神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合
1Auditory Neuroscience Laboratory, Institute of Neuroscience of Castilla y León,University of Salamanca, 2Neural Systems Laboratory, Institute for Systems Research,University of Maryland, 3Medical Research Council Institute of Hearing Research, 4Department of Cell Biology and Pathology, Faculty of Medicine,University of Salamanca
Summary
We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.
Abstract
神经递质和调质，因此不同的神经反应的活性差异，可以麻醉和清醒的动物之间找到。因此，允许突触系统的清醒动物的操作方法，需要在以确定的突触输入的神经元处理由麻醉剂的影响的贡献。这里，我们提出的方法用于电极的结构，以同时记录细胞外神经活动和在清醒小鼠的记录点的附近释放多个神经活性物质。通过结合这些过程，我们进行了gabazine的微电泳注射，以选择性阻断GABA A受体头抑制小鼠的下丘神经元。 Gabazine成功修改神经反应性能如频率响应区域和特定刺激的适应。因此，我们证明我们的方法适用于recordin克单单元活动和解剖在听觉处理特定神经递质受体的作用。
所描述的过程的主要限制是在相对较短的记录时间（〜3小时），这是由动物到记录会话的习惯的水平来确定。另一方面，多个记录会话可以在相同的动物中进行。这种技术在用于操纵神经传递或神经调节（如全身注射或使用光遗传学模型）的水平的其他实验程序的优点是，药物的效果被限制在本地突触输入到目标神经元。此外，电极的定制制造允许根据神经结构和感兴趣（如用于提高记录的信噪比的尖端电阻）神经元的类型的具体参数的调整。
Introduction
神经兴奋和抑制的相互作用是感觉信息1的处理的基础。它也已知麻醉对皮层激活和突触输入2,3的时间模式的动态的强烈冲击。例如，已经观察到，麻醉剂改变的视觉诱发反应在皮质神经元3,4的持续时间。此外，兴奋性和抑制性突触输入之间的比例是在麻醉和清醒动物4,5-不同，改变既诱发和自发活动率6,7。通过测量突触电导，海德尔和他的同事发现4在振幅抑制匹配激励麻醉下而清醒时，抑制作用大于激发更强。这些发现提示的实验程序的开发，研究具体的突触输入感官处理在清醒动物的影响。
<P类=“jove_content”>通过施加小电流注射的（NA的数量级上）带电神经活性物质的受控喷射已被广泛用于研究的突触输入的贡献，并推定细胞受体的感觉处理8-13的作用。这种技术被称为微电泳，允许药物在记录神经元，这有助于快速和密闭效果附近的应用。这个过程是更适合于学习的神经活性物质局部作用，相比于其它实验操作如全身注射，微透析或使用光遗传学技术引起了广泛的影响。通常，一个背驮式电极结构14,15被用于同时记录所述目标神经元，并提供所关注的神经活性物质。它由附接到承载神经活性物质的多管吸移管的记录电极。邻的修改通过Havey和Caspary 14描述riginal程序已付诸实施。例如，钨电极，而不是玻璃之一，可以用来记录神经活动16。对钨电极17,18的制造先前公布的方法包括三个基本步骤：钨丝提示，玻璃绝缘，并且末端曝光调整电解蚀刻，以满足记录的要求。听觉神经科学的一个有趣的和紧急字段是特定刺激的适应（SSA 19）的研究。 SSA是在给不推广到其他，很少呈现声音重复声音的神经反应的特定下降。 SSA的重要性在于它在听觉大脑神经机制底层偏差检测，以及对听觉的后期失匹配负成分的可能相关的神经元的潜在作用诱发电位20,21。 ØSSA从IC ccurs到听觉皮层19,22-24。 GABA A介导的抑制已被证实作为对SSA 7,16,25的增益控制机制，这也被证明是由麻醉26的影响。这里，我们提出，结合先前描述的方法之前和清醒小鼠的GABA A -受体的选择性拮抗物的应用过程中记录的IC神经元的单单元活动的协议。首先，我们描述背驮式电极和下，手术和记录方法中的用途。为了测试药物释放的功效，我们比较了感受域以及集成电路神经元SSA之前和gabazine的微电泳喷射期间的水平。
Protocol
所有试验程序都在萨拉曼卡大学与批准进行，使用符合标准的方法，萨拉曼卡动物保健委员会的大学，以及欧盟的标准（指令63分之2010/ EU）对在神经科学研究中使用动物。 1.钨电极注意：钨电极的制造是基于在美林和安斯沃思27和安斯沃思等人 28所述的原技术和使用布洛克等人 17所述的工作站设置进行。 以记录的IC神经元的单单元外…
Representative Results
我们记录4以及隔离IC的神经元的单单元的活动。期间在清醒小鼠外记录获得的典型的信号-噪声比示于图3B。 图4A示出每个神经元的频率响应区域（FRA）之前和期间的GABA A的封锁与gabazine受体拮抗剂。未观察到响应强度（峰值/刺激）以及光谱调谐的扩大的增量。在诱发反应的增加也很明显在从显示（ 图4B）对所有的频率和强度?…
Discussion
的神经活性物质在清醒的动物的微电泳是一个功能强大的技术来探测并解剖上单个神经元40,41的活性的特定的突触输入的作用。更重要的是，这个过程可以对神经回路的神经递质和调质无麻醉剂的潜在干扰的影响的决心。这里，我们表明，在清醒小鼠的集成电路gabazine的应用鲁棒改变频率调谐（ 图4A），时间的反应模式（ 图4B）和SSA（ 图5）。
