This work was funded by the Whitehall Foundation and the NIH. We thank Clifford Dax (University of Oregon Technical Support Administration) for his help and expertise in designing a circuit for light delivery.

참고 : 오레곤 동물 관리 및 사용위원회의 대학 승인 한 다음 프로토콜은 건강 지침의 국립 연구소에 따른다. 1. 급성 수술 <ol><li> 복강 내 (IP) 주사 (표 1)를 통해, 케타민 - medetomidine 칵테일 동물을 마취. 참고 :이 실험에 사용 된 마우스는 드라이버 선 (; SST-iCre12, SOM의 +, CR-iCre12, CR + Pvalb-iCre11, PV +)를 interneuron하는 CRE에 의존 ChR2 - EYFP 형질 전환 line10 교차에 의해 생성됩니다. ChR2 또는 관련 opsins의 바이러스 배달 유사한 발현 수준이 얻어진다 가정, 동일하게 작동합니다. </li><li> 수술을 시작하기 전에, 확인 동물 전시 부드러운 발가락 핀치에 대한 응답이. 이 깊이의 마취를 유지하기 위해 필요에 따라 실험을하는 동안 마취제를 다시 투여. 주 사용 마취제를 사용하는 경우, 선택적으로 유지 보수 주사에 대한 IP 카테터를 이식. </li><li> 식염수 또는 유지 보수 주사 (표 1)에 대해 적절하게 희석 마취제 칵테일을 사용하여 예를 들어 실험을하는 동안 락 테이트 링거액 (약 3 ㎖ / ㎏ / 시간), 수화 동물을 유지합니다. </li><li> 정위 또는 다른 헤드 유지 장치에서 동물을 놓습니다. 두개골이 잘 고정되어 있는지 확인합니다. 이는 안정적인 단일 세포 기록을 유지하기 위해 필수적이다. </li><li> 건조를 방지하기 위해 눈에 opthalamic 연고를 적용합니다. 36.5-37 ° C의 체온을 유지한다. </li><li> 추가 기록 안정성 조에 고인 드레인을 수행합니다. 메스 블레이드를 사용하여 치스 테르 나디 라티 나 마그나을 노출 두개골의 후방 얼굴에서 조직을 제거합니다. 뇌척수액을 배출하기 위해 두라에 작은 흠을 확인합니다. 블레이드 만 매우 팁을 사용하여 소뇌 나 뇌간에 문의하지 마십시오. </li><li> 메스를 사용하여, 청각에 대한 (관심있는 대뇌 피질의 영역 위에 작은 개두술 (~ 2mm 2)을 수행피질, 브레 그마의 약 -2.3 mm 후방, 정중선의 4.5 mm의 측면). </li><li> 경질을 제거하고 따뜻한 0.5 아가로 오스의 층으로 노출 된 피질 커버 - 1mm 두께 (식염수에 1.5 % 아가로 오스, 0.9 %의 NaCl을;에 적용 ~ 37 ° C). 주기적으로 생리 식염수를 몇 방울을 적용하여 실험을하는 동안 아가로 오스 습기를 유지합니다. </li></ol> 2. 녹화 환경 <ol><li> 텅스텐 전극 준비 (7-14 MΩ, 127 μm의 직경, 12 ° 테이퍼 팁, 에폭시 코팅). 유리 모세관 튜브에 전극을 Superglue 그립 (그림 1)를위한 열 수축 튜브를 추가합니다. </li><li> 대뇌 피질의 표면에 직각 여행 세트 모터 (또는 유압) 미세 조작기에 전극을 장착합니다. 두개골에 대해, 피부 아래에 지선을 밀어 넣습니다. 그들은 근전도 아티팩트를 생성 할 수있는 머리의 측면과 목 뒤쪽에 근육과의 접촉을 피하십시오. </li><li> 세포 외 A를 이용하여 전기 신호를 증폭단일 단위 녹음에 적합한 리 파이 어은, 바람직하게는 임피던스 검사 모드를 갖추고 있습니다. 두 개의 오실로스코프 및 전원 스피커 세트 - 지속적인 스파이크 활동 (5,000 Hz의 대역 통과 300)를 모니터링합니다. 