이 연구는 FMMU 새로운 재단과 부분적으로 NIH의 기금을 찾는 의해 지원되었다.

1. Glia 세포 문화 Glial 세포는 중추 신경계의 다른 지역에서 교양 수 있습니다. 전체 프로세스는 프로세스 그림에 표시됩니다. <img src="/files/ftp_upload/2111/2111process.jpg" alt="프로세스 1" /> 1 일 코팅 문화 플레이트와 coverslips <ol><li> 건조 멸균 유리 현미경의 원형은 압력솥에 coverslips. </li><li> 멸균 24 웰 플레이트에 문화 플레이트 살균 coverslips. </li><li> 코팅 폴리 - 라이신과 루트 온도에 따라 2 시간 동안 부화와 coverslips. </li><li> 3 단계와 같은 방식으로 코트 6 - 웰 문화 번호판입니다. </li><li> 증류수와 문화 후드의 건조 공기를 두 번 coverslips 6 - 글쎄 문화 플레이트 씻으십시오. </li><li> 5 % CO 2와 37 정도에 따라 6 자 판 24 - 잘 판 각 잘 2 ML 각 물론 200 μL DMEM 매체를 (10 % FBS)를 추가하고 인큐베이터에 넣어 </li></ol> 2 일 분리 피질과 glial 세포 배양 <ol><li> 긍정적인 흐름 절개 후드를 소독. </li><li> 20 분 대한 자외선을 켭니다. </li><li> 70 % 에탄올 모든 표면을 스프레이하고 사용하기 전에 15 분 기다립니다. </li><li> 시제 변화 : 마취 쥐 저체온증에 의해 새끼 (P2 - P6), 및 운영 가위를 사용하여 framen 매그넘의 기지에서 목을 베다. </li><li> 두개골에서 이리스 가위와 껍질을 사용하여 화살 봉합 따라 두개골을 엽니다. </li><li> forebrain를 제거하고 stereomicroscope에서 차게 L15 중간 넣어, 신중하게, 혈관과 함께 두라와 피아 멤브레인을 청소 피질을 분리하고 L15 매체를 여러 번 씻는다. </li><li> 미세의 가위와 작은 조각으로 피질 컷. </li><li> 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) 15 분 37 급 피질의 조각과 부화에 L15 매체를 사용한 0.125 %를 추가합니다. </li><li> 최종 농도 10ug/mL에 DNase 주식 솔루션을 추가 조직 솔루션을 대기음 아래 화재 광택 유리 Pasteure 피펫 20 번, 단일 세포 현탁액을 수집, 50mL 튜브에 최고 20 % FBS와 20mL DMEM 매체로 조직 블록을 전송합니다. </li><li> DMEM 매체를 다시 중지 10% FBS과 DMEM의 세포를 5000-10000/cm 2 hemocytometer, 씨앗 세포와 현미경으로 세포를 계산합니다.와 세포 현탁액 한 번 씻으십시오 37 학위 5% 배양기에서 세포를 유지, 주당 2 회 매체의 절반을 변경합니다. </li></ol> 2. 도설 루트 신경 뉴런의 절연, 문화 및 정제 1 일 문화의 자료를 준비 <ol><li> 폴리 - 라이신, 세차 coverslips와 살균 coverslips 두 번과 물과 공기 건조를 증류 코트는 24 잘 접시에 넣어 두었어요. </li><li> 2퍼센트 B27와 2.5S NGF (50 NG / ML)로 100μL Neurobasal 매체를 추가, 37도 5 % CO 2에 접시를 넣어. </li></ol> 일 2 배아에서 DRGs을 분리 <ol><li> glial 세포 배양과 같은 방법으로 흐름 후드를 소독. </li><li> CO 2, 70 % 에탄올로 소독 복부와 과다 복용하여 임신 쥐 (E15)를 안락사, 배아는 자궁에서 분리 차게 L15 중간에 투입되었다. </li><li> 냉장 L15 매체와 35mm 요리 stereomicroscope 및 전송 아래에 연결 등의 루트 신경로 척수 코드를 분리. </li><li> 단일 세포 현탁액를 수집하고 NBF 매체 (Neurobasal 매체 2% B27와 2.5S NGF (50 NG / ML) 포함)을 한 번 씻는다. </li></ol> 3 일 정화 DRG 뉴런에게 <ol><li> 18H - 24 신경 세포의 시딩 후, 20 μm의 200 μL의 최종 볼륨의 최종 농도에 신경 세포의 문화에 1 ㎜ 집중 FUDR 주식 솔루션을 추가합니다. </li><li> 72h는 나중에, FUDR없이 2퍼센트 B27와 2.5S NGF (50ng/mL)이있는 Neurobasal 매체로 절반 매체를 교체하십시오. 