The Journal of Physiology｜脊髓损伤后感觉剥夺后神经元活

　　The Journal of Physiology ArticleOct 01, 2021: 599 (20),4643-466910.1113/JP281901本文由“天纳”临床学术信息人工智能系统自动翻译点击文末“阅读原文”下载本文PDFCortical areas have the capacity of large-scale reorganization following sensory deafferentation. However, it remains unclear whether this phenomenon is a unique process that homogeneously affects the entire deprived cortical region or whether it is susceptible to changes depending on neuronal networks across distinct cortical layers. Here, we studied how the local circuitry within each layer of the deafferented cortex forms the basis for neuroplastic changes after immediate thoracic spinal cord injury (SCI) in anaesthetized rats. In vivo electrophysiological recordings from deafferented hindlimb somatosensory cortex showed that SCI induces layer-specific changes mediating evoked and spontaneous activity. In supragranular layer 2/3, SCI increased gamma oscillations and the ability of these neurons to initiate up-states during spontaneous activity, suggesting an altered corticocortical network and/or intrinsic properties that may serve to maintain the excitability of the cortical column after deafferentation. On the other hand, SCI enhanced the infragranular layers’ ability to integrate evoked sensory inputs leading to increased and faster neuronal responses. Delayed evoked response onsets were also observed in layer 5/6, suggesting alterations in thalamocortical connectivity. Altogether, our data indicate that SCI immediately modifies the local circuitry within the deafferented cortex allowing supragranular layers to better integrate spontaneous corticocortical information, thus modifying column excitability, and infragranular layers to better integrate evoked sensory inputs to preserve subcortical outputs. These layer-specific neuronal changes may guide the long-term alterations in neuronal excitability and plasticity associated with the rearrangements of somatosensory networks and the appearance of central sensory pathologies usually associated with spinal cord injury.皮层区域在感觉去传入后具有大规模重组的能力。然而，目前尚不清楚这种现象是否是一种独特的过程，均匀地影响整个被剥夺的皮层区域，或者它是否易受不同皮层神经元网络的影响。在这里，我们研究了麻醉大鼠立即胸脊髓损伤（SCI）后，去传入皮质各层内的局部回路如何形成神经可塑性变化的基础。来自去传入后肢体感皮层的活体电生理记录显示，SCI诱导介导诱发和自发活动的层特异性变化。在粒上2/3层，SCI增加了伽马振荡和这些神经元在自发活动期间启动状态的能力，表明皮质皮质网络和/或内在特性发生了改变，可能有助于在去传入后维持皮质柱的兴奋性。另一方面，SCI增强了颗粒下层整合诱发感觉输入的能力，从而增加和加快神经元反应。在第5/6层也观察到延迟诱发反应，表明丘脑皮质连接发生改变。总之，我们的数据表明，SCI立即改变去分化皮质内的局部电路，使粒上层更好地整合自发的皮质皮质信息，从而改变柱兴奋性，粒下层更好地整合诱发的感觉输入，以保留皮质下输出。这些层特异性神经元变化可能指导与体感网络重排相关的神经元兴奋性和可塑性的长期改变，以及通常与脊髓损伤相关的中枢感觉疾病的出现。Spinal cord injury (SCI) produces a physical disconnection between the brain and spinal cord regions below the injury level. Such deafferentation interrupts the sensory information ascending towards the somatosensory cortex, which promotes extensive cortical reorganization (CoRe) of sensorimotor areas (Jain et?al. [1998, 2008]; Curt et?al. [2002]; Endo et?al. [2007]; Ghosh et?al. [2010]). Similar cortical rearrangements following sensory deprivation have also been described in the visual (Griffen et?al. [2017]) and auditory (Bola et?al. [2017]) cortex, indicating that CoRe remapping is a physiological phenomenon across brain regions. In the case of a SCI, the importance of CoRe lies in the crucial role that it plays in functional recovery (Kao et?al. [2009]; Manohar et?al. [2017]). On the other hand, an excessive or maladaptive remapping is implicated in the generation of associated pathologies such as neuropathic pain and spasticity (Siddall et?al. [2003]; Wrigley et?al. [2009]). Therefore, a deep and fine scale understanding of the complexity of CoRe is still needed to improve our knowledge of the plastic process in order to find new strategies to modulate the strength of the reorganization and to improve functional recovery.脊髓损伤（SCI）在损伤程度以下的大脑和脊髓区域之间产生物理断开。这种去传入干扰了向躯体感觉皮层提升的感觉信息，从而促进感觉运动区的广泛皮层重组（核心）（Jain et al.[1998,2008]；Curt et al.[2002]；Endo et al.[2007]；Ghosh et al.[2010]）。视觉（Griffen et al.[2017]）和听觉（Bola et al.[2017]）皮层中也描述了感觉剥夺后类似的皮层重排，表明核心重映射是跨大脑区域的一种生理现象。就SCI而言，核心的重要性在于其在功能恢复中发挥的关键作用（Kao等人[2009]；Manohar等人[2017]）。