Name: recording gamma band oscillations in pedunculopontine nucleus neurons
Uploaded: 2016-09-14T15:00:00.000+00:00
Duration: 9 min 4 s
Description: Watch this Scientific Journal Video about Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons at JoVE.com

This work was supported by core facilities of the Center for Translational Neuroscience supported by NIH award P20 GM103425 and P30 GM110702 to Dr. Garcia-Rill. This work was also supported by grants from FONCYT-Agencia Nacional de Promoci&#242;n Cient&#236;fica y Tecnol&#242;gica; BID 1728 OC.AR. PICT-2012-1769 and UBACYT 2014-2017 #20120130101305BA (to Dr. Urbano).

所有的实验方案得到阿肯色大学医学科学（协议号＃3593）的机构动物护理和使用委员会批准，均符合卫生准则的国家机构的协议照顾和使用实验动物。 1.标准人工脑脊液的制备（学联） <ol><li> 原液的制备 <ol><li>添加化学品前加入700毫升蒸馏水至一个干净的1升烧杯中。 </li><li>同时连续搅拌下在500毫升体积，添加136.75克的NaCl，6.99克氯化钾，2.89克硫酸镁 ，并2.83克的NaH 2 PO 4的。 </li><li>添加更多的蒸馏水中以达到1升的最终体积稀释后，将各化合物的最终浓度为117毫摩尔NaCl，4.69毫摩尔的KCl，1.2毫硫酸镁 ，和1.18毫摩尔的NaH 2 PO 4。 </li><li>在4℃冷藏保存长达2周。 </li></ol></li><li> B原液的制备 <ol><li>添加化学品前加入700毫升蒸馏水到一个干净的1升烧杯中。 </li><li>同时连续搅拌下在500毫升体积，添加41.45克D-葡萄糖和41.84克的NaHCO 3。 </li><li>添加更多的蒸馏水中以达到1升的最终体积稀释后，将各化合物的最终浓度为11.5毫D-葡萄糖和24.9毫碳酸氢钠 。 </li><li>在4℃冷藏保存长达2周。 </li><li>每个实验混合之前50ml料A的用50ml库存B和带来到1L蒸馏水中，得到最终浓度脑脊液溶液并在RT离开而连续地卡波金（95％O 2 - 5％CO 2的混合物）充氧它至少30分钟。仅在每个实验开始制备最终浓度脑脊液和在一天结束时丢弃。 </li></ol></li></ol> 2.蔗糖人工脑脊液的制备（蔗糖学联） <ol><li> 库存溶液 C 的制备 <ol><li>添加化学品前加入700毫升蒸馏水至一个干净的1升烧杯中。 </li><li>同时连续搅拌500毫升蒸馏水，加入240克蔗糖，6.55克碳酸氢钠的，0.671克氯化钾，4.88克的 MgCl 2，0.22克CaCl 2和0.21g的抗坏血酸。 </li><li>添加更多的蒸馏水中以达到1升的最终体积稀释后，将各化合物的最终浓度为701.1毫蔗糖，78毫碳酸氢钠 ，9毫米氯化钾，24毫氯化镁 ，1.5mM的氯化钙和1.2mM的抗坏血酸。 </li><li>保持股票溶液C在4℃下长达一个星期。 </li><li>每次实验混合之前300毫升50 ml储备下用蒸馏水以获得终浓度的蔗糖学联溶液并在RT离开而连续地卡波金（95％O 2 - 5％CO 2的混合物）充氧它将600ml至少30分钟。 </li></o升&gt; </li></ol> 3.切片准备<ol><li>放置一个干净的烧杯在冰100毫升蔗糖学联溶液中，同时用卡波金充氧，并使用pH计，同时加入0.1M的NaOH溶液几滴7.4固定pH值（在pH &lt;7.4）或0.1M的HCl溶液（当pH值&gt; 7.4）。 </li><li>填补了蔗糖学联一个vibratome的切割室和氧化它。转联接到所述切割室的基于甘油的冷冻装置上，并等待15分钟，以允许它冷却到0 - 4℃。 </li><li>麻醉大鼠幼仔用氯胺酮（成年定时怀孕的Sprague-Dawley大鼠9岁至12）（70毫克/ kg， 腹腔 ;使用&lt;50微升最终体积）。当小狗很平静，仔细检查尾巴捏反射不存在。 </li><li>斩首幼崽。 <ol><li>从前面纵向切割头部皮肤用碳钢手术刀刀片向后，和皮肤拉向两侧使用镊子。切骨保护大脑移动SC横向issors，完全删除它暴露大脑。 </li><li>然后，迅速去除用刮刀（最初置于嗅球下以轻轻地从最喙朝向最尾区压出大脑）的大脑。轻轻推脑入冰冷却的含氧蔗糖人工脑脊液（蔗糖ACSF）。 </li></ol></li><li>使在右侧半球一个矢状切口（除去半球的大约三分之一），和大脑的修整侧胶上，将被磁性固定到vibroslicer的切割室切割含有矢状400微米的部分的金属盘脚桥核（PPN）。保持PPN切片在RT之前全细胞膜片钳记录45分钟。 </li></ol> 4. PPN切片录音伽玛波段振荡<ol><li> 钾细胞内葡萄糖溶液的制备（高K +解决方案） <ol><li>放入冰一个干净的烧杯用10毫升蒸馏水。在不断搅拌下加入:K + -葡萄糖，90.68毫克磷酸迪的Tris盐，47.66毫克HEPES，1.5毫克EGTA，40.58毫克镁离子 -ATP和4毫克的Na + 2 -GTP。 </li><li>调节pH至7.3，用KOH（100毫在蒸馏水中）。加蒸馏水至达到20毫升的最终体积。如果需要的话，调节摩尔渗透压浓度与蔗糖（100mM的蒸馏水中）为280 - 290毫渗透摩尔浓度。稀释后，将各化合物的最终浓度为124毫K + -葡萄糖，10mM的磷酸二的Tris盐，10毫米的HEPES，0.2mM的EGTA，4mM的镁离子 -ATP和0.3毫米的 Na + 2 -GTP。 </li><li>在1ml试管等分试样的细胞内溶液并冷冻在-20℃。使用一个等份每一天，在实验过程中保持在4℃。 </li></ol></li><li> 全细胞膜片钳记录 <ol><li>放置切片中的浸渍室和含氧灌注他们（1.5毫升/分钟）（95％ 的 O 2 - ，5％CO 2）含有下列受体拮抗剂脑脊液:选择性NMDA受体拮抗剂的2-氨基-5- phosphonovaleric酸（APV，40μM），竞争性AMPA /红藻氨酸盐型谷氨酸受体拮抗剂6-氰基-7-硝基喹喔啉-2,3-二酮（CNQX，10μM），甘氨酸受体拮抗剂士的宁（STR，10μM），具体的 GABA A受体拮抗剂gabazine（GBZ，10μM）和钠通道阻滞剂河豚毒素（TTX，3μM） 注:在结果和附图，这些拮抗剂统称为突触阻滞剂或SBS。 </li><li>填记录补丁移液器;与细胞内高-K +溶液使用与市售的膜片吸管填料（电阻2-7兆欧从常规，可商购的厚壁硼硅玻璃1.0毫米外径和0.6内径的毛细管制造）解决方案过滤器。插入其放大器的持有人的吸管。取一小POS机可持续的竞争压力用1ml注射器连接到使用连接到一个三通阀的硅管的吸移管支架。连接吸管保持器到一个膜片钳放大器的背面。 </li><li>移动使用使用4X物镜相结合，近红外微分干涉对比光学的PPN核附近的机械显微记录吸管。 注:PPN核可在背片可以观察到小脑上脚（SCP;观察到轴突的厚束）。