그 후, 매일 매체를 변경합니다. </li></ol> 3. glial 세포와 Coculture DRG 뉴런 <ol><li> glial 세포 배양이 합류를 (시딩 이후 20 일 정도)에 도달하면, 그들은 뉴런과 coculture 준비했다. </li><li> 24시간 정화 뉴런을 추가하기 전에, glial 세포 매체는 2퍼센트 B27와 2.5S NGF (50 NG / ML)이있는 Neurobasal 매체로 변경되었습니다. </li><li> 정화 뉴런이 문화 우물에서 수집된 단일 세포 현탁액은 화재 광택 Pasteure의 피펫을 통해 기계적 전달하여 얻은되었습니다. </li><li> 숫자를 계산하면, 뉴런은 2퍼센트 B27와 2.5S NGF (50ng/mL)이있는 Neurobasal 매체 합류 glial 세포에서 500/cm 2 씨앗을 품고 있었다. </li><li> glial 세포에서 뉴런의 부착 및 neurite 성장이 기록과 세포 유형의 특정 항체를 사용하여 이미지 분석 소프트웨어와 immunocytochemistry에 의해 서로 다른 시간 시점에서 분석할 수 있습니다. </li></ol> 4. 대표 결과 <ol><li> Glial 세포 배양이 : 시딩 후, glial 세포는 20 일 정도 합류하게 될 것입니다. 위상 콘트라스트 microsc에서ope, 그것은 쉽게 다른 형태학의 glial 세포 subpopulations 다른 성장 패턴 substructures를 결성하고 immuocytochemisty 기술 (그림 1, 그림 2) 그림과 같이 GFAP 긍정적인 astrocytes가 90 % 이상 차지하는 것이 확인되었다. </li><li> DRG 뉴런의 문화 : 우리의 방법에서 72 시간 FUDR 치료 후, DRG 뉴런의 순도 6 일 이내 99%만큼 높이 도달할 것이며, DRG 뉴런들은 독특한 morphologies을 보여주 높은 밀도 neurite 성장 (그림 3, 그림 4). </li><li> Glial 세포와 뉴런 coculture : DRG 뉴런의 부착 및 neurite의 파생물이 시딩 후 4 시간 이내 glial 세포에서 쉽게 발생,주의 관찰이 신경 세포의 부착 및 neurite 성장이 특별한 성장 패턴 substructures을 형성 glial 세포 subpopulations에 의해 영향을 것을 보여주었다, 이것은 쉽게 확인 수 위상 콘트라스트 현미경과 immunocytochemistry (그림 5, 그림 6)에서. </li></ol><img src="/files/ftp_upload/2111/2111fig1.jpg" alt="그림 1" /> 그림 1. 합류 glial 세포의 형태가. 두피 glial 세포가 polylysine 코팅 coverslip에 도금 20 일 동안 교양 있었다, glial 세포의 서로 다른 성장 패턴을 참고, 왼쪽에있는 세포는 방사 방법으로 배열. <img src="/files/ftp_upload/2111/2111fig2.jpg" alt="그림 2" /> 그림 2. Glial fibrillary 산성 단백질 (GFAP) 항체에 의해 표시 콘플루앙 glial 세포가. 오른쪽에 GFAP (적색) 긍정적인 세포의 방사 배열을 확인합니다. <img src="/files/ftp_upload/2111/2111fig3.jpg" alt="그림 3" /> 그림 3. 도설 루트 신경의 뉴런은 FUDR 치료하지 않고 체외에서 성장했다. 훼손 세포가 뉴런 &#39;배경을 DRG 형성. <img src="/files/ftp_upload/2111/2111fig4.jpg" alt="그림 4" /> 그림 4. 도설 루트 신경의 뉴런은 FUDR 치료 후 체외에서 성장했다. 배경 오염 세포가 완전히 제거했다. <img src="/files/ftp_upload/2111/2111fig5.jpg" alt="그림 5" /> 그림 5. glial 세포에서 성장 도설 루트 신경의 뉴런은. 뉴런의 부착 및 neurite 성장은 방사 배열 세포에 저해과 오른쪽 glial 세포에 국한되었다. <img src="/files/ftp_upload/2111/2111fig6.jpg" alt="그림 6" /> 그림 6. neurofilament 항체에 의해 분류 glial 세포에서 성장 도설 루트 신경의 신경이. neurite (녹색)가 방사 배열 왼쪽 glial 세포에서 저해하고 오른쪽에 제한되었다, 모든 glial 세포는 GFAP 항체 (적색)으로 표시했다.

