Name: Optogenetic 쥐 행동에 Hippocampal 세타 진동의 유입 (동영상) | JoVE Journal
Uploaded: 2018-06-29T13:30:00.000+00:00
Duration: 7 min 33 s
Description: 배우세요 생쥐 해마 세타 진동을 광유전학과 전기생리학 기록으로 실시간 조절하는 방법을. 공간 탐색 행동에서 신경 동기화 메커니즘을 연구하는 데 활용할 수 있습니다.

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We would like to thank Maria Gorbati for expert help with data analysis and Jennifer Kupferman for comments on the manuscript. This work was supported by Deutsche Forschungsgemeinschaft (DFG; Exc 257 NeuroCure, TK and AP; Priority Program 1665, 1799/1-1(2), Heisenberg Programme, 1799/2-1, AP), the German-Israeli Foundation for Scientific Research and Development (GIF; I-1326-421.13/2015, TK) and the Human Frontier Science Program (HFSP; RGY0076/2012, TK).

The authors have nothing to disclose.

Here we presented a widely accessible methodology to entrain and elicit hippocampal theta oscillations in the behaving animal. This approach can be useful for studies of theta rhythm&#39;s functions in information processing and behavior. Critical aspects of this method include: (1) choice of the opsin and targeting of ChR2 to axons of MS cells in the hippocampus, (2) robust optical and electrical features of implanted optic fiber-wire array assemblies to ensure continuous stimulation and LFP recording in behaving mice, ...

Neuronal activity in mammals is coordinated by network oscillations, which assist information transfer within and between brain regions1,2,3,4. Brain rhythms include oscillations ranging from very slow (&#60; 0.8 Hz) up to ultrafast (&#62; 200 Hz) frequencies. A large body of evidence supports involvement of network oscillations in diverse brain functions, including cognition5,6,7,8,9,10, innate behaviors11,12 as well as neuropsychiatric disorders such as Parkinson&#39;s disease and epilepsy13,14,15. Selective and temporally precise methods for experimental manipulation of network oscillations are therefore essential for the development of physiologically plausible models of synchronization and for establishing causal links with behavior.
Network synchronization is mediated by diverse biological substrates and processes, ranging from molecular identity of ion channels and their kinetics to neuromodulation of excitability and network connectivity. The biological design of rhythm generators16 has been revealed for many brain rhythms, distinct aspects of which (e.g., frequency, amplitude) are often brought about by dynamics of distinct cell types and networks. For instance, inhibitory interneurons targeting the somata of principal cells are the most important players across frequency bands and brain regions17,18, including theta19,20, gamma20,21, and ripple (140-200 Hz)22 oscillations. In turn, phase synchronization of distant cells is ensured by robust feed-forward signaling of pyramidal cells, which resets the firing of interneurons. A crucial parameter of oscillations, the size of the synchronized neuronal population, is closely related to the measured LFP oscillation&#39;s amplitude and, at least for fast oscillations, depends on the excitatory drive onto interneurons2. In contrast, slower oscillations, like delta and theta rhythms, are generated by long-range reentrant loops, formed by cortico-thalamic23,24 and hippocampal-medial septal projections25,26,27, respectively. Oscillations in such circuits are brought about by interactions of signal propagation delays, excitable responses, and their frequency preference in participating cells28,29,30,31,32. Inhibitory projections from GABAergic parvalbumin (PV)-positive cells of the medial septum (MS) to interneurons in the hippocampus25,33, parahippocampal regions and entorhinal cortex26 are essential for the generation of theta oscillations in the medial temporal lobe. Thus, physiological mechanisms of network oscillations and neuronal synchronization can be manipulated using optogenetics with a real-time precision.
Cell type-specific optogenetic manipulations have been applied for studies of the hippocampal and cortical oscillations in vitro34,35,36,37,38 and in vivo30,39,40,41,42,43,44,45, including functional investigations of gamma5,12,36,46,47,48,49,50,51,52 and ripple oscillations40,53,54 and sleep spindles55,56. Recently we expressed a Cre-dependent ChR2 virus in the MS, a key region for the generation of the hippocampal theta rhythm, of PV-Cre mice. Using this preparation, features of the hippocampal theta oscillations (frequency and temporal stability) were controlled by optogenetic stimulation of inhibitory projections of the MS in the hippocampus11. Furthermore, theta-frequency optogenetic stimulation of inhibitory septo-hippocampal projections evoked theta rhythm during awake immobility. The optogenetically entrained theta rhythm displayed properties of spontaneous theta oscillations in the mouse at LFP and neuronal activity levels.
Key features of this protocol include: (1) utilization of an inhibitory pathway that is physiologically critical for spontaneous theta oscillations while avoiding unspecific effects on hippocampal excitability; (2) axonal, i.e., projection-specific stimulation to minimize a direct influence on non-hippocampal MS efferents; (3) local theta-rhythmic light stimulation, ensuring a minimal direct interference with theta-rhythmic septo-hippocampal dynamics and a global bilateral entrainment of theta oscillations; (4) parametric control of theta oscillations frequency and regularity; and (5) quantification of entrainment fidelity with high temporal resolution using LFP to enable quantitative causality analysis in behaving animals. Since this preparation essentially capitalizes on a well-known role of the septo-hippocampal disinhibition in theta generation25,30, it enables robust control over several parameters of theta oscillations in behaving mice. Studies where other less investigated pathways and cell types of the septo-hippocampal circuitry were manipulated38,39,47,49,50,51,52,53,54,55,56,57,58 reveal further mechanisms of the theta rhythm.

