다. 우리가 조직 culturing 실시간 bioluminescence 기록에 대한 수행 프로토콜은 철저하게 야마자키와 다카하시 11 설명했습니다.

Watch this Scientific Journal Video about Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus at JoVE.com

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

성인 마우스 hypothalamic suprachiasmatic 핵 (SCN), 그리고 문화에 빠른 방법 organotypic 문화 상태에있는 SCN의 조직을 포함 준비 슬라이스의 절차가보고됩니다. 또한, 동적 루시페라제 리포터 기술을 사용하여 oscillatory 시계 유전자 단백질 표현의 측정이 설명되어 있습니다.

슬라이스 준비, Organotypic 조직 Culturing과 Suprachiasmatic 핵의 시계 유전자 활동의 루시페라제 녹음

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

slice-preparation-organotypic-tissue-culturing-luciferase-recording

Research

JoVE Journal

Neuroscience

35.1K 조회수.  Karolinska Institutet. 성인 마우스 hypothalamic suprachiasmatic 핵 (SCN), 그리고 문화에 빠른 방법 organotypic 문화 상태에있는 SCN의 조직을 포함 준비 슬라이스의 절차가보고됩니다. 또한, 동적 루시페라제 리포터 기술을 사용하여 oscillatory 시계 유전자 단백질 표현의 측정이 설명되어 있습니다.

비디오: Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

의 연결을 부상을 검사에 대한 Organotypic Hippocampal 슬라이스 문화 모델

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

연구

신경과학

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

Organotypic Hippocampal 슬라이스 문화

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

네크로 텍스의 Organotypic 슬라이스 준비에서 전체 셀 녹음

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

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. ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

Organotypic 슬라이스 문화의 Purkinje 세포 수지상의 형태학의 분석

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

쥐에 활동 륜 실행에 녹음 및 Circadian 리듬 분석

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Organotypic 슬라이스는 문화 희소 돌기 아교 세포 역학 및 수초화을 공부하기

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

에서 신경 혈관 리모델링의 분석 Entorhino - 해마 Organotypic 슬라이스 문화

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.