另一方面，过度或不适应的重新映射与相关病理学的产生有关，如神经病理性疼痛和痉挛（Siddall等人[2003]；Wrigley等人[2009]）。因此，我们仍然需要对核的复杂性进行深入细致的理解，以提高我们对塑性过程的认识，从而找到新的策略来调节重组的强度，并改善功能恢复。Over the past two decades, the use of large-scale experimental approaches such as extracranial electroencephalographic recordings (Green et?al. [1998]), voltage sensitive dyes (Ghosh et?al. [2010]) and functional magnetic resonance imaging (Endo et?al. [2007]) has revealed important insights about the process of CoRe in the time period ranging from days to months after the injury. In addition, fine-scale techniques using in vivo electrophysiological recordings demonstrate increased neuronal activity in the deafferented cortex in response to peripheral stimulation of body regions located above lesion level (Jain et?al. [1998], 2008). Interestingly, changes in the cortical activity after SCI have been mostly studied by using electrophysiological recordings from layer 5 neurons (Aguilar et?al. [2010]; Ghosh et?al. [2012]; Ganzer et?al. [2013]; Humanes-Valera et?al. [2013]; Humanes-Valera et?al. [2017]; Manohar et?al. [2017]). Important reasons support this selection: layer 5 pyramidal neurons receive direct thalamic inputs that are reduced after SCI; its apical dendrite spans multiple layers to integrate inputs along the vertical column axis; layer 5 corticospinal neurons are directly axotomized after a SCI, thereby altering the columnar spontaneous activity; and finally it is the main cortical output to control behaviour and sensory ascending inputs (Canedo & Aguilar [2000]; Manohar et?al. [2017]; Larkum et?al. [2018]). In this regard, SCI has been described as: (1) altering the neuronal excitability at both thalamic level and cortical layer 5 activity (Jain et?al. [2008]; Liang & Mendell [2013]; Alonso-Calvi?o et?al. [2016]), (2) inducing spine loss in the proximal segments of the apical dendrite of axotomized and non-axotomized corticospinal layer 5 neurons in the deafferented cortex (Ghosh et?al. [2012]), and (3) increasing the strength of corticocortical connections (Endo et?al. [2007]; Ganzer et?al. [2013]; Humanes-Valera et?al. [2017]; Manohar [2017]). However, the neocortex is a complex structure composed of six horizontal layers that are spatially and functionally defined by vertical columns. Each layer is defined by different functional properties, cellular composition, and input/output from and to cortical and subcortical regions that under physiological conditions work together to generate and control the pattern of cortical activity. Therefore, the effects that a SCI will produce at the cortical level might not be restricted to layer 5 but might span different cortical layers as a result of altered input statistics that take into account changes in the spontaneous excitability, altered retrograde messaging due to the axotomy, and changes in thalamic and cortical connectivity. In this study, we determine whether each cortical layer may reflect different aspects of CoRe after SCI based in the local network, columnar connections and corticocortical connections, in addition to a direct effect on layer 5 pyramidal neurons.在过去20年中，使用大规模实验方法，如颅外脑电图记录（Green等人[1998]），电压敏感染料（Ghosh et al.[2010]）和功能磁共振成像（Endo et al.[2007]）揭示了在损伤后几天到几个月的时间段内对核心过程的重要见解。此外，使用活体电生理记录的精细技术表明，在损伤水平以上的身体区域受到外周刺激时，去传入皮质中的神经元活动增加（Jain等人[1998]，2008年）。有趣的是，SCI后皮层活动的变化主要通过使用第5层神经元的电生理记录进行研究（Aguilar等人[2010]；Ghosh等人[2012]；Ganzer等人[2013]；Humanes Valera等人[2013]；Humanes Valera等人[2017]；Manohar等人[2017]）。支持这种选择的重要原因是：脊髓损伤后，第5层锥体神经元接收到的丘脑直接输入减少；其顶端树突跨越多层，沿垂直柱轴整合输入；脊髓损伤后第5层皮质脊髓神经元被直接轴切，从而改变柱状自发活动；最后，它是控制行为和感觉提升输入的主要皮质输出（Canedo&Aguilar[2000]；Manohar等人[2017]；Larkum等人[2018]）。在这方面，SCI被描述为：（1）改变丘脑水平和皮层第5层活动的神经元兴奋性（Jain等人[2008]；Liang&Mendell[2013]；Alonso Calvi?o等人[2016]），（2）在去传入皮质的轴切和非轴切皮质脊髓第5层神经元的顶端树突的近端节段诱导脊柱缺失（Ghosh等人[2012]），（3）增加皮质-皮质连接的强度（Endo等人[2007]；Ganzer等人[2013]；Humanes Valera等人[2017]；Manohar[2017]）。然而，新皮质是一个复杂的结构，由六个水平层组成，这些水平层在空间和功能上由垂直柱定义。每一层都由不同的功能特性、细胞组成以及来自和到皮质和皮质下区域的输入/输出来定义，这些区域在生理条件下共同产生和控制皮质活动的模式。因此，SCI在皮层水平产生的效应可能不局限于第5层，而是可能跨越不同的皮层层，这是由于考虑到自发兴奋性变化、轴突切断引起的逆行信息改变以及丘脑和皮层连通性变化的输入统计改变的结果。在这项研究中，除了对第5层锥体神经元产生直接影响外，我们还确定了每个皮质层是否可以反映SCI后局部网络、柱状连接和皮质-皮质连接中核心的不同方面。