دليل ل<em> في الجسم الحي</em> تسجيل وحدة واحدة من التي تم تحديدها Optogenetically القشرية التثبيطي Interneurons
Summary
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.
Abstract
وثمة تحد رئيسي في الفسيولوجيا العصبية لتوصيف خصائص الاستجابة وظيفة من العديد من أنواع الخلايا المثبطة في القشرة الدماغية. نحن هنا نشارك إستراتيجيتنا للحصول مستقرة، معزولة جيدا تسجيلات وحدة واحدة من interneurons المثبطة التي تم تحديدها في القشرة الماوس تخدير باستخدام الطريقة التي وضعتها ليما و1 الزملاء. وتجرى التسجيلات في الفئران معربا عن Channelrhodopsin 2 (ChR2) فى فئة محددة العصبية. يتم التعرف على أفراد من السكان من قبل ردهم إلى مضة قصيرة من الضوء الأزرق. هذه التقنية – يطلق عليه "PINP"، أو تحديد ضوئي بمساعدة من السكان العصبية – يمكن تنفيذها مع المعدات القياسية تسجيل خارج الخلية. يمكن أن تكون بمثابة بديل رخيص الثمن وفي متناول التصوير الكالسيوم أو الترقيع الموجهة بصريا، لغرض استهداف التسجيلات خارج الخلية إلى الخلايا المحددة وراثيا. Hيحرث نحن نقدم مجموعة من الإرشادات لتحسين الأسلوب في الممارسة اليومية. نحن المكرر استراتيجيتنا خصيصا لاستهداف (PV +) خلايا parvalbumin إيجابي، ولكن وجدت أنه يعمل لأنواع عصبون أخرى أيضا، مثل (CR +) interneurons، معربا عن calretinin (SOM +)، معربا عن السوماتوستاتين و.
Introduction
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 “PINP” – 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.
Protocol
Representative Results
Discussion
على الرغم من PINP واضح ومباشر من الناحية النظرية، يمكن أن يكون صعبا من الناحية العملية. والمحدد الرئيسي للنجاح هو اختيار القطب. في دائرة نصف قطرها الاستماع الكهربائي المعلمة حرجة. يجب أن تكون كبيرة بما فيه الكفاية للكشف عن أثار المسامير الضوئية عند الطرف لا يزال على مس?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Name of Material/Equipment | Company | Product/Stock Number | Comments/Description |
| ChR2-EYFP Line | Jackson Colonies | 12569 | |
| Pvalb-iCre (PV) Line | Jackson Colonies | 8069 | |
| Sst-iCre (SOM) Line | Jackson Colonies | 13044 | |
| Cr-iCre (CR) Line | Jackson Colonies | 10774 | |
| Agarose | Sigma-Aldrich | A9793 | Type III-A, High EEO |
| Micro Point (dural hook) | FST | 10066-15 | |
| Surgical Scissors | FST | 14084-09 | |
| Scalpel | FST | 10003-12 (handle), 10011-00 (blades) | |
| Puralube Ophthalmic Ointment | Foster & Smith | 9N-76855 | |
| Homeothermic Blanket | Harvard Apparatus | 507220F | |
| Tungsten Microelectrodes | A-M Systems | 577200 | 12 MΩ AC resistance, 127 μm diameter, 12° tapered tip, epoxy-coated |
| Capillary Glass Tubing | Warner Instruments | G150TF-3 | |
| Heat Shrink Tubing | DigiKey | A332B-4-ND | |
| Zapit Accelerator | DVA | SKU ZA/ZAA | Use with standard Super Glue. |
| Microelectrode AC Amplifier 1800 | AM Systems | 700000 | |
| MP-285 Motorized Micromanipulator | Sutter | MP-285 | |
| 4-channel Digital Oscilloscopes | Tektronix | TDS2000C | |
| Powered Speakers | Harman | Model JBL Duet | |
| Manual Manipulator | Scientifica | LBM-7 | |
| 800 µm Fiber Optic Patch Cable | ThorLabs | FC/PC BFL37-800 | |
| Power Meter | ThorLabs | PM100D (Power Meter), S121C (Standard Power Sensor) | |
| 475 nm Cree XLamp XP-E | DigiKey | XPEBLU-L1-R250-00Y01DKR-ND | LED power and efficiency are continually increasing, so we recommend checking for the latest products (www.cree.com). |
| Arduino UNO | DigiKey | 1050-1024-ND |
References
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