录音移液器均位于PPN 的致密部 ，这是直接位于背侧脚的后端。 </li><li>带来与PPN神经元接触记录吸管，同时用40X水浸透镜可视化，并迅速施加负抽吸，以形成与电池的密封。 </li><li>使用电压钳密封使用的软件制造商的协议来监测负压吸引过程中吸管阻力。 <ol><li>当负小​​号，减税缓慢增加，电阻值通过在吸液管的尖端的膜片钳显示器读达到80 - 100兆欧姆，快速改变保持电位至-50毫伏并释放负压。开始连续施加负抽吸直至破裂神经元的膜和电访问在全细胞构造来实现的。 </li><li>如果由电压钳密封软件来测量接入电阻值为10兆欧或更高，然后继续在较小量施加负吸力。 </li></ol></li><li>补偿电容和串联电阻在电压钳模式（ 即 ，慢速和快速瞬变破裂细胞的细胞膜后观察到的）。切换录音模式电流钳，并迅速弥补桥值（ 例如 ，移动放大器旋钮或按使用鼠标computer&#39;s自动补偿菜单上）。 <ol><li> PPN神经元的连续监测静息电位被记录ū唱制造商的协议。如果休息朝去极化或超极化值的膜电位的移位，然后使用少量的直流（高达100帕），以保持最佳-50毫伏最终值。 注:仅保留PPN神经元-48 mV的稳定的静息膜电位（RMP）以上超极化（ 即 RMP &lt;-48 mV的）。 </li></ol></li></ol></li></ol>

<ol>
	<li>Profice, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurophysiological+evaluation+of+the+pedunculopontine+nucleus+in+humans.">Neurophysiological evaluation of the pedunculopontine nucleus in humans.</a> J. Neural. Transm (Vienna). 118 (10), 1423-1429 (2011).</li><li>Steriade, M., Datta, S., Pare, D., Oakson, G., Curro Dossi, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+activities+in+brain-stem+cholinergic+nuclei+related+to+tonic+activation+processes+in+thalamocortical+systems.">Neuronal activities in brain-stem cholinergic nuclei related to tonic activation processes in thalamocortical systems.</a> J. Neurosci. 10 (8), 2541-2559 (1990).</li><li>Steriade, M., Dossi, R. C., Pare, D., Oakson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+oscillations+(20-40+Hz)+in+thalamocortical+systems+and+their+potentiation+by+mesopontine+cholinergic+nuclei+in+the+cat.">Fast oscillations (20-40 Hz) in thalamocortical systems and their potentiation by mesopontine cholinergic nuclei in the cat.</a> Proc. Natl. Acad. Sci. U.S.A. 88 (10), 4396-4400 (1991).</li><li>Deurveilher, S., Hennevin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lesions+of+the+pedunculopontine+tegmental+nucleus+reduce+paradoxical+sleep+(PS)+propensity:+evidence+from+a+short-term+PS+deprivation+study+in+rats.">Lesions of the pedunculopontine tegmental nucleus reduce paradoxical sleep (PS) propensity: evidence from a short-term PS deprivation study in rats.</a> Eur. J. Neurosci. 13 (10), 1963-1976 (2001).</li><li>Steriade, M., Pare, D., Datta, S., Oakson, G., Curro Dossi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+cellular+types+in+mesopontine+cholinergic+nuclei+related+to+ponto-geniculo-occipital+waves.">Different cellular types in mesopontine cholinergic nuclei related to ponto-geniculo-occipital waves.</a> J. Neurosci. 10 (8), 2560-2579 (1990).</li><li>Steckler, T., Inglis, W., Winn, P., Sahgal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pedunculopontine+tegmental+nucleus:+a+role+in+cognitive+processes?.">The pedunculopontine tegmental nucleus: a role in cognitive processes?.</a> Brain Res. Brain Res. Rev. 19 (3), 298-318 (1994).</li><li>Garcia-Rill, E., Simon, C., Smith, K., Kezunovic, N., Hyde, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pedunculopontine+tegmental+nucleus:+from+basic+neuroscience+to+neurosurgical+applications:+arousal+from+slices+to+humans:+implications+for+DBS.">The pedunculopontine tegmental nucleus: from basic neuroscience to neurosurgical applications: arousal from slices to humans: implications for DBS.</a> J. Neural. Transm. 118 (10), 1397-1407 (2011).