Functional imaging and electrophysiological studies have revealed that CoRe is a physiological phenomenon in which the time elapsed after the injury may be considered a key factor. In this regard, functional changes have been observed immediately after SCI in different animal models as rodents (Aguilar et?al. [2010]; Yagüe et?al. [2011, 2014]; Humanes-Valera et?al. [2013]) and pigs (Jutzeler et?al. [2018]). Previous data from our group show that, a few minutes after the injury, both intact and deprived somatosensory cortex became more responsive to peripheral sensory stimulation above the lesion level (Humanes-Valera et?al. [2013]; Yagüe et?al. [2014]) and spontaneous activity is drastically reduced (Aguilar et?al. [2010]; Fernández-López et?al. [2019]). In addition to the cortical changes, SCI also modifies thalamic and brainstem spontaneous and evoked neuronal excitability, which may be linked to the changes observed in the somatosensory cortex (Jain et?al. [2008]; Alonso-Calvi?o et?al. [2016]). Therefore, for a precise understanding of the physiological basis of CoRe initiation across cortical layers in deafferented cortex, here we used an acute model of SCI in which simultaneous electrophysiological recordings from six cortical layers were obtained. For this purpose, the use of a vertical array spanning the full cortical depth allows us to determine how SCI affects the neuronal activity within individual layers, the vertical relationship between layers, as well as horizontal connections from adjacent intact cortical columns.功能成像和电生理研究表明，CoRe是一种生理现象，损伤后经过的时间可能被认为是一个关键因素。在这方面，在不同的动物模型中，如啮齿类动物（Aguilar et al.[2010]；Yagüe et al.[2011,2014]；Humanes Valera et al.[2013]）和猪（Jutzeler et al.[2018]），在脊髓损伤后立即观察到功能变化。我们小组先前的数据显示，受伤几分钟后，完整和缺失的体感皮层对病变水平以上的外周感觉刺激反应更强（Humanes Valera等人[2013]；Yagüe等人[2014]），自发活动显著减少（Aguilar等人[2010]；Fernández-López等人[2019]）。除了皮层变化外，SCI还会改变丘脑和脑干自发和诱发的神经元兴奋性，这可能与体感皮层中观察到的变化有关（Jain等人[2008]；Alonso Calvi?o等人[2016]）。因此，为了准确理解去分化皮质中跨皮质层核心起始的生理基础，我们使用了一个急性脊髓损伤模型，在该模型中，同时获得了六个皮质层的电生理记录。为此，使用跨越整个皮质深度的垂直阵列，我们可以确定SCI如何影响各个层内的神经元活动、层间的垂直关系，以及相邻完整皮质柱的水平连接。Using in vivo electrophysiological recordings from anaesthetized rats, we studied how neuronal activity mediated by corticocortical and thalamic connections as well as the local circuitry in the hindlimb cortex (HLCx) is immediately affected by deafferentation after a thoracic SCI. For this, we used a vertical multielectrode array to determine the neuronal excitability across layers of the deprived HLCx during evoked and spontaneous activity. Peripheral stimulation of the contralateral forelimb showed a layer-dependent increase in sensory-evoked local field potential (LFP) responses across HLCx layers indicating that changes in neuronal network properties of the deprived cortical column may favour excitability. However, a striking heterogeneity was observed when other physiological parameters were analysed. Infragranular L5/6, but not L2/3, exhibited increased LFP slope, increased multiunit activity and delayed onset latencies. Contrary to evoked responses, spontaneous activity was mostly affected in supragranular layers as observed by increased high rhythm frequencies and probability of initiating up-states. Altogether, our data indicate that SCI immediately modifies the local circuitry within the deprived cortex allowing supragranular layers to better integrate spontaneous corticocortical information, thus modifying the excitability of the column, and infragranular layers to better integrate evoked sensory inputs to preserve subcortical outputs.利用麻醉大鼠的活体电生理记录，我们研究了在胸部SCI后，由皮质皮质和丘脑连接以及后肢皮质（HLCx）局部回路介导的神经元活动如何立即受到去传入的影响。为此，我们使用垂直多电极阵列来确定在诱发和自发活动期间剥夺的HLCx各层的神经元兴奋性。对对对侧前肢的外周刺激显示，HLCx各层的感觉诱发局部场电位（LFP）反应呈层依赖性增加，表明被剥夺的皮层柱神经元网络特性的变化可能有利于兴奋性。然而，当分析其他生理参数时，观察到了一种显著的异质性。粒下L5/6（而非L2/3）表现出LFP斜率增加、多单位活动增加和起效潜伏期延迟。与诱发反应相反，通过增加高节律频率和开始上升状态的概率观察到，自发活动主要受粒上层的影响。总之，我们的数据表明，SCI会立即改变被剥夺皮质内的局部回路，使粒上层更好地整合自发的皮质皮质信息，从而改变柱的兴奋性，粒下层更好地整合诱发的感觉输入，以保留皮质下输出。Experiments were performed on two groups of male Wistar rats of age 2–6 months, mean weight 395 (SD 45)?g. Group 1 was intended for the cortical layer study (n?