여기서, 기록 된 데이터는 10 kHz의 샘플링 속도로 연속적으로 디지털화되고; 스파이크는 오프라인 추출한다. </li><li> 팁 피질의 표면에 도달 할 때까지 아가 통해 전극을 전진. 현미경을 통해이 단계를 관찰한다. &quot;제로&quot;미세 조작기에이 위치. </li><li> 최고 기록의 깊이를 근사하기 위해, 시각적 침투 끝에 전극을 인출 할 때, 전극 팁은 제로의 깊이에 대뇌 피질을 종료 확인. 참고 : 조작하는 읽기와 관찰 된 전극의 위치 사이의 불일치가 조직에 상대적으로 표류 나타냅니다. 이것은 뇌이나 동물, 또는 조작하는 드리프트의 불안정으로 인해 발생할 수 있습니다. <ol><li> 있는지 확인하기 위해 추가 검사로조직의 첫 번째 수백 마이크로 미터를 통해 진행하는 동안이 제로 세트 포인트가 대뇌 피질의 표면에 해당하고, 자극 유발 필드 전위를 모니터링 할 수 있습니다. 필드 전위를 모니터링 할 때, 10 Hz로 세포 외 증폭기의 하부 대역 통과 필터 컷오프 낮은. 로컬 필드 전위의 극성이 레이어 I / II 국경 근처에 13 ~ 100 ㎛의 깊이 주위에 반전 있는지 확인합니다. 이는 청각 피질 신뢰성 기준점이지만, 다른 피질 다를 수있다. </li></ol></li><li> 수동 미세 조작기에 푸른 빛 소스 (그림 2)에 결합 된 광섬유를 탑재합니다. 현미경을 사용하여 가능한 한 아가의 표면에 가깝게 섬유 팁의 위치. 전극이 조직을 입력 할 빔을 센터입니다. </li><li> 지정된 폭과 duratio의 TTL (트랜지스터 - 트랜지스터 로직) 펄스열을 제공 할 수있는 제어 장치와, 광원의 출력을 레귤N- 예 아두 이노 (도 3), (도시 된 바와 같이) 컴퓨터 I / O 카드, 또는 시판 펄스 발생기. <ol><li> 모두 오실로스코프에 신호를 모니터링합니다. </li></ol></li><li> 파워 미터를 이용하여 광섬유의 선단부 전체 광선 전력 (MW)을 측정한다. 섬유 코어의 단면적으로 나누어 방사도 (Mw / mm 2)을 계산하기 위해이 값을 사용한다. 10의 범위의 강도 값으로 시작 - 15 mW의 / mm이 필요한 경우 생성물이 제거 될 때까지, 하향으로 조정한다. </li><li> 조직을 입력 할 태세 아가로 오스의 전극, 빛 이슈를 확인합니다. 검색 펄스열을 시작합니다 (예를 들어, 30 밀리 광 펄스, 500 밀리 초 interstimulus 간격 (ISI)). <ol><li> 입사광의 각도를 변경하는 전극에 대하여 광섬유를 재배치함으로써 과도 광 아티팩트 (도 4)을 제거한다. 유물이 계속되면, 빛 전력을 감소하려고합니다. 일에즉 아티팩트가 완전히 (에만 최소화)을 제거 할 수없는 드문 경우에는, 탐색 펄스의 지속 기간을 연장. 이것은 사실이 신경 세포의 스파이크를 식별하는 데 도움이 될 수 있습니다. </li></ol></li></ol> 3. 스트레이트 떠있을-에 &#39; <ol><li> 약 1 ㎛ / 초의 속도로 뇌를 통해 천천히 전극을 전진. 지속적인 활동을 모니터링 한 오실로스코프를 사용합니다. 다른 한편으로, 레이저 펄스를 발생시킨다. </li><li> 전극이 ChR2 + 셀을 접근 종종 전기 신호 오실로스코프에 분명 잘하기 전에 감지 할 수 있음을 나타냅니다 오디오 모니터에 빛을 유발 스파이크의 희미한 &#39;해시&#39;를 수신합니다. 사전의 속도를 느리게하는 효과가 있습니다. NOTE : 셀 전극의 경로에 직접적으로 놓여있는 경우, 광 - 유발 활성이 더 큰 (더 크게) 성장할 것이다. 