</li><li>Mazzone, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantation+of+human+pedunculopontine+nucleus:+a+safe+and+clinically+relevant+target+in+Parkinson's+disease.">Implantation of human pedunculopontine nucleus: a safe and clinically relevant target in Parkinson's disease.</a> Neuroreport. 16 (17), 1877-1881 (2005).</li><li>Simon, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamma+band+unit+activity+and+population+responses+in+the+pedunculopontine+nucleus.">Gamma band unit activity and population responses in the pedunculopontine nucleus.</a> J. Neurophysiol. 104 (1), 463-474 (2010).</li><li>Kezunovic, N., Urbano, F. J., Simon, C., Hyde, J., Smith, K., Garcia-Rill, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+behind+gamma+band+activity+in+pedunculopontine+nucleus+(PPN).">Mechanism behind gamma band activity in pedunculopontine nucleus (PPN).</a> Eur. J. Neurosci. 34 (3), 404-415 (2011).</li><li>Hyde, J. R., Kezunovic, N., Urbano, F. J., Garcia-Rill, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+properties+of+high+speed+calcium+oscillations+in+the+pedunculopontine+nucleus.">Spatiotemporal properties of high speed calcium oscillations in the pedunculopontine nucleus.</a> J. Appl. Physiol (1985). 115 (9), 1402-1414 (2013).</li><li>Llinas, R. R., Leznik, E., Urbano, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+binding+via+cortical+coincidence+detection+of+specific+and+nonspecific+thalamocortical+inputs:+a+voltage-dependent+dye-imaging+study+in+mouse+brain+slices.">Temporal binding via cortical coincidence detection of specific and nonspecific thalamocortical inputs: a voltage-dependent dye-imaging study in mouse brain slices.</a> Proc. Natl. Acad. Sci. U.S.A. 99 (1), 449-454 (2002).</li><li>Boucetta, S., Cisse, Y., Mainville, L., Morales, M., Jones, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discharge+profiles+across+the+sleep-waking+cycle+of+identified+cholinergic,+gabaergic,+and+glutamatergic+neurons+in+the+pontomesencephalic+tegmentum+of+the+rat.">Discharge profiles across the sleep-waking cycle of identified cholinergic, gabaergic, and glutamatergic neurons in the pontomesencephalic tegmentum of the rat.</a> J. Neurosci. 34 (13), 4708-4727 (2014).</li><li>Datta, S., Siwek, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+activity+patterns+of+pedunculopontine+tegmentum+neurons+across+the+sleep-wake+cycle+in+the+freely+moving+rats.">Single cell activity patterns of pedunculopontine tegmentum neurons across the sleep-wake cycle in the freely moving rats.</a> J. Neurosci. Res. 70 (4), 79-82 (2002).</li><li>Datta, S., Siwek, D. F., Stack, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+cholinergic+and+non-cholinergic+neurons+in+the+pons+expressing+phosphorylated+cyclic+adenosine+monophosphate+response+element-binding+protein+as+a+function+of+rapid+eye+movement+sleep.">Identification of cholinergic and non-cholinergic neurons in the pons expressing phosphorylated cyclic adenosine monophosphate response element-binding protein as a function of rapid eye movement sleep.</a> Neuroscience. 163 (1), 397-414 (2009).</li><li>Kayama, Y., Ohta, M., Jodo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Firing+of+'possibly'+cholinergic+neurons+in+the+rat+laterodorsal+tegmental+nucleus+during+sleep+and+wakefulness.">Firing of 'possibly' cholinergic neurons in the rat laterodorsal tegmental nucleus during sleep and wakefulness.