=?29, Ctr:WI Charles River, Barcelona, RRID:RGD_737929); and group 2 was intended for the thalamo-cortical study (n?=?7; Ctr:WI Charles-River RRID:RGD_737929). All animals were housed two per cage in standardized cages, with?ad libitum?access to food and water and maintained at 23°C on a 12-h light/dark cycle. All steps of the experimental procedure were performed in such a way to minimize the animals’ pain and suffering and were approved by the Ethical Committee for Animal Research at the Hospital Nacional de Parapléjicos (reference number for group 1: 152CEEA/2016; group 2: 85CEEA/2012). Note that in group 2, the cortical dataset used in the present work was obtained simultaneously with the dataset regarding the thalamic activity after SCI that has been previously published in Alonso-Calvi?o et?al. ([2016]). However, the cortical dataset has been analysed for the first time for the present work, in line with the rule of three Rs in order to reduce the number of experimental animals. Approximately 14% of the total number of animals died before or during the experimental procedure for SCI, due to anaesthesia intolerance (Field et?al. [1993]). All researchers involved in the study were aware of the ethical principles under which The Journal operates and complied with the animals’ ethics checklist set out by?The Journal of Physiology, the International Council for Laboratory Animal Science, the European Union 2010/63/EU and the ARRIVE guidelines.实验在两组年龄为2-6个月、平均体重395（SD 45）的雄性Wistar大鼠上进行；g、 第一组用于皮质层研究（n=29，Ctr:WI查尔斯河，巴塞罗那，RRID:RGD737929）；第2组用于丘脑皮质研究（n=7；Ctr:WI查尔斯河RRID:RGD737929）。所有动物都被关在标准化的笼子里，每个笼子两只，每只笼子；随意；获得食物和水，并在12小时的光/暗循环中保持在23°C。实验程序的所有步骤都是以尽量减少动物疼痛和痛苦的方式进行的，并得到了国立帕雷吉科斯医院动物研究伦理委员会的批准（第一组参考号：152CEEA/2016；第二组：85CEEA/2012）。请注意，在第2组中，本研究中使用的皮质数据集与之前发表在Alonso Calvi?o等人的SCI后丘脑活动数据集同时获得；al.（[2016]）。然而，为了减少实验动物的数量，本研究首次对皮质数据集进行了分析，符合三R法则。大约14%的动物在SCI实验程序之前或期间死于麻醉不耐受（Field等人[1993]）。参与该研究的所有研究人员都了解该杂志的伦理原则，并遵守了；《生理学杂志》、国际实验动物科学理事会、欧盟2010/63/EU和ARRIM指南。The general experimental approach (anaesthesia, surgery and peripheral stimulation) was similar to that used in our previous studies (Aguilar et?al. [2010]; Humanes-Valera et?al. [2013]; Alonso-Calvi?o et?al. [2016]; Humanes-Valera et?al. [2017]). Briefly, animals were anaesthetized with an intraperitoneal injection of urethane (1.5 g/kg i.p., Sigma-Aldrich, Spain, U2500-100G), placed in a stereotaxic frame (SR-6 Narishige Scientific Instruments, Tokyo, Japan) passively ventilated at 2 litres O2/min by a mask (Medical Supplies & Services, Int. Ltd, Keighley, UK) and body temperature kept constant at 36.5°C using a homeothermic blanket (Cibertec SL, Madrid, Spain). The optimal level of anaesthesia was settled at stage III-4 of cortical activity described by Friedberg et?al. ([1999]). The absence of reflexes to forelimb stimuli, spontaneous whisker movements and corneal reflex was also used to verify the level of anaesthesia. Constant verification of EEG activity and reflexes took place throughout the experiment to guarantee the optimal level of anaesthesia. Then, lidocaine 5% (Normon, Madrid, cat. no. P06B1) was applied subcutaneously into the areas of the incision, and thoracic laminectomy?(at T9–T10 vertebra) was performed keeping the dura mater?intact and protected until the moment of performing a complete transection of the?spinal cord. Next, the skull was exposed, and a craniotomy was performed on the right hemisphere over the hindlimb representation of the primary somatosensory cortex (AP 0 to ?3 mm; ML 1 to 4 mm; Paxinos & Watson, [2007]) to allow the lowering of a vertical array for further recording of neuronal activity. The stability of recordings was improved by drainage of the cisterna magna and covering the exposed cortex with agar at 4% (Sigma-Aldrich, cat. no. A7002-250G). The exact location of the probe was optimized by assessing the responses to tactile stimulation of the rat's hindlimb with a cotton swab while listening to the recorded signal through a pair of loudspeakers.一般实验方法（麻醉、手术和外周刺激）与我们之前的研究（Aguilar等人[2010]；Humanes Valera等人[2013]；Alonso Calvi?o等人[2016]；Humanes Valera等人[2017]）中使用的方法相似。