使用光遗传学逆转大鼠的神经可塑性并抑制可卡因的寻找
Summary
这里描述的方法概述了用于在大鼠行为相关回路中以光遗传学逆转可卡因诱导的可塑性的程序。丘脑-杏仁核突触的持续低频光刺激可诱发长期抑郁（LTD）。在经历可卡因的大鼠中 体内光遗传 诱导的LTD导致随后的线索动机药物寻求减弱。
Abstract
该协议展示了使用光遗传学工具在丘脑 – 杏仁核回路中逆转可卡因诱导的可塑性以减少随后在大鼠中寻找可卡因行为所需的步骤。在我们的研究中，我们发现，当大鼠自我静脉注射可卡因与视听线索配对时，随着线索 – 可卡因关联的学习，丘脑内侧膝状核（MGN）输入到侧杏仁核（LA）主要神经元处形成的突触变得更强。我们假设在这些突触上逆转可卡因诱导的可塑性将减少线索动机的可卡因寻求行为。为了在 体内完成这种类型的神经调控，我们想诱导突触长期抑制（LTD），这会降低MGN-LA突触的强度。为此，我们使用了光遗传学，它允许使用光神经调控大脑回路。通过将含有oChiEF的AAV注入MGN在LA的突触前MGN末端表达兴奋性视蛋白oChiEF。然后将光纤植入LA，并以1 Hz的频率脉冲473nm激光15分钟，以诱导LTD和反向可卡因诱导的可塑性。这种操作导致与可卡因相关的线索诱导药物寻求行动的能力长期降低。
Introduction
药物滥用在美国和全世界都是一个非常严重的公共卫生问题。尽管进行了数十年的深入研究，但有效的治疗选择很少1，2。治疗的一个主要挫折是，长期使用药物在环境线索和药物本身之间产生了长期的联想记忆。再次暴露于与药物相关的线索会推动生理和行为反应，从而激发持续吸毒和复发3。一种新的治疗策略是制定基于记忆的治疗方法，旨在操纵调节药物线索关联的回路。最近，观察到外侧杏仁核 （LA） 中的突触，特别是丘脑内侧膝状核 （MGN） 产生的突触，通过重复提示相关的可卡因自我给药而得到加强，并且这种增强作用可以支持可卡因寻找行为4，5。因此，有人提出，提示诱导的恢复可以通过逆转MGN-LA突触的可塑性来减弱。
精确靶向特定大脑回路的突触可塑性的能力一直是该领域的一个主要挑战。传统的药理学工具在减少复发行为方面取得了一些成功，但由于无法操纵单个突触而受到限制。然而，体内光遗传学的最新发展提供了克服这些限制和以时间和空间精度控制神经通路所需的工具6，7，8。通过在特定的脑回路中表达光敏视蛋白，激光可用于激活或抑制回路。频率依赖性光刺激可用于特异性地操纵行为动物中电路的突触可塑性。
本手稿概述了使用 体内 光遗传学操纵行为相关的MGN-LA回路的程序。首先，兴奋性视蛋白oChIEF在MGN中表达，光纤在LA中双侧植入。然后训练动物以线索依赖的方式自我管理可卡因，这增强了MGN-LA途径。接下来，使用473nm激光的持续低频刺激来产生电路特定的LTD.逆转可卡因使用引起的可塑性导致线索触发与药物寻求行为相关的行动的能力长期降低。
Protocol
该协议中描述的实验符合美国国立卫生研究院实验动物护理和使用指南规定的指南，并得到匹兹堡大学机构 动物护理和使用 委员会的批准。所有程序均使用成年幼稚的Sprague-Dawley大鼠进行，到达时重275-325克。 1. 光纤植入物和跳线的构造 按照先前发布的协议准备光纤植入物9.本协议中描述的实验使用200μm芯光纤（0.5 NA）和Ø1.25 mm多…
Representative Results
概述实验顺序的时间线如图 1 所示。在整个行为实验中，可卡因输注的次数以及在主动杠杆上做出的反应数量可以衡量可卡因寻求行为的强度。在可卡因自我给药的最初几天，积极反应的数量应在每个采集日逐渐增加，然后在第二周稳定下来。相反，在整个实验过程中，非活动杠杆响应应保持较低水平（图2A）。在仪器性灭绝的第一天，主动杠杆反应?…
Discussion
如上所述，有几个关键步骤对于获得适当的实验结果很重要。该方案可能只对正确获得可卡因自我给药的动物有效，迄今为止，它仅使用上述参数进行了测试。可卡因剂量、强化计划和提示参数可能会被修改，但对行为结果的影响可能很小，除了二阶强化方案可能导致杏仁核非依赖性可卡因的寻找，这可能会降低手术的有效性，尽管这尚未经过直接测试14.在整个协议中有几个要?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者希望感谢USPHS拨款K01DA031745（MMT），R01DA042029（MMT），DA035805（YHH），F31DA039646（MTR），T32031111（MTR）和宾夕法尼亚州卫生部的支持。
Materials
| 0.9% Saline | Fisher Scientific | NC0291799 | |
| A.M.P.I. Stimulus Isolator | Iso-Flex | ||
| AAV5.hSyn.oChIEF.tdTomato | Duke Viral Vector Core (via Roger Tsien) | #268 | See Lin et al., 2009; Nabavi et al., 2014 |
| AAV5.hSyn.tdTomato (Control) | Duke Viral Vector Core Control | See Lin et al., 2009; Nabavi et al., 2014 | |
| Artificial Tears (Opthalmic Ointment) | Covetrus | 70349 | |
| ATP Magnesium Salt | Fisher Scientific | A9187 | |
| Betadine | Butler Schein | 38250 | |