전극은 단일 interneuron 접근하면, 해시는 균일 한 크기와 형태의 작지만 잘 정의 스파이크로 해결한다. </li><li> 즉시 리튬 등GHT-유발 스파이크 개별적으로 오실로스코프의 오프 트리거 할만큼 큰, 그래서 일을 시작합니다. 스파이크 파형의 정확한 형상을 관찰 횡축 배율을 조정한다. </li><li> 중지하고 조직이 정착 될 때까지 기다립니다. 가까운 셀에 전극을 이동하는 유혹에 저항. 이 단계는 안정된 기록 중요하다. 몇 분 후, 신호 대 잡음비가 개선되지 않은 경우 - 즉, 티슈가 가라 앉을되지만 가​​까이 전극의 선단에 세포를 가져 못한 - 미리 5 더 μm의 다시 기다린다. <ol><li> 피크 또는 활동 전위의 물마루 어느 확실 잡음 플로어 (예를 들어, +/- 300 μV 이상)보다 충분히 위에 설정된 전압 임계치로 캡처 될 수있을 때까지이 과정을 반복한다. 참고 : 억제의 interneurons을 PINPing 때 가양 또는 모호함의 작은 위험이있다. 대부분의 세포는 쉽게 30 밀리의 기간 간 PUL과 펄스의 기차를 따를 수있다SE 간격 2-5 밀리 초 (그림 5)의 신뢰할 수있는 최초의 스파이크 대기 시간이 500 밀리 초, 또는 빠르고. PV + 세포의 대부분은, 특히, 전체 1 초 (그림 6)에 대한 발사 유지할 수 있습니다. 이러한 억제 뉴런 때문에, disynaptic (경유) 활성화는 큰 문제가되지 않습니다. </li></ol></li><li> 녹음하는 동안, 최초의 오실로스코프에 지속적인 활동을 모니터링 할 수 있습니다. 크기와 두 번째 오실로스코프를 사용하여 스파이크의 모양에 눈을 떼지. 스피커에 자신의 소리의 품질을합니다. <ol><li> 셀이 중 하나 (이 찔려 죽은 또는 손상 위험이) 전극에 너무 가까이 도면을 나타냅니다 급격한 변화에 대해주의 깊게 듣거나 떨어져 표류한다. 스파이크가 크고 왜곡되는 경우, 백 아웃; 이들이 작은 커지면, 전극을 전진. 2 μm의 단계로 천천히 이동합니다. </li><li> 정점 또는 저점에 정렬 된 모든 스파이크 파형을 중첩하여 기록 사후의 품질을 확인합니다. 전압 Thres를 비바리보유. 임계 값의 넓은 범위에 걸쳐, 오직 일정한 높이 (도 5a)의 일관된 스파이크 형상이 있어야한다. </li></ol></li><li> 어떤 지점에서 신호가 인접 신경 세포에서 스파이크에 오염 될 경우, 이동합니다. 전극은 인접 셀의 범위 밖에 통과하지만, 표적 뉴런의 범위 내에 남아있을 오프 기회에 천천히 전진. (이것은 일반적으로 성공하지 못합니다.) </li><li> 각 침투 피질 (900+ μm의)의 전체 깊이를 통해 전극을 이동합니다. 더 빛 응답 뉴런 반대로, 녹음 정기적으로 이웃 ChR2- 세포의 스파이크에 오염 된 많은 침투 또는 후에 발생하지 않으면, 새로운 전극을보십시오. 참고 : 단 한 번의 일괄 처리 내 개별 전극의 임피던스 및 팁 형상 상당히 다를 수 있습니다. 두 요인 ㅁ - 빛 유발 스파이크를 감지 할만큼 충분히 커야합니다 전극의 유효 &quot;듣고 반경&quot;에 기여ILE 검색은 아직 분리에서 하나의 interneuron을 기록 수 있도록 충분히 제한. </li><li> 원하는대로 전극의 임피던스를 테스트합니다. 조직의 손상을 방지하기 위해, 아니라 뇌 밖에, 아가로 오스 층의 상부에 전극의 끝으로이 작업을 수행. 7-14 MΩ의 범위 내에서, 정확한 임피던스는이 애플리케이션에 대한 성능, 또는 수율의 확실한 예측 아니다. 즉, 수신 반경을위한 합리적인 프록시의 말했다 : 낮은 임피던스 전극 이상의 유닛을 선택, 높은 임피던스, 적은. </li><li> 실험의 끝에, 이러한 마취 깊이 평면 아래 마취제 과다, 또는 경추 탈구 등 제도적으로 승인 된 수단에 의해 동물을 안락사. </li></ol>