简单地说，通过腹腔注射乌拉坦（1.5 g/kg i.p.，Sigma-Aldrich，西班牙，U2500-100G）对动物进行麻醉，放置在立体定向框架（SR-6 Narishige Scientific Instruments，日本东京）中，用口罩（英国凯利国际医疗用品服务有限公司）以2升氧气/分钟的速度被动通风，并使用恒温毯（西班牙马德里Cibertec SL）将体温保持在36.5°C的恒定温度。最佳麻醉水平在Friedberg等人描述的第III-4阶段皮质活动中确定；al.（[1999]）。无前肢刺激反射、自发胡须运动和角膜反射也用于验证麻醉水平。在整个实验过程中，对脑电图活动和反射进行持续验证，以确保最佳麻醉水平。然后，将5%利多卡因（诺蒙，马德里，目录号P06B1）皮下注射到切口区域，并进行胸椎椎板切除术；（在T9–T10椎体处）保持硬膜；完整且受保护，直到完成整个横切面；脊髓。接下来，暴露头骨，并在右侧大脑半球初级体感皮层后肢代表处进行开颅手术（AP 0至?3毫米；毫升1至4毫米；Paxinos&Watson[2007]），允许降低垂直阵列以进一步记录神经元活动。通过引流大池并用4%的琼脂覆盖暴露的皮层（Sigma-Aldrich，目录号A7002-250G），记录的稳定性得到了改善。通过评估大鼠后肢对棉签触觉刺激的反应，同时通过一对扬声器收听记录的信号，优化了探针的准确位置。Extracellular recordings were obtained from 24 rats by a linear vertical probe of 32 iridium contacts of 177?μm2 spaced at 50 μm (impedance 1–4?MΩ at 1?kHz; ref: A1X32-6mm-50-177-A32, NeuroNexus Technologies Inc., Ann Arbor, MI, USA). The array was slowly introduced (1–2?μm/s; Fiáth et?al. [2019]) through the craniotomy into the HLCx and a ground electrode was placed in the parietal muscular tissue. The reference electrode was built in the vertical probe, 0.5?mm above the superficial recording site and outside the cortex (area 4200?μm2). The recording protocol started ～40?min after the end of the electrode insertion to allow recovery of cortical tissue following the time line in Fig.?1A. Spontaneous activity was recorded during 10?min. The stimulation protocol (0.5?ms duration at 0.5?Hz) was applied through bipolar?needle electrodes (30G, B. Braun, Melsungen, Germany) located subcutaneously in the?wrist?of contralateral forelimb and hindlimb extremities. Two different intensities were applied: (1) low intensity (0.5?mA) to activate only a fraction of the available peripheral fibres, mainly low-threshold primary fibres running through the lemniscal pathway from the dorsal column to the?brainstem, and (2) high intensity (5?mA) to activate the maximum number of fibres including high-threshold primary fibres that synapse in the?dorsal horns of the spinal cord including the?spinothalamic tract (Lilja et?al. [2006]; Yague et?al. [2011]). However, note that all data analysed throughout this study were obtained from responses showing an initial latency below 15?ms, which corresponds to low-threshold peripheral fibres (tactile and proprioceptive) from the entire paw (digits and palm). After recordings of evoked and spontaneous activity in control conditions, complete transection of the spinal cord was performed using spring scissors. Immediately after transection, pulses of 10?mA electrical stimulation were applied to the contralateral hindlimb to confirm that no physiological responses were evoked by stimuli delivered below the level of the lesion. Complete spinal cord transection was also visually confirmed under the surgical microscope by the total separation of the borders with the help of a small piece of an absorbable haemostatic gelatin sponge (Spongostan, Ferrosan Medical Devices, Denmark). Recordings were continuously acquired during the transection to confirm the stability of the recordings. Approximately 20–30?min after the transection, the same protocol as before SCI was applied. All recording data were converted into digital data at a 40?kHz sampling rate (16/24 rats) and 1?kHz (8/24 rats), with 16-bit quantization by an OmniPlex System controlled by OmniPlex Software (RRID:SCR_014803, Plexon Inc., Dallas, TX, USA). All the 40?kHz signals were offline-filtered into two signals: LFP (low-frequency band: up to 1?kHz) and multiunit activity (MUA; finite impulse response (FIR) bandpass: 0.3–3?kHz, gap 0.12?kHz) by using Spike2.v7 (RRID:SCR_000903, Cambridge Electronic Design (CED), Cambridge, UK).通过线性垂直探针从24只大鼠身上获取177个铱触点的细胞外记录；μm2，间距为50μm（1 kHz时的阻抗为1–4 mΩ；参考：A1X32-6mm-50-177-A32，美国密歇根州安娜堡NeuroExus Technologies Inc.）。通过开颅手术将阵列缓慢地引入HLCx（1–2μm/s；Fiáth等人[2019]），并在顶叶肌肉组织中放置接地电极。参比电极内置在垂直探头中，0.5；表面记录部位上方和皮质外部（面积4200μm2）mm。录制协议启动了～40；电极插入结束后的分钟，以便按照图中的时间线恢复皮质组织；1A。在10个月内记录了自发活动；最小刺激方案（0.5赫兹时持续0.5毫秒）通过双极性；针状电极（30G，B.Braun，Melsungen，德国）位于皮下；手腕；指对侧前肢和后肢。应用了两种不同的强度：（1）低强度（0.5 mA）仅激活一小部分可用的外周纤维，主要是穿过从背柱到背柱的丘系通路的低阈值初级纤维；（2）高强度（5 mA）激活最大数量的纤维，包括在大脑中突触的高阈值初级纤维；脊髓的背角，包括；脊髓丘脑束（Lilja等人[2006]；Yague等人[2011]）。