| Calcium chloride | Fisher Scientific | C1016 | |
| Cesium chloride | Fisher Scientific | 289329 | |
| Cesium hydroxide | Fisher Scientific | 516988 | |
| Cesium methanesulfonate | Fisher Scientific | C1426 | |
| Cocaine HCl | NIDA Drug Supply Center | 9041-001 | |
| Cryostat | Leica | CM1950 | |
| D-Glucose | Sigma-Aldrich | G8270 | |
| DMSO | Fisher Scientific | BP231-1 | |
| Dual-Channel Temperature Controller | Warner Instruments | TC-344C | |
| EGTA | Fisher Scientific | E3889 | |
| Ethanol | University of Pittsburgh Chemistry Stockroom | 200C5000 | |
| Ferrule Dust Caps | Thor Labs | CAPL | White plastic dust caps for 1.25 mm Ferrules |
| Ferrule Mating Sleeves | Doric Lenses | F210-3011 | Sleeve_BR_1.25, Bronze, 1.25 mm ID |
| Ferrules | Precision Fiber Products | MM-FER2007C-2300 | Ø1.25 mm Multimode LC/PC Ceramic ferrule, Ø230 μm hole size |
| Fiber Optic | Thor Labs | FP200URT | 200 μm core multimode fiber (0.5 NA) |
| Fiber Optic Rotary Joint | Prizmatix | (Ordered from Amazon) | 18 mm diameter, FC-FC connector for fiber |
| Fiber Stripping Tool | Thor Labs | T12S21 | |
| Fluoroshield with DAPI | Sigma-Aldrich | F6057 | |
| Gentamicin | Henry Schein | 6913 | |
| GTP Sodium Salt | Fisher Scientific | G8877 | |
| Hamilton syringe | Hamilton | 80085 | 10 μL volume, 26 gauge, 2 inch, point style 3 |
| Heat Gun | Allied Electronics | 972-6966 | 250 V, 750-800 °F |
| Heat-Curable Epoxy | Precision Fiber Products | PFP-353ND-8OZ | |
| Heparin | Henry Schein | 55737 | |
| HEPES | Sigma-Aldrich | H3375 | |
| Hydrochloric Acid | Fisher Scientific | 219405490 | |
| Isoflurane | Henry Schein | 29405 | |
| Ketamine HCl | Henry Schein | 55853 | Ketamine is a controlled substance and should be handled according to institutional guidelines |
| Lactated Ringer’s | Henry Schein | 9846 | |
| Laser, driver, and laser-to-fiber coupler | OEM Laser Systems | BL-473-00100-CWM-SD-xx-LED-0 | 100 mW, 473-nm, diode-pumped solid-state laser (One option) |
| L-glutathione | Fisher Scientific | G4251 | |
| Lidocaine | Butler Schein | 14583 | |
| Light Sensor | Thor Labs | PM100D | Compact energy meter console with digital display |
| Loctite instant adhesive | Grainger | 5E207 | |
| Magnesium sulfate | Sigma-Aldrich | 203726 | |
| Microelectrode Amplifier/Data Acquisition | Molecular Devices | MULTICLAMP700B / Digidata 1440A | |
| Microinjector pump | Harvard Apparatus | 70-4501 | Dual syringe |
