指南<em>在体内</em>单单元从Optogenetically鉴定皮质抑制性录像
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
在神经生理学的一个主要挑战是在大脑皮质中表征的许多抑制性细胞类型的响应特性和功能。 在这里，我们分享我们的战略得到稳定的，良好的分离鉴定，从抑制性的单单元记录在麻醉的小鼠皮层利用利马和他的同事开发出1的方法。录音小鼠在特定的神经元亚群表达Channelrhodopsin-2（的ChR2）上进行。该群体的成员是通过它们的响应于蓝光的短暂闪光。这种技术 – 被称为“PINP”，或神经元群体的光刺激辅助识别 – 可以与标准的细胞外记录设备来实现。它可以作为一种廉价的和可替代的钙成像或目视制导修补，用于靶向细胞外记录到确定的转基因细胞中的用途。 ħERE我们提供了一套指引，以优化在日常实践中的方法。我们具体地细化我们的策略用于靶向小清蛋白阳性（PV +）细胞，但已经发现，它适用于其它中间神经元的类型，以及，如促生长素抑制素表达（SOM +）和钙网膜蛋白表达（CR +）的interneurons。
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
虽然PINP是从概念上简单的，也可以是在实践中具有挑战性。成功的一个主要因素是电极的选择。电气听音半径是关键的参数。它必须足够大，以检测光诱发尖峰时的前端还有一段距离远离的ChR2 +细胞，这样就可以相应地调整前进的速度。同时，它必须被限制，足以使良好的单单元隔离。也就是说，电极不能同时拾取尖峰来自相邻-CHR 2 – 单元。达成适当的平衡，听力半径而言将是任何靶细胞类型?…
Declarações
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 |
Referências
- Lima, S. Q., Hromadka, T., Znamenskiy, P., Zador, A. M. PINP: a new method of tagging neuronal populations for identification during in vivo electrophysiological recording. PLoS One. 4, (2009).
- Moore, A. K., Wehr, M. Parvalbumin-expressing inhibitory interneurons in auditory cortex are well-tuned for frequency. J Neurosci. 33, 13713-13723 (2013).
- Merchant, H., de Lafuente, V., Pena-Ortega, F., Larriva-Sahd, J. Functional impact of interneuronal inhibition in the cerebral cortex of behaving animals. Prog Neurobiol. 99, 163-178 (2012).
- Markram, H., et al. Interneurons of the neocortical inhibitory system. Nat Rev Neurosci. 5, 793-807 (2004).
- Atallah, B. V., Bruns, W., Carandini, M., Scanziani, M. Parvalbumin-expressing interneurons linearly transform cortical responses to visual stimuli. Neuron. 73, 159-170 (2012).
- Wilson, N. R., Runyan, C. A., Wang, F. L., Sur, M. Division and subtraction by distinct cortical inhibitory networks in vivo. Nature. 488, 343-348 (2012).
- Letzkus, J. J., et al. A disinhibitory microcircuit for associative fear learning in the auditory cortex. Nature. 480, 331-335 (2011).
- Pi, H. J., et al. Cortical interneurons that specialize in disinhibitory control. Nature. 503, 521-524 (2013).
- Adesnik, H., Bruns, W., Taniguchi, H., Huang, Z. J., Scanziani, M. A neural circuit for spatial summation in visual cortex. Nature. 490, 226-231 (2012).
- Madisen, L., et al. A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing. Nat Neurosci. 15, 793-802 (2012).
- Hippenmeyer, S., et al. A developmental switch in the response of DRG neurons to ETS transcription factor signaling. PLoS Biol. 3, 159 (2005).
- Taniguchi, H., et al. A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex. Neuron. 71, 995-1013 (2011).
- Christianson, G. B., Sahani, M., Linden, J. F. Depth-dependent temporal response properties in core auditory cortex. J Neurosci. 31, 12837-12848 (2011).
- Povysheva, N. V., Zaitsev, A. V., Gonzalez-Burgos, G., Lewis, D. A. Electrophysiological heterogeneity of fast-spiking interneurons: chandelier versus basket cells. PLoS One. 8, 70553 (2013).