然而，请注意，本研究中分析的所有数据均来自初始潜伏期低于15的回复；ms，对应于整个爪子（手指和手掌）的低阈值外周纤维（触觉和本体感受）。在对照条件下记录诱发和自发活动后，使用弹簧剪刀进行脊髓完全横断。横断后立即进行10次脉冲；对对对侧后肢进行mA电刺激，以确认在损伤水平以下的刺激不会引起生理反应。在手术显微镜下，借助一小块可吸收性止血明胶海绵（Spongostan，Ferrosan Medical Devices，丹麦）完全分离脊髓边界，也可目视确认脊髓完全横断。在横断过程中连续采集记录，以确认记录的稳定性。大约20-30；在横断后min，应用与SCI前相同的方案。所有记录数据以40秒的速度转换为数字数据；kHz采样率（16/24只大鼠）和1；kHz（8/24只大鼠），由OmniPlex软件控制的OmniPlex系统进行16位量化（RRID:SCR_014803，Plexon Inc.，达拉斯，德克萨斯州，美国）。全部40个；kHz信号被离线过滤为两个信号：LFP（低频段：高达1 kHz）和多单元活动（MUA；有限脉冲响应（FIR）带通：0.3–3；千赫，间隙0.12；kHz）通过使用Spike2。v7（RRID:SCR_000903，剑桥电子设计公司，英国剑桥）。A, schematic illustration of the experimental protocol. Extracellular recordings were obtained using a multielectrode probe inserted in the hindlimb of the primary somatosensory cortex (HLCx) from anaesthetized rats. Complete transection of the spinal corOnce the experimental protocol for electrophysiological recordings was completed, animals were prepared for transcardial perfusion to preserve the brain tissue, which was submitted to posterior histological procedures (see ‘Histology’ section). For this purpose, an overdose of anaesthetic (urethane 1.5?g/kg i.p., Sigma-Aldrich, U2500-100G) was applied to induce anaesthetic level IV (Friedberg et?al. [1999]), which was confirmed by a flat EEG, while heart beating and respiration were optimally preserved.一旦完成了电生理记录的实验方案，动物就准备好进行经心灌注以保存脑组织，并将其提交给后组织学程序（见“组织学”一节）。为此，应用过量麻醉剂（乌拉坦1.5 g/kg i.p.，Sigma-Aldrich，U2500-100G）诱导IV级麻醉剂（Friedberg et al.[1999]），并通过平面脑电图确认，同时心脏跳动和呼吸得到最佳保存。At the end of the experiments and after receiving an overdose of urethane (1.5?g/kg) as described above, animals were transcardially perfused with heparinized saline followed by 4% paraformaldehyde. Then the brain was removed and post-fixed in the same fixative solution for 24 h at 4°C. After fixation, brain tissue was cryopreserved in a 30% sucrose solution until it sank and coronal sections at 50?μm thickness were obtained with a sliding microtome (Microm HM 450 V; Microm International GmbH, Dreieich, Germany). Following washing in 0.1?M phosphate buffer, sections were mounted on gelatin slides, air-dried, processed for tionine (Nissl) staining (T7029-5G, Sigma-Aldrich), dehydrated in xylene and coverslipped with DePeX (cat. no. 18243.01, SEVA, Heidelberg, Germany).在实验结束时，在如上所述接受过量的氨基甲酸乙酯（1.5 g/kg）后，用肝素化生理盐水和4%多聚甲醛经心脏灌注动物。然后取出大脑，并在4°C的相同固定液中后固定24小时。固定后，脑组织在30%蔗糖溶液中冷冻保存，直到其下沉，并在50°C的温度下进行冠状切片；使用滑动切片机（Microm HM 450 V；Microm International GmbH，Dreiich，德国）获得μm厚度。在0.1洗涤后；M磷酸盐缓冲液，切片安装在明胶载玻片上，风干，处理以进行离子（Nissl）染色（T7029-5G，Sigma-Aldrich），在二甲苯中脱水，并用DePeX覆盖玻片（目录号18243.01，德国海德堡塞瓦）。Simultaneous electrophysiological recordings from forelimb cortex (FLCx) and HLCx in response to peripheral forelimb stimulation were obtained by using two single tungsten electrodes located on infragranular layer 5 of both cortical regions under control conditions and after SCI. Note that this dataset was obtained simultaneously to the thalamic data previously published in Alonso-Calvi?o et?al. ([2016]), but the cortical dataset has been for the first time analysed for the present work. Experimental protocol was the same as for experiments described above. Briefly, experiments were performed on male Wistar rats (n?=?7, Ctr:WI Charles River RRID:RGD_737929) aging 2–6 months. Animals were housed two per cage in standardized cages, with ad libitum access to food and water and maintained at 23°C on a 12-h light/dark cycle. All experiments were performed in accordance with the International Council for Laboratory Animal Science and the European Union 2010/63/EU and were approved by the Ethical Committee for Animal Research at the Hospital Nacional de Parapléjicos (85/2012) and funded by Spanish Ministry of Economy and Competitiveness and co-funded by FEDER (SAF2012-40109).在对照条件下和脊髓损伤后，使用位于两个皮质区颗粒下第5层的两个单钨电极，获得前肢皮质（FLCx）和HLCx对周围前肢刺激的同时电生理记录。请注意，该数据集与之前发表在Alonso Calvi?o et上的丘脑数据同时获得；al.（[2016]），但本研究首次对皮质数据集进行了分析。实验方案与上述实验相同。简而言之，实验是在2-6个月大的雄性Wistar大鼠（n=7，Ctr:WI查尔斯河RRID:RGD737929）上进行的。每笼两只动物被安置在标准化笼中，可自由获取食物和水，并在12小时的光/暗循环中保持在23°C。