<table><tbody><tr><td>PV-Cre mice</td><td>The Jackson Laboratory</td><td>B6;129P2-Pvalbtm1(cre)Arbr/J</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Surgery&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>Stereotaxis</td><td>David Kopf Instruments, Tujunga, CA, USA</td><td>Model 963</td><td>Ultra Precise Small Animal Stereotaxic Instrument</td></tr><tr><td>Drill bits, 0.8 mm</td><td>Bijoutil, Allschwil, Switzerland</td><td>49080HM</td><td></td></tr><tr><td>0.01-1 ml syringe</td><td>Braun, Melsungen, Germany</td><td>9161406V</td><td></td></tr><tr><td>Sterican cannulas</td><td>Braun</td><td>26 G, 0.45x25 mm BL/LB</td><td></td></tr><tr><td>Fine and sharp scissors</td><td>Fine Science Tools Inc., Vancouver, Canada</td><td>14060-09</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools Inc.</td><td>11210-10</td><td>Dumont AA - Epoxy Coated Forceps</td></tr><tr><td>Blunt stainless steel scissors</td><td>Fine Science Tools Inc.</td><td>14018-14</td><td></td></tr><tr><td>Soldering station</td><td>Weller Tools GmbH, Besigheim, Germany</td><td>WSD 81</td><td></td></tr><tr><td>Erythromycin</td><td>Rotexmedica GmbH, Trittau, Germany</td><td>PZN: 10823932</td><td>1g Powder for Solution for Infusion</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Optogenetics&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>Hamilton pump</td><td>PHD Ultra, Harvard Apparatus, Holliston, MA, USA</td><td>model 703008</td><td>PHD Ultra Syringe Pump with push/pull mechanism</td></tr><tr><td>Hamilton 5 &amp;micro;L Syringe, 26 gauge</td><td>PHD Ultra, Harvard Apparatus</td><td>Model 75 RN SYR</td><td></td></tr><tr><td>Hamilton 5 &amp;micro;L Plunger</td><td>PHD Ultra, Harvard Apparatus</td><td>Model 75 RN SYR</td><td></td></tr><tr><td>Tubing</td><td>Fisher Scientific, Pittsburgh, USA</td><td>PE 20</td><td>Inner diameter 0.38 mm (.015&quot;), Outer diameter 1.09 mm (.043&quot;)</td></tr><tr><td>Sterican cannulas</td><td>Braun, Melsungen, Germany</td><td>27 G, 25x0.40 mm, blunt</td><td></td></tr><tr><td>Precision drill/grinder</td><td>Proxxon, Wecker, Luxemburg</td><td>fbs 240/e</td><td></td></tr><tr><td>Cutting disks</td><td>Proxxon</td><td>NO 28812</td><td></td></tr><tr><td>Cre dependent channelrhodopsin</td><td>Penn Vector Core, Philadelphia, PA, USA</td><td>AV-1-18917P</td><td>Contruct name: AAV2/1.CAGGS.flex.ChR2.tdTomato, titer: 1.42x1013 vg/ml</td></tr><tr><td>Cam kinase dependent halorhodopsin</td><td>Penn Vector Core</td><td>AV-1-26971P</td><td>Construct name: eNpHR3.0, AAV2/1.CamKIIa.eNpHR3.0-EYFP.WPRE.hGH, titer: 2.08_1012 vg/ml</td></tr><tr><td>Multimode optic fiber</td><td>ThorLabs, Dachau, Germany</td><td>FG105LCA</td><td>0.22 NA, Low-OH, &amp;Oslash;105 &amp;micro;m Core, 400 - 2400 nm</td></tr><tr><td>Ceramic stick ferrule</td><td>Precision Fiber Products, Milpitas, CA, USA</td><td>CFLC126</td><td>Ceramic LC MM Ferrule, ID 126um</td></tr><tr><td>Polishing paper</td><td>Thorlabs</td><td>LF3D</td><td>6&quot; x 6&quot; Diamond Lapping (Polishing) Sheet</td></tr><tr><td>Power