| Micromanipulator | Sutter Instruments | MPC-200/ROE-200 | |
| Microscope | Olympus | BX51WI | Upright microscope for electrophysiology |
| Microscope | Olympus | BX61VS | Epifluorescent slide-scanning microscope |
| N-methyl-D-glucamine | Sigma-Aldrich | M2004 | |
| Orthojet dental cement, liquid | Lang Dental | 1504BLK | black |
| Orthojet dental cement, powder | Lang Dental | 1530BLK | Contemporary powder, black |
| Paraformaldehyde | Sigma-Aldrich | P6148 | |
| Patch Cables | Thor Labs | FP200ERT | Multimode, FT030 Tubing |
| Picrotoxin | Fisher Scientific | AC131210010 | |
| Polishing Disc | Thor Labs | D50FC | |
| Polishing Pad | Thor Labs | NRS913 | 9" x 13" |
| Polishing Paper | Thor Labs | LFG5P | 5 μm grit |
| Polishing Paper | Thor Labs | LFG3P | 3 μm grit |
| Polishing Paper | Thor Labs | LFG1P | 1 μm grit |
| Polishing Paper | Thor Labs | LFG03P | 0.3 μm grit |
| Potassium chloride | Sigma-Aldrich | P9333 | |
| Potassium hydroxide | Fisher Scientific | P5958 | |
| Potassium methanesulfonate | Fisher Scientific | 83000 | |
| QX-314-Cl | Alomone Labs | Q-150 | |
| Rimadyl (Carprofen) | Henry Schein | 24751 | |
| Self-Administration Chambers/Software | Med Associates | MED-NP5L-D1 | |
| Sodium bicarbonate | Sigma-Aldrich | S5761 | |
| Sodium chloride | Sigma-Aldrich | S7653 | |
| Sodium Hydroxide | Sigma-Aldrich | 1064980500 | |
| Sodium L-Ascorbate | Sigma-Aldrich | A7631 | |
| Sodium Pentobarbital | Henry Schein | 24352 | |
| Sodium phosphate | Sigma-Aldrich | S9638 | |
| Sodium phosphocreatine | Fisher Scientific | P7936 | |
| Sodium pyruvate | Sigma-Aldrich | P2256 | |
| Stainless steel machine screws | WW Grainger | 6GB25 | M2-0.40mm Machine Screw, Pan, Phillips, A2 Stainless Steel, Plain, 3 mm Length |
| Stereotaxic adapter for ferrules | Thor Labs | XCL | |
| Stereotaxic Frame | Stoelting | 51603 | |
| Sucrose | Sigma-Aldrich | S8501 | |
| Suture Thread | Fine Science Tools | 18020-50 | Silk thread; Size: 5/0, Diameter: 0.12 mm |
| TEA-Chloride | Fisher Scientific | T2265 | |
| Thiourea | Sigma-Aldrich | T8656 | |
| Vetbond Tissue Adhesive | Covetrus | 001505 | |
| Vibratome | Leica | VT1200S | |
| Xylazine | Butler Schein | 33198 |
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Cite This Article
Rich, M. T., Huang, Y. H., Torregrossa, M. M. Using Optogenetics to Reverse Neuroplasticity and Inhibit Cocaine Seeking in Rats. J. Vis. Exp. (176), e63185, doi:10.3791/63185 (2021).