所有实验均按照国际实验动物科学理事会和欧盟2010/63/EU的要求进行，并得到了国家帕雷吉科斯医院动物研究伦理委员会（85/2012）的批准，由西班牙经济和竞争力部资助，由FEDER共同资助（SAF2012-40109）。Extracellular recordings were obtained using tungsten electrodes (TM31C40KT, 4?MΩ impedance at 1?kHz or TM31A50KT, 5?MΩ impedance at 1?kHz; World Precision Instruments Inc., Sarasota FL, USA). All recordings were pre-amplified in the DC mode, low pass filtered (<3?kHz) and amplified using a modular system (Neurolog, Digitimer Ltd, Welwyn Garden City, UK). Analog signals were converted into digital data at a 20?kHz with 16-bit quantization via CED Power 1401 apparatus (RRID:SCR_017282) controlled by Spike2. The data were analysed using Spike2 software. Onset latency of cortical responses was obtained using the same method as for the HLCx multielectrode recordings phase (Fedchyshyn & Wang [2007]). In order to obtain comparable data, animals used for this analysis presented a deep state of anaesthesia (III-4; Friedberg et?al. [1999]; Erchova et?al. [2002]) corresponding to slow-wave cortical activity, and evoked cortical responses to peripheral forelimb stimulation were used at 5?mA (0.5?Hz).使用钨电极（TM31C40KT，4 MΩ阻抗，1?kHz或TM31A50KT，5；1时的MΩ阻抗?千赫；世界精密仪器公司（美国佛罗里达州萨拉索塔）。所有记录均在直流模式下预放大，低通滤波（<；3?使用模块化系统进行放大（Neurolog，Digitimer Ltd，Welwyn Garden City，UK）。模拟信号以20秒的速度转换成数字数据?通过Spike2控制的CED Power 1401设备（RRID:SCR_017282）进行16位量化的kHz。使用Spike2软件对数据进行分析。使用与HLCx多电极记录阶段相同的方法获得皮质反应的起始潜伏期（Fedchyshyn&Wang[2007]）。为了获得可比数据，用于该分析的动物呈现出与慢波皮层活动相对应的深度麻醉状态（III-4；Friedberg et al.[1999]；Erchova et al.[2002]），并在5；毫安（0.5赫兹）。Once the experimental protocol for electrophysiological recordings was completed, animals were prepared for transcardial perfusion to preserve the brain tissue with an overdose of anaesthetic (urethane 1.5?g/kg i.p., Sigma-Aldrich, U2500-100G) applied to induce anaesthetic level IV (Friedberg et?al. [1999]), which was confirmed by a flat EEG, while heart beat and respiration were optimally preserved.一旦完成了电生理记录的实验方案，动物就准备好进行经心灌注，以用过量的麻醉剂（乌拉坦1.5 g/kg i.p.，Sigma-Aldrich，U2500-100G）保存脑组织，以诱导IV级麻醉（Friedberg等人[1999]），平板脑电图证实了这一点，同时心跳和呼吸得到了最佳保护。For laminar profile analysis, LFP-evoked responses from each electrode were averaged across 100 stimuli (0.5?Hz) and measured as the maximum amplitude to negative peak (mV) in the local fast response in a time window corresponding to 5–60?ms or 5–30?ms following sensory stimulation of hindimb or forelimb, respectively. In order to quantify MUA, local field potentials were bandpass filtered (FIR bandpass 0.3–3?kHz, gap 0.12?kHz) to obtain multi-unit activity. MUA was then rectified (rMUA), downsampled to 2?kHz and averaged across 100 stimuli to measure the total voltage resulting from the averaged area of responses (μV). The background voltage corresponding to basal activity was subtracted from response voltage (equal time to analysis window but preceding stimulus). For layer analysis, electrode sites were grouped according to layer thickness following these depths: layer 2/3 (150–650?μm), layer 4 (700–1000?μm), layer 5 (1050–1450?μm) and layer 6 (1500–2000?μm; Fiáth et?al. [2016]). For LFP data analysis, electrode measures were averaged across layers while neuronal signals obtained from individual channels (rMUA) were summed within a layer to allow robust detection of the neuronal activity.对于层流剖面分析，每个电极的LFP诱发反应在100个刺激（0.5 Hz）上取平均值，并在对应于5–60的时间窗口内测量为局部快速反应的最大负峰值振幅（mV）；ms或5-30；分别在感觉刺激后肢或前肢后出现ms。为了量化MUA，对局部场电位进行带通滤波（FIR带通0.3–3 kHz，间隙0.12 kHz）以获得多单位活性。然后对MUA进行了纠正（rMUA），将样本减少到2个；100个刺激的平均值，以测量平均响应面积（μV）产生的总电压。从反应电压中减去与基础活动相对应的背景电压（与分析窗口时间相等，但在刺激之前）。对于层分析，根据以下深度的层厚度对电极位置进行分组：第2/3层（150-650μm）、第4层（700-1000μm）、第5层（1050-1450μm）和第6层（1500-2000μm，Fiáth等人[2016]）。对于LFP数据分析，电极测量值在各层之间取平均值，而从单个通道（rMUA）获得的神经元信号在一层内求和，以便对神经元活动进行可靠检测。