<ol>
	<li>Rossignol, S., Schwab, M., Schwartz, M., Fehlings, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+injury:+time+to+move?.">Spinal cord injury: time to move?.</a> J Neurosci. 27 (44), 11782-11792 (2007).</li><li>Yiu, G., He, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+inhibition+of+CNS+axon+regeneration.">Glial inhibition of CNS axon regeneration.</a> Nat Rev Neurosci. 7 (8), 617-627 (2006).</li><li>Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mystery+and+magic+of+glia:+a+perspective+on+their+roles+in+health+and+disease.">The mystery and magic of glia: a perspective on their roles in health and disease.</a> Neuron. 60 (3), 430-440 (2008).</li><li>Matyash, V., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+astrocyte+morphology+and+physiology.">Heterogeneity in astrocyte morphology and physiology.</a> Brain Res Rev. , (2009).</li><li>Wanner, I. B., Deik, A., Torres, M., Rosendahl, A., Neary, J. T., Lemmon, V. P., Bixby, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+in+vitro+model+of+the+glial+scar+inhibits+axon+growth.+Glia.">A new in vitro model of the glial scar inhibits axon growth. Glia.</a> 56 (15), 1691-1709 (2008).</li><li>Kuffler, D. P., Sosa, I. J., Reyes, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+chondroitin+sulfate+proteoglycan+inhibits+dorsal+root+ganglion+neuron+neurite+outgrowth+and+substrate+specificity+via+a+soma+and+not+a+growth+cone+mechanism.">Schwann cell chondroitin sulfate proteoglycan inhibits dorsal root ganglion neuron neurite outgrowth and substrate specificity via a soma and not a growth cone mechanism.</a> J Neurosci Res. 87 (13), 2863-2871 (2009).</li><li>Kleitman, N., Wood, P. M., Bunge, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+culture+method+for+the+study+of+myelination.">Tissue culture method for the study of myelination.</a> Culturing nerve cells. , P559-P559 (1998).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이 실험 프로토콜은 glial 세포, 신경 세포의 부착 및 neurite 성장에 특히 astrocyte 이질의 영향을 연구하는 두 목표를 달성하도록 설계되었습니다. 첫 번째 목표는이 실험에서 astrocyte 이질 최대한을 유지하는 것이었다, 합류 glial 세포 배양 농축 astrocytes는 더욱 세포를 손상 astrocytes의 부상 반응을 유발 수있는 화학 치료 및 소화 전파없이 기본 문화를 혼합했다. 최종 astrocyte 순도가 fibrobasts FN 의해 검?...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM(high glucose)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10313-039</td>
<td>&nbsp;</td>
<tr>
<td>L15 medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11415-114</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10437-077</td>
<td>&nbsp;</td>
<tr>
<td>Neurobasal Medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21103-049</td>
<td>&nbsp;</td>
<tr>
<td>poly-lysine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4832</td>
<td>culture grade</td>
</tr>
<tr>
<td>NGF(2.5S)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13257-019</td>
<td>&nbsp;</td>
<tr>
<td>B27 supplyment</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>0.25% trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25200-056</td>
<td>&nbsp;</td>
<tr>
<td>FUDR</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F0503</td>
<td>&nbsp;</td>
<tr>
<td>neurofilament antibody</td>
<td>&nbsp;</td>
<td>Abcam</td>
<td>ab24575</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

study glial cell heterogeneity influence on axon growth using a new coculture method

모든 포유 동물의 중추 신경계에 부상 후 절단 axons 1 매우 가난 원래의 목표와 기능 회복에 재생 수 없습니다. 축삭 재생의 실패는 적대적 glial 세포 환경 억제 myelin 관련 분자 및 내장 신경 세포 재생 능력이 감소 등 여러 가지 요인의 복합 결과입니다. Astrocytes는 중추 신경계에서 가장 지배 glial 세포 유형이며, 생리학 및 병리 조건 3 아래의 축삭 기능에 중요한 역할을한다. 동종 oligodendrocytes에 대비, astrocytes는 다양한 morphologies 및 유전자 발현 4 가지 astrocyte subpopulations로 구성된 이종 세포 인구입니다. 같은 축삭의 성장에 대한 자신의 영향으로이 이질의 기능적 의미는, 대부분 알 수 있습니다. 특히 glial 세포, 신경 세포의 동작 astrocyte 이질의 기능을 연구하기 위해, 우리는 쥐의 피질에서 얻은 glial 세포와 공동 culturing 높은 정화 등의 루트 신경의 신경에 의해 새로운 방법을 만들었습니다. 이 기법으로, 우리는 직접적으로 동일한 조건하에 다른 astrocytes의 subpopulations에서 신경 세포의 부착 및 축삭의 성장을 비교할 수 있었다. 이 보고서에서 우리는 astrocytes 절연 및 문화 등의 루트 신경 뉴런의 절연 및 정화하고, astrocytes와 뉴런을 DRG의 공동 문화에 대해이 방법의 상세한 프로토콜을 제공합니다. 이 방법은 뉴런과 glial 세포 사이의 세포 또는 지역 특정 상호 작용을 연구하는 다른 뇌 영역 확장 수 있습니다.