<ol>
	<li>Lima, S. Q., Hromadka, T., Znamenskiy, P., Zador, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINP:+a+new+method+of+tagging+neuronal+populations+for+identification+during+in+vivo+electrophysiological+recording.">PINP: a new method of tagging neuronal populations for identification during in vivo electrophysiological recording.</a> PLoS One. 4, (2009).</li><li>Moore, A. K., Wehr, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parvalbumin-expressing+inhibitory+interneurons+in+auditory+cortex+are+well-tuned+for+frequency.">Parvalbumin-expressing inhibitory interneurons in auditory cortex are well-tuned for frequency.</a> J Neurosci. 33, 13713-13723 (2013).</li><li>Merchant, H., de Lafuente, V., Pena-Ortega, F., Larriva-Sahd, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+impact+of+interneuronal+inhibition+in+the+cerebral+cortex+of+behaving+animals.">Functional impact of interneuronal inhibition in the cerebral cortex of behaving animals.</a> Prog Neurobiol. 99, 163-178 (2012).</li><li>Markram, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interneurons+of+the+neocortical+inhibitory+system.">Interneurons of the neocortical inhibitory system.</a> Nat Rev Neurosci. 5, 793-807 (2004).</li><li>Atallah, B. V., Bruns, W., Carandini, M., Scanziani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parvalbumin-expressing+interneurons+linearly+transform+cortical+responses+to+visual+stimuli.">Parvalbumin-expressing interneurons linearly transform cortical responses to visual stimuli.</a> Neuron. 73, 159-170 (2012).</li><li>Wilson, N. R., Runyan, C. A., Wang, F. L., Sur, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Division+and+subtraction+by+distinct+cortical+inhibitory+networks+in+vivo.">Division and subtraction by distinct cortical inhibitory networks in vivo.</a> Nature. 488, 343-348 (2012).</li><li>Letzkus, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+disinhibitory+microcircuit+for+associative+fear+learning+in+the+auditory+cortex.">A disinhibitory microcircuit for associative fear learning in the auditory cortex.</a> Nature. 480, 331-335 (2011).</li><li>Pi, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+interneurons+that+specialize+in+disinhibitory+control.">Cortical interneurons that specialize in disinhibitory control.</a> Nature. 503, 521-524 (2013).</li><li>Adesnik, H., Bruns, W., Taniguchi, H., Huang, Z. J., Scanziani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neural+circuit+for+spatial+summation+in+visual+cortex.">A neural circuit for spatial summation in visual cortex.</a> Nature. 490, 226-231 (2012).</li><li>Madisen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+toolbox+of+Cre-dependent+optogenetic+transgenic+mice+for+light-induced+activation+and+silencing.">A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing.</a> Nat Neurosci. 15, 793-802 (2012).</li><li>Hippenmeyer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+developmental+switch+in+the+response+of+DRG+neurons+to+ETS+transcription+factor+signaling.">A developmental switch in the response of DRG neurons to ETS transcription factor signaling.</a> PLoS Biol. 3, 159 (2005).</li><li>Taniguchi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+resource+of+Cre+driver+lines+for+genetic+targeting+of+GABAergic+neurons+in+cerebral+cortex.">A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex.</a> Neuron. 71, 995-1013 (2011).</li><li>Christianson, G. B., Sahani, M., Linden, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depth-dependent+temporal+response+properties+in+core+auditory+cortex.">Depth-dependent temporal response properties in core auditory cortex.</a> J Neurosci. 31, 12837-12848 (2011).</li><li>Povysheva, N. V., Zaitsev, A. V., Gonzalez-Burgos, G., Lewis, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+heterogeneity+of+fast-spiking+interneurons:+chandelier+versus+basket+cells.">Electrophysiological heterogeneity of fast-spiking interneurons: chandelier versus basket cells.</a> PLoS One. 8, 70553 (2013).</li></ol>