<p cla…Declarações
The authors have nothing to disclose.
Acknowledgements
该项目是由MINECO资助授予BFU201343608-P和PSI2013-49348-EXP和JCYL授予SA343U14男男性行为者和MRC核心资金ARP。 YAA举行了国家科学技术委员会（216106）和SEP奖学金。
Materials
| Tungsten wire | Harvard Apparatus LTD | 33-0099 | 0.005 inches x 3 inches |
| Borosilicate glass capillary | Harvard Apparatus LTD | 30-0053 | Borosilicate standard wall without filament, 1.5 mm OD, 0.86 mm ID, 100 mm long |
| Multibarrel glass capillaries | World Precision Instruments | 5B120F-4 | 5-barrel capillary, 4 inches long, 1.2 mm OD, with filament |
| Diaplus | DiaDent | 2001-2101 | Light-curing adhesive, used to attach the tungsten electrode to the glas multibarrel pipette |
| G-Bond | GC Corporation | 2277 | Light-curing adhesive, used to attach the headpost to the animal's skull |
| Charisma | Heraeus Kulzer | 66000087 | Light-curing composite, used to reinforce the bond of the headpost with the skull |
| Araldit Cristal | Ceys | 2-component expoxy, used to further secure the attachment of the tungsten electrode to the glass multibarrel pipette | |
| Heating blanket | Cibertec | RTC1 | |
| Stereotactic frame | Narishige | SR-6N | Modified for mice |
| Microiontophoretic device | Harvard Apparatus LTD | Neurophore BH-2 | Including IP-2 iontophoresis pumps (one for each drug delivery channel) and a balance module |
| Multibarrel glass pipette puller | Narishige | Model PE-21 | |
| LED lamp | Technoflux | CV-215 | 5 W, 430-485 nm |
| MicroFil | World Precision Instruments | MF34G-5 | Flexible plastic needle, 34 AWG |
| Imalgene | Merial | Ketamine, 100 mg/mL | |
| Rompun | Bayer | Xylazine, 20 mg/mL | |
| Gabazine / SR-95531 | Sigma | S106 | Prepare ~ 1000µl of 20 mM gabazine in distilled water and adjust the pH to 4 |
Referências
- Harris, K. D., Thiele, A. Cortical state and attention. Nat Rev Neurosci. 12 (9), 509-523 (2011).
- Constantinople, C. M., Bruno, R. M. Effects and mechanisms of wakefulness on local cortical networks. Neuron. 69 (6), 1061-1068 (2011).
- Sellers, K. K., Bennett, D. V., Hutt, A., Williams, J. H., Frohlich, F. Awake vs. anesthetized: layer-specific sensory processing in visual cortex and functional connectivity between cortical areas. J Neurophysiol. 113 (10), 3798-3815 (2015).
- Haider, B., Hausser, M., Carandini, M. Inhibition dominates sensory responses in the awake cortex. Nature. 493 (7430), 97-100 (2013).