</a> Brain Res. 569 (2), 210-220 (1992).</li><li>Sakai, K., El Mansari, M., Jouvet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+by+carbachol+microinjections+of+presumptive+cholinergic+PGO-on+neurons+in+freely+moving+cats.">Inhibition by carbachol microinjections of presumptive cholinergic PGO-on neurons in freely moving cats.</a> Brain Res. 527 (2), 213-223 (1990).</li><li>Lindsley, D. B., Bowden, J. W., Magoun, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+upon+the+EEG+of+acute+injury+to+the+brainstem+activating+system.">Effect upon the EEG of acute injury to the brainstem activating system.</a> Electroenceph. Clin. Neurophysiol. 1 (4), 475-486 (1949).</li><li>Moruzzi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sleep-waking+cycle.">The sleep-waking cycle.</a> Ergeb. Physiol. 64, 1-165 (1972).</li><li>Moruzzi, G., Magoun, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+stem+reticular+formation+and+activation+of+the+EEG.">Brain stem reticular formation and activation of the EEG.</a> Electroenceph. Clin. Neurophysiol. 1 (4), 455-473 (1949).</li><li>Steriade, M., Constantinescu, E., Apostol, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlations+between+alterations+of+the+cortical+transaminase+activity+and+EEG+patterns+of+sleep+and+wakefulness+induced+by+brainstem+transections.">Correlations between alterations of the cortical transaminase activity and EEG patterns of sleep and wakefulness induced by brainstem transections.</a> Brain Res. 13 (1), 177-180 (1969).</li><li>Ishibashi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orexin+receptor+activation+generates+gamma+band+input+to+cholinergic+and+serotonergic+arousal+system+neurons+and+drives+an+intrinsic+Ca2++-dependent+resonance+in+LDT+and+PPT+cholinergic+neurons.">Orexin receptor activation generates gamma band input to cholinergic and serotonergic arousal system neurons and drives an intrinsic Ca2+ -dependent resonance in LDT and PPT cholinergic neurons.</a> Frontiers Neurol. 6, e120 (2015).</li><li>Brown, R. E., Winston, S., Basheer, R., Thakkar, M. M., McCarley, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+characterization+of+neurons+in+the+dorsolateral+pontine+REM+sleep+induction+zone+of+the+rat:+intrinsic+membrane+properties+and+responses+to+carbachol+and+orexins.">Electrophysiological characterization of neurons in the dorsolateral pontine REM sleep induction zone of the rat: intrinsic membrane properties and responses to carbachol and orexins.</a> Neuroscience. 143 (3), 739-755 (2006).</li><li>Goetz, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+role+of+the+pedunculopontine+nucleus+and+mesencephalic+reticular+formation+in+locomotion+in+non-human+primates.">On the role of the pedunculopontine nucleus and mesencephalic reticular formation in locomotion in non-human primates.</a> J. Neurosci. 36 (18), 4917-4929 (2016).</li><li>Fraix, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pedunculopontine+nucleus+area+oscillations+during+stance,+stepping+and+freezing+in+Parkinson's+disease.">Pedunculopontine nucleus area oscillations during stance, stepping and freezing in Parkinson's disease.</a> PLOS ONE. 8 (12), e83919 (2013).</li><li>Luster, B., Hyde, J., D'Onofrio, S., Urbano, F. J., Garcia-Rill, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+behind+gamma+band+