The authors have no competing financial interests.

떠있을 개념적으로 간단하지만, 실제로 문제가 될 수 있습니다. 성공의 주요 결정은 전극의 선택입니다. 전기 청취 반경은 중요한 파라미터이다. 이것은 선단이 약간 먼 거리 ChR2 + 셀로부터 정지되었을 때에 하나 이에 따라 어드밴스의 속도를 조정할 수 있도록, 광 유발 스파이크를 검출하기에 충분히 커야한다. 동시에, 양호한 단일 유닛 분리 가능하도록 충분히 제한되어야한다. 즉, 전극은 이웃...

Characterizing the myriad cell types that comprise the mammalian brain has been a central, but long-elusive goal of neurophysiology. For instance, the properties and function of different inhibitory cell types in the cerebral cortex are topics of great interest but are still relatively unknown. This is in part because conventional blind in vivo recording techniques are limited in their ability to distinguish between different cell types. Extracellular spike width can be used to separate putative parvalbumin-positive inhibitory neurons from excitatory pyramidal cells, but this method is subject to both type I and type II errors2,3. Alternatively, recorded neurons can be filled, recovered, and stained to later confirm their morphological and molecular identity, but this is a pain-staking and time-consuming process. Recently, genetically identified populations of inhibitory interneurons have become accessible by means of calcium imaging or visually guided patch recordings. In these approaches, viral or transgenic expression of a calcium reporter (such as GCaMP) or fluorescent protein (such as GFP) allows identification and characterization of cell types defined by promoter expression. These approaches use 2-photon microscopy, which requires expensive equipment, and are also limited to superficial cortical layers due to the light scattering properties of brain tissue. 
Recently, Lima and colleagues1 developed a novel application of optogenetics to target electrophysiological recordings to genetically identified neuronal types in vivo, termed &#8220;PINP&#8221; &#8211; or Photostimulation-assisted Identification of Neuronal Populations. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. Unlike many other optogenetic applications, the goal is not to manipulate circuit function but simply to identify neurons belonging to a genetically-defined class, which can then be characterized during normal brain function. The technique can be implemented with standard extracellular recording equipment and can therefore serve as an accessible and inexpensive alternative to calcium imaging or visually-guided patching. Here we describe an approach to PINPing specific cell types in the anesthetized auditory cortex, with the expectation that the more general points can be usefully applied in other preparations and brain regions.
In cortex, PINP holds particular promise for investigating the in vivo response properties of inhibitory interneurons. GABAergic interneurons comprise a small, heterogeneous subset of cortical neurons4. Different subtypes, marked by the expression of particular molecular markers, have recently been shown to perform different computational roles in cortical circuits5-9. As genetic tools improve it may eventually be possible to distinguish morphologically- and physiologically-separable types that fall within these broad classes. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. This strategy was developed specifically for targeting parvalbumin-positive (PV+) cells, but we have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons. Although PINPing is conceptually straightforward, it can be surprisingly unyielding in practice. We learned a number of tips and tricks through trial-and-error that may be useful to others attempting the method.