Watch this Scientific Journal Video about Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method at JoVE.com

Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

이 프로토콜에서는, 우리는 함께 축삭의 성장에 glial 세포 이질의 영향을 연구하는 새로운 방법을 설명<em&gt; 체외에서</em&gt; 공동 문화 시스템. 쥐 대뇌 피질의 glial 세포가 합류하는 교양과 고도의 쥐 등의 루트 신경의 신경을 정화와 cocultured되었습니다. 뉴런의 부착 및 축삭의 성장에 다른 glial 세포에 영향을 같은 문화에 직접적으로 비교했다. 이 방법은 직접적으로 신경 세포의 부착 및 축삭의 성장에 glial 세포 이질의 영향을 연구하는 새로운 방법을 제공합니다.

새 Coculture 방법을 사용 액슨 성장에 대한 연구 Glial 세포 이질 영향

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is ...

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JoVE Journal

Neuroscience

17.2K 조회수.  Cedars Sinai Medical Center, UCLA. 이 프로토콜에서는, 우리는 함께 축삭의 성장에 glial 세포 이질의 영향을 연구하는 새로운 방법을 설명 체외에서 공동 문화 시스템. 쥐 대뇌 피질의 glial 세포가 합류하는 교양과 고도의 쥐 등의 루트 신경의 신경을 정화와 cocultured되었습니다. 뉴런의 부착 및 축삭의 성장에 다른 glial 세포에 영향을 같은 문화에 직접적으로 비교했다. 이 방법은 직접...

비디오: Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

쥐 뇌 조각에서 뉴런의 Vibrodissociation는 시냅틱 전송 및 이미지 Presynaptic 터미널 공부

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

연구

신경과학

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

교양 성인 지느러미 루트 신경절의 신경 세포와 축삭 재생의 유전 연구

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

<em&gt; Caenorhabditis의 엘레</emAmylopathy 연구에 대한&gt; 모델 시스템

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

신생아 설치류의 복부 두개골에 개두를 확인하는 방법

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

신경 발사 환율의 조작에 의해 성상 세포 수용체의 가소성을 유도

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

성인 마우스 척수 조직에서 차 혼합 폐해 문화의 준비

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

선형 아가로 오스 채널을 사용하여 공부하기<em&gt; 초파리</em&gt; 애벌레 크롤링 동작

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

미세 전극 배열에서 신경 세포 배양의 자극에 대한 네트워크 반응의 시간 의존적 증가

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

New Methods to Study Gustatory Coding

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons (GRNs) detect tastant molecules, they encode information about the identity and concentration of the tastant as patterns of electrical activity that then propagate to follower neurons in the brain. These patterns constitute internal representations of the tastant, which then allow the animal to select actions and form memories. The use of relatively simple animal models has been a powerful tool to study basic principles in sensory coding. Here, we propose three new methods to study gustatory coding using the moth Manduca sexta. First, we present a dissection procedure for exposing the maxillary nerves and the subesophageal zone (SEZ), allowing recording of the activity of GRNs from their axons. Second, we describe the use of extracellular electrodes to record the activity of multiple GRNs by placing tetrode wires directly into the maxillary nerve. Third, we present a new system for delivering and monitoring, with high temporal precision, pulses of different tastants. These methods allow the characterization of neuronal responses in vivo directly from GRNs before, during and after tastants are delivered. We provide examples of voltage traces recorded from multiple GRNs, and present an example of how a spike sorting technique can be applied to the data to identify the responses of individual neurons. Finally, to validate our recording approach, we compare extracellular recordings obtained from GRNs with tetrodes to intracellular recordings obtained with sharp glass electrodes.

We present three new methods to study gustatory coding. Using a simple animal, the moth Manduca sexta (Manduca), we describe a dissection protocol, the use of extracellular tetrodes to record the activity of multiple gustatory receptor neurons, and a system for delivering and monitoring precisely timed pulses of tastants.

새로운 방식으로 맛보기 코딩 공부하기

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons ...

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system.

새 Coculture 방법을 사용 액슨 성장에 대한 연구 Glial 세포 이질 영향

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