meter</td><td>Thorlabs</td><td>PM100D</td><td>Compact Power and Energy Meter Console, Digital 4&quot; LCD</td></tr><tr><td>Multimode fiber optic coupler</td><td>Thorlabs</td><td>FCMM50-50A-FC</td><td>1x2 MM Coupler, 50:50 Split Ratio, 50 &amp;micro;m GI Fibers, FC/PC</td></tr><tr><td>Fiberoptic patch cord</td><td>Thorlabs</td><td>FG105LCA CUSTOM-MUC</td><td>custom made, 3 m long, with protective tubing, Tubing: FT030, Connector 1: FC/PC, Connector 2: 1.25mm (LC) Ceramic Ferrule</td></tr><tr><td>Sleeve</td><td>Precision Fiber Products, Milpitas, CA, USA</td><td>ADAL1</td><td>Ceramic Split Mating Sleeve for &amp;Oslash;1.25 mm (LC/PC) Ferrules</td></tr><tr><td>473 nm DPSS laser</td><td>Laserglow Technologies, Toronto, ON, Canada</td><td>R471005FX</td><td>LRS-0473 Series</td></tr><tr><td>593 nm DPSS laser</td><td>Laserglow Technologies</td><td>R591005FX</td><td>LRS-0594 Series</td></tr><tr><td>MC_Stimulus II</td><td>Multichannel Systems, Reutlingen, Germany</td><td>STG 4004</td><td></td></tr><tr><td>Impedance conditioning module</td><td>Neural microTargeting worldwide, Bowdoin, USA</td><td>ICM</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Electrophysiology&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>Tungsten wires</td><td>California Fine Wire Company, Grover Beach, CA, USA</td><td>CFW0010954</td><td>40 &amp;micro;m, 99.95%</td></tr><tr><td>Capillary tubing</td><td>Optronics</td><td>1068150020</td><td>ID: 100.4 &amp;micro;m</td></tr><tr><td>Omnetics nanoconnector</td><td>Omnetics Connector Corporation, Minneapolis, USA</td><td>A79038-001</td><td></td></tr><tr><td>Screws</td><td>Bilaney, D&amp;uuml;sseldorf, Germany</td><td>00-96x1/16</td><td>stainless-steel</td></tr><tr><td>Silicone probe</td><td>NeuroNexus Technologies, Ann Arbor, MI, USA</td><td>B32</td><td></td></tr><tr><td>Headstage</td><td>Neuralynx, Bozeman, Montana USA</td><td>HS-8</td><td>miniature headstage unity gain preamplifiers</td></tr><tr><td>Silver conductive paint</td><td>Conrad electronics, Germany</td><td>530042</td><td></td></tr><tr><td>Liquid flux</td><td>Felder GMBH L&amp;ouml;ttechnik, Oberhausen, Germany</td><td>L&amp;ouml;t&amp;ouml;l ST</td><td>DIN EN 29454.1, 3.2.2.A (F-SW 11)</td></tr><tr><td>LED</td><td>Neuralynx</td><td>HS-LED-Red-omni-10V</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Software&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>MATLAB</td><td>Mathworks, Natick, MA, USA</td><td></td><td></td></tr><tr><td>MC_Stimulus software</td><td>Multichannel, Systems</td><td></td><td></td></tr><tr><td>Neurophysiological Data Manager</td><td></td><td></td><td>NDManager, http://neurosuite.sourceforge.net</td></tr><tr><td>Klusters</td><td></td><td></td><td>http://neurosuite.sourceforge.net, Hazan et al., 2006</td></tr><tr><td>Software of the recording system</td><td>Neuralynx</td><td>Cheetah</td><td>https://neuralynx.com/software/cheetah</td></tr><tr><td>Multi-channel data analysis software</td><td>Cambridge Electronic Design Limited, Cambridge, GB</td><td>Spike2</td><td></td></tr></tbody></table>