- Rudolph, M., Pospischil, M., Timofeev, I., Destexhe, A. Inhibition determines membrane potential dynamics and controls action potential generation in awake and sleeping cat cortex. J Neurosci. 27 (20), 5280-5290 (2007).
- Buran, B. N., von Trapp, G., Sanes, D. H. Behaviorally gated reduction of spontaneous discharge can improve detection thresholds in auditory cortex. J Neurosci. 34 (11), 4076-4081 (2014).
- Duque, D., Malmierca, M. S., Caspary, D. M. Modulation of stimulus-specific adaptation by GABA(A) receptor activation or blockade in the medial geniculate body of the anaesthetized rat. J Physiol. 592, 729-743 (2014).
- Stone, T. W. . Microiontophoresis and Pressure Ejection. , (1985).
- Lalley, P. M., Windhorst, U., Johansson, H. . Modern techniques in neuroscience research). , 193-212 (1999).
- Foeller, E., Celikel, T., Feldman, D. E. Inhibitory sharpening of receptive fields contributes to whisker map plasticity in rat somatosensory cortex. J Neurophysiol. 94 (6), 4387-4400 (2005).
- Foeller, E., Vater, M., Kossl, M. Laminar analysis of inhibition in the gerbil primary auditory cortex. J Assoc Res Otolaryngol. 2 (3), 279-296 (2001).
- Kurt, S., Crook, J. M., Ohl, F. W., Scheich, H., Schulze, H. Differential effects of iontophoretic in vivo application of the GABA(A)-antagonists bicuculline and gabazine in sensory cortex. Hear Res. 212 (1-2), 224-235 (2006).
- Sivaramakrishnan, S., et al. GABA(A) synapses shape neuronal responses to sound intensity in the inferior colliculus. J Neurosci. 24 (21), 5031-5043 (2004).
- Havey, D. C., Caspary, D. M. A simple technique for constructing ‘piggy-back’ multibarrel microelectrodes. Electroencephalogr Clin Neurophysiol. 48 (2), 249-251 (1980).
- Dondzillo, A., Thornton, J. L., Tollin, D. J., Klug, A. Manufacturing and using piggy-back multibarrel electrodes for in vivo pharmacological manipulations of neural responses. J Vis Exp. (71), e4358 (2013).
- Perez-Gonzalez, D., Hernandez, O., Covey, E., Malmierca, M. S. GABA(A)-Mediated Inhibition Modulates Stimulus-Specific Adaptation in the Inferior Colliculus. PLoS ONE. 7 (3), e34297 (2012).
- Bullock, D. C., Palmer, A. R., Rees, A. Compact and easy-to-use tungsten-in-glass microelectrode manufacturing workstation. Med Biol Eng Comput. 26 (6), 669-672 (1988).
- Sugiyama, K., Dong, W. K., Chudler, E. H. A simplified method for manufacturing glass-insulated metal microelectrodes. J Neurosci Methods. 53 (1), 73-80 (1994).
- Ulanovsky, N., Las, L., Nelken, I. Processing of low-probability sounds by cortical neurons. Nat Neurosci. 6 (4), 391-398 (2003).
- Escera, C., Malmierca, M. S. The auditory novelty system: An attempt to integrate human and animal research. Psychophysiology. 51 (2), 111-123 (2014).
- Malmierca, M. S., Sanchez-Vives, M. V., Escera, C., Bendixen, A. Neuronal adaptation, novelty detection and regularity encoding in audition. Front Syst Neurosci. 8, 111 (2014).
- Malmierca, M. S., Cristaudo, S., Perez-Gonzalez, D., Covey, E. Stimulus-specific adaptation in the inferior colliculus of the anesthetized rat. J Neurosci. 29 (17), 5483-5493 (2009).
- Antunes, F. M., Nelken, I., Covey, E., Malmierca, M. S. Stimulus-specific adaptation in the auditory thalamus of the anesthetized rat. PLoS ONE. 5 (11), 14071 (2010).
- von der Behrens, W., Bauerle, P., Kossl, M., Gaese, B. H. Correlating stimulus-specific adaptation of cortical neurons and local field potentials in the awake rat. J Neurosci. 29 (44), 13837-13849 (2009).
- Perez-Gonzalez, D., Malmierca, M. S. Variability of the time course of stimulus-specific adaptation in the inferior colliculus. Front Neural Circuits. 6, 107 (2012).