Onset latency of evoked LFP and rMUA was calculated for each layer by fitting the averaged response with an equation of the form of the Boltzmann charge–voltage function. This equation was solved for its fourth derivative giving a highly accurate measure of the response onset independent of the slope rise phase (Fedchyshyn & Wang [2007]). Slopes were measured by using the formula ΔV/(t1 ? t2), where ΔV is the LFP amplitude, t1 is the onset and t2 is the time of the negative peak. For analysis of the spontaneous cortical activity, up-states within the slow-wave activity (SWA) were first analysed in periods of 5 min of spontaneous HLCx recordings for each subject to compare cortical state immediately before and between 10 and 30?min after SCI. Raw signals were downsampled to 500?Hz and then a fast Fourier transform analysis was performed to confirm that the maximum peak frequency of the recordings was below 1?Hz. Frequency power (mV2) was extracted for each electrode by summing power within same frequency band: SWA (0.1–1?Hz), delta (1–4?Hz), theta (4–8?Hz), alpha (8–12?Hz), beta (12–25?Hz), low gamma (25–50?Hz) and high gamma (50–80?Hz). LFP power obtained from individual electrodes within a layer was averaged to obtain a layer frequency power. Finally, the power of each frequency band was normalized to the total power of the LFP recording (0.1–80?Hz) to obtain the relative power of each band. Additionally, individual up-states were selected within periods of 60?s of LFP for each subject to obtain the onset in control and acute SCI using the same methodology as for the onset of evoked-LFP responses (Fedchyshyn & Wang [2007]). To calculate the velocity of propagation, the electrode presenting the earliest onset was taken as reference, and the velocity rate calculated as the distance in the cortical depth as a function of time.通过使用Boltzmann电荷-电压函数形式的方程拟合平均响应，计算每层诱发LFP和rMUA的起始潜伏期。对该方程的四阶导数进行了求解，给出了独立于坡度上升阶段的高精度响应起始测量（Fedchyshyn&Wang[2007]）。使用公式ΔV/（t1）测量斜率? t2），其中ΔV是LFP振幅，t1是开始，t2是负峰值的时间。为了分析自发皮层活动，首先对每个受试者在5分钟自发HLCx记录期间的慢波活动（SWA）中的上升状态进行分析，以比较10到30分钟之前和之间的皮层状态；SCI后分钟。原始信号的采样率降至500；然后进行快速傅里叶变换分析，以确认记录的最大峰值频率低于1；赫兹。通过将同一频带内的功率相加，提取每个电极的频率功率（mV2）：SWA（0.1–1 Hz）、delta（1–4 Hz）、theta（4–8 Hz）、alpha（8–12 Hz）、beta（12–25 Hz）、低伽马（25–50 Hz）和高伽马（50–80 Hz）。将从层内单个电极获得的LFP功率进行平均，以获得层频率功率。最后，将每个频带的功率归一化为LFP记录的总功率（0.1–80 Hz），以获得每个频带的相对功率。此外，在60年内选择了各个up州；使用与诱发LFP反应相同的方法（Fedchyshyn&Wang[2007]）获得对照组和急性SCI的发病率。为了计算传播速度，以出现最早发作的电极为参考，速度率计算为皮质深度中的距离作为时间的函数。Statistical analyses were performed using Statistica.Ink (RRID:SCR_014213, Statsoft Ibérica, Lisbon, Portugal). The Shapiro–Wilk test was used to test normality of the distribution (P?>?0.05). If normality was violated (non-Gaussian distribution P?<?0.05, rMUA response magnitude, Fig.?3), data were transformed to a normal distribution by using the square root. Grubb's test was used to identify and remove outliers (α?=?0.05). Our main hypothesis (differences across layers) was first tested in our control data set (pre-lesion) using one-way analysis of variance (ANOVA) with Layer as the independent factor. Dependent differences between pre- and post-injury among animals and layers were determined by two-way ANOVA, with Layer as an independent factor and Time as a repeated measures factor (two levels, Pre- and Post-lesion). When significant differences in analysis of variance where found, groups were further compared using Tukey's post hoc test. For statistical analysis of the dual recording site from intact and deafferented cortices (Fig.?6), we used a two-tailed paired Student's t-test with a Bonferroni correction for multiple comparisons (P?<?0.025). The threshold for statistical significance was P?<?0.05 throughout. Group measurements are expressed as means ± standard deviation (SD). Graphs and figures were made using IgorPro (WaveMetrics, Lake Oswego, OR, USA, RRID:SCR_000325) and Adobe Illustrator (RRID: SCR_010279).采用统计学方法进行统计分析。墨水（RRID:SCR_014213，Statsoft Ibérica，葡萄牙里斯本）。Shapiro–Wilk检验用于检验分布的正态性（P>0.05）。如果违反了正态性（非高斯分布P<0.05，rMUA响应幅度，图3），则使用平方根将数据转换为正态分布。Grubb检验用于识别和去除异常值（α=0.05）。我们的主要假设（各层之间的差异）首先在我们的对照数据集（病变前）中使用单向方差分析（ANOVA）进行检验，层作为独立因素。通过双向方差分析确定动物和蛋鸡损伤前后的依赖性差异，蛋鸡作为独立因素，时间作为重复测量因素（两个水平，损伤前和损伤后）。当方差分析中发现显著差异时，使用Tukey的事后检验对各组进行进一步比较。为了对完整和去分化皮质的双重记录位点进行统计分析（图6），我们使用了双尾配对Student t检验和Bonferroni校正进行多重比较（P<0.025）。统计显著性的阈值为P< ；全程0.05。组测量值以平均值±标准偏差（SD）表示。使用IgorPro（WaveMetrics，Lake Oswego，OR，USA，RRID:SCR_000325）和Adobe Illustrator（RRID:SCR_010279）制作图表。全文超过5万字符微信字数限制无法显示全文扫描左侧二维码查看全文?The Journal of Physiology ArticleOct 01, 2021: 599 (20),4643-466910.1113/JP281901