optogenetic entrainment of hippocampal theta oscillations in behaving mice

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

Targeting of ChR2 to GABAergic cells in the MS as described in section 1 is illustrated in Figure 2A. Optogenetic stimulation of axons of MS GABAergic cells in the dorsal hippocampus via an optic fiber which is implanted above the CA1 area entrains theta oscillations at the frequency of the stimulus in the ipsilateral (Figure 2B) as well as contralateral hemisphere (Figure 2C). Theta oscillations cou...

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

This method can help answer key questions in the neurophysiology field, such as causal role of theta oscillation in special navigation and associated behaviors. The main advantage of this technique is that it enables a real-time control over the frequency and regularity of hippocampal theta oscillations in behaving mice. Demonstrating the procedure will be Dr.Franziska Bender, a former grad student in my laboratory.Begin with the fully anesthetized mouse secured in the stereotaxic frame. Drill a hole above the medial septum. Submerge the tip of the injection needle in a droplet of virus on a piece of paraffin film and carefully draw up the virus at about 500 nanoliters per minute while observing the level of the fluid.To prevent air from entering the injection needle, stop withdrawal before the virus is entirely taken up. Clean the needle with a paper tissue. Check that the virus and oil are separated by an air bubble and mark the level of the virus on the tube.Position the needle above the craniotomy and slowly insert it into the brain at the first injection point. Inject 450 nanoliters of the virus at a rate of 100 to 150 nanoliters per minute. Following the injection, wait for 1

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Optogenetic 쥐 행동에 Hippocampal 세타 진동의 유입

출처: Kodani, S., et.al. 마우스의 수면/각성 상태를 모니터링하는 동안 신경 회로의 광유전학적 조작. J. Vis. 경험. (2019년)이 비디오는 생쥐의 수면에서 각성으로의 전환에 관여하는 신경 회로의 광유전학적 조작을 보여줍니다. 이 절차에는 광섬유와 전극을 뇌와 근육에 외과적으로 이식하여 빛 자극과 전기생리학적 기록을 가능하게 하는 것이 포함됩니다. 말단 줄조선(BNST)의 침대 핵에서 빛에 민감한 양이온 채널을 활성화하기 위해 빛을 전달함으로써 신경 활동이 조절되어 연구자들은 각성 유도와 관련된 뇌 및 근육 신호를 분석할 수 있습니다.

마우스의 수면에서 각성으로의 전환을 위한 신경 회로의 광유전학적 조작

이 비디오는 GABAergic 내측 중격 뉴런에 빛에 민감한 이온 채널이 있는 유전자 조작 마우스 모델에서 세타 진동을 조절하는 프로토콜을 보여줍니다. 이 프로토콜은 광 펄스를 해마에 전달함으로써 뉴런 활동을 동기화하고 국소 자기장 전위로 기록되는 세타 진동을 생성하는 것을 목표로 합니다.