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a guide to in vivo single-unit recording from optogenetically identified cortical inhibitory interneurons

신경 생리에서 중요한 과제는 대뇌 피질의 다수의 억제 세포 유형의 응답 특성과 기능을 특성화하고있다. 우리는 여기 리마 1 동료들에 의해 개발 된 방법을 사용하여 마취 마우스 피질에서 확인 된 억제의 interneurons 안정적인, 잘 격리 된 단일 단위 기록을 얻기위한 우리의 전략을 공유 할 수 있습니다. 녹음은 특정 신경 세포의 소집단에 Channelrhodopsin-2 (ChR2)를 표현 생쥐에서 수행됩니다. 인구의 회원은 푸른 빛에 대한 간단한 플래시에 대한 반응으로 식별됩니다. &quot;PinP의&quot;이라, 또는 신경 세포 집단의 Photostimulation 이용한 식별 - -이 기술은 표준 세포 외 기록 장치로 구현 될 수있다. 이것은 유전자 식별 세포 외 기록 타겟팅 목적, 칼슘 이미징 또는 시각 유도 패치에 저렴하고 대안적인 접근이 될 수있다. H오히려 우리는 매일 연습하는 방법을 최적화하기위한 지침을 제공합니다. 우리는 parvalbumin 양성 (태양 광 +) 세포를 대상으로 특별히 우리의 전략을 정제하지만 이러한 소마토​​스타틴 (somatostatin) 발현 (SOM +)를하고 calretinin 발현 (CR +)의 interneurons으로뿐만 아니라 다른 interneuron 유형에 대해 작동하는 것을 발견했다.

우리는 여기에 &quot;리마 등. 한. 표 1 세부 제안 마취 칵테일, 케타민-Medetomidine - 아세 프로 마진을 (가 개발 한 optogenetic 방법을 사용하여, 마취 마우스 피질에서 유전 분류 억제의 interneurons에서 단일 단위 기록을 얻기위한 우리의 전략을 공유 KMA &quot;).도 1은 도표 4. 3 아두 이노 마이크로 컨트롤러와 광 출력 게이팅위한 구성 및 코드를 포함하는도.도 ...

Watch this Scientific Journal Video about A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons at JoVE.com

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

에 가이드<em&gt; 생체</emOptogenetically 확인 된 대뇌 피질의 억제의 interneurons에서&gt; 단일 단위 기록

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

a-guide-to-vivo-single-unit-recording-from-optogenetically-identified

Research

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Neuroscience

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

19.0K 조회수.  University of Oregon. Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching. 

비디오: A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

단일 유닛 생체내에서</em암흑의 광학 Chiasm에서> 레코딩

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

연구

신경과학

Recording Behavioral Responses to Reflection in Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

크레이 피쉬에 리플렉션하는 행동 응답 녹음

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

단일 유닛 활동과 Electrocorticographic 신호를 녹음하기 위해 만성 신경 전극 외과 이식

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

확인된 뉴런과 먹이 행동에서 칼슘 신호의 동시 녹화 Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

역행 형광 라벨이 확인 된 신경 세포의 표적 세포 외 단일 단위 기록을 허용<em&gt; 생체 내</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

형질 전환 생선의 본래 뇌에서 발견 뉴런의 전기적 활동을 기록

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

세포 내 기록, 감각이 필드를 매핑하고, 거머리의 배양 확인 된 뉴런, Hirudo의 medicinalis</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

두피의 interneurons의 하위 유형 선택적 일렉트로

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Morphologically- 및 Neurochemically 확인 된 해마의 interneurons에서 전체 세포 패치 클램프 녹음

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

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

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

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

동시 전기 생리 녹화 및 설치류의 뇌에서 억제 에이전트의 마이크로 주사

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