- Duque, D., Malmierca, M. S. Stimulus-specific adaptation in the inferior colliculus of the mouse: anesthesia and spontaneous activity effects. Brain Struct Funct. , (2014).
- Merrill, E. G., Ainsworth, A. Glass-coated platinum-plated tungsten microelectrodes. Med Biol Eng. 10 (5), 662-672 (1972).
- Ainsworth, A., Dostrovsky, J. O., Merrill, E. G., Millar, J. An improved method for insulating tungsten micro-electrodes with glass [proceedings]. J Physiol. 269 (1), 4-5 (1977).
- Bryant, J. L., Roy, S., Heck, D. H. A technique for stereotaxic recordings of neuronal activity in awake, head-restrained mice. J Neurosci Methods. 178 (1), 75-79 (2009).
- Portfors, C. V., Roberts, P. D., Jonson, K. Over-representation of species-specific vocalizations in the awake mouse inferior colliculus. Neurociência. 162 (2), 486-500 (2009).
- Portfors, C. V., Mayko, Z. M., Jonson, K., Cha, G. F., Roberts, P. D. Spatial organization of receptive fields in the auditory midbrain of awake mouse. Neurociência. 193, 429-439 (2011).
- Muniak, M. A., Mayko, Z. M., Ryugo, D. K., Portfors, C. V. Preparation of an awake mouse for recording neural responses and injecting tracers. J Vis Exp. (64), (2012).
- Deacon, R. M. Housing, husbandry and handling of rodents for behavioral experiments. Nat Protoc. 1 (2), 936-946 (2006).
- Malmierca, M. S., et al. A discontinuous tonotopic organization in the inferior colliculus of the rat. J Neurosci. 28 (18), 4767-4776 (2008).
- Izquierdo, M. A., Gutierrez-Conde, P. M., Merchan, M. A., Malmierca, M. S. Non-plastic reorganization of frequency coding in the inferior colliculus of the rat following noise-induced hearing loss. Neurociência. 154 (1), 355-369 (2008).
- Palmer, A. R., Shackleton, T. M., Sumner, C. J., Zobay, O., Rees, A. Classification of frequency response areas in the inferior colliculus reveals continua not discrete classes. J Physiol. 591 (16), 4003-4025 (2013).
- Ayala, Y. A., Malmierca, M. S. Stimulus-specific adaptation and deviance detection in the inferior colliculus. Front Neural Circuits. 6, 89 (2013).
- Duque, D., Perez-Gonzalez, D., Ayala, Y. A., Palmer, A. R., Malmierca, M. S. Topographic distribution, frequency, and intensity dependence of stimulus-specific adaptation in the inferior colliculus of the rat. J Neurosci. 32 (49), 17762-17774 (2012).
- Ayala, Y. A., Perez-Gonzalez, D., Duque, D., Nelken, I., Malmierca, M. S. Frequency discrimination and stimulus deviance in the inferior colliculus and cochlear nucleus. Front Neural Circuits. 6, 119 (2013).
- Perkins, M. N., Stone, T. W. In vivo release of [3H]-purines by quinolinic acid and related compounds. Br J Pharmacol. 80 (2), 263-267 (1983).
- Lalley, P. M., Windhorst, U., Johansson, H. . Modern Techniques in Neuroscience Research. , 193-212 (1999).
- Candy, J. M., Boakes, R. J., Key, B. J., Worton, E. Correlation of the release of amines and antagonists with their effects. Neuropharmacology. 13 (6), 423-430 (1974).
- Martins, A. R., Froemke, R. C. Coordinated forms of noradrenergic plasticity in the locus coeruleus and primary auditory cortex. Nat Neurosci. , (2015).
- LeBeau, F. E., Rees, A., Malmierca, M. S. Contribution of GABA- and glycine-mediated inhibition to the monaural temporal response properties of neurons in the inferior colliculus. Journal of Neurophysiology. 75 (2), 902-919 (1996).
- Ayala, Y. A., Malmierca, M. S. Cholinergic modulation of stimulus-specific adaptation in the inferior colliculus. The Journal of Neuroscience. 35 (35), 12261-12272 (2015).
Citar este artigo
Ayala, Y. A., Pérez-González, D., Duque, D., Palmer, A. R., Malmierca, M. S. Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice. J. Vis. Exp. (111), e53914, doi:10.3791/53914 (2016).