마우스 모델에서 Theta Oscillations의 선택적 조작

이 비디오는 마우스 모델의 해마에 와이어 전극 어레이와 광섬유를 정위 이식하는 방법을 보여줍니다.

전극 배열과 광섬유를 마우스 해마에 이식

이 비디오는 고립된 해마에서 세타 진동이라고도 하는 리드미컬한 뉴런 네트워크 활동을 기록하는 방법을 보여줍니다. 전극은 세타 진동을 감지하고 분석하기 위해 다양한 층과 공간 위치에 삽입됩니다. 이것은 독특한 뉴런 구성과 해마의 여러 층에 걸친 뉴런 네트워크의 연결성을 보여줍니다.

마우스 해마의 세타 진동에 대한 시험관 내 기록

이 비디오는 쥐의 뇌 절편에서 유전적으로 변형된 빛에 민감한 인터뉴런을 활성화하여 세타 진동 동기화를 향상시키고 신경 회로 동작을 연구함으로써 광유전학을 사용하여 해마 네트워크 역학을 조절하는 방법을 보여줍니다.

해마 네트워크 역학의 광유전학적 제어

진동은 기본적인 네트워크 속성이며 질병과 약물에 의해 조절된다. 뇌 슬라이스 진동을 공부하는 것은 통제 된 조건에서 격리 된 네트워크의 특성을 수 있습니다. 프로토콜은 CA1의 γ 진동을 불러 일으키는 급성 뇌 조각의 준비를 위해 제공됩니다.

파상풍의 자극에 의​​해 지역의 CA1 γ 진동의 발생

해마의 세타 활동은 특정인지 및 행동 단계와 관련이 있습니다. 여기에서는 시간-주파수(즉, 웨이블릿 분석) 기반 접근 방식을 사용하여 해마 내에서 고도로 조직화된 세타 진동을 감지하는 분석 방법을 설명합니다.

쥐 EEG에서 매우 조직 된 세타 진동의 자동 감지

여기에서, 우리는 격리 된 전체 해마 제조물로부터 리듬 뉴런 네트워크 세타 및 감마 진동을 기록하기위한 프로토콜을 제시한다. 우리는 해마 추출에서 현장, 단일 및 전체 세포 패치 클램프 녹음뿐만 아니라 theta 리듬의 광 발생기 페이싱에 이르기까지의 실험 단계를 설명합니다.

Hippocampal 쎄타 밴드의 튜닝<em&gt; 체외에서</em&gt; : 격리 된 설치류 Septohippocampal Circuit에서 기록하는 방법론

이 프로토콜 로컬 필드 전위 선형 실리콘 프로브 멀티 생크와의 녹음을 설명 합니다. 현재 소스 밀도 분석을 사용 하 여 신호 변환 마우스 해 마에 지역 전기 활동의 개조를 허용 한다. 이 기술을 공간적으로 제한 된 뇌 진동 자유롭게 마우스를 이동에 공부 될 수 있다.

행동 하는 쥐의 해 마에서 진동 제한 공간 기록

여기에서, 우리는 마우스에 있는 잠/각성 상태의 감시 도중 뉴런의 특정 모형의 광유전학 조작의 방법을 기술합니다, 예를 들면 stria 말단의 침대 핵에 우리의 최근 일을 제시하.

마우스에서 수면/각성 상태를 모니터링하는 동안 신경 회로의 광유전학 적 조작

이 문서는 주입 캐뉼라와 마우스의 해마의 CA1 영역에 이식으로 보충 된 맞춤형 이미징 창의 준비를 보여줍니다.

마우스를 면도하는 해마에서 신경 활동을 시각화하기위한 두개골 절제술 절차

배우세요 생쥐 해마 세타 진동을 광유전학과 전기생리학 기록으로 실시간 조절하는 방법을. 공간 탐색 행동에서 신경 동기화 메커니즘을 연구하는 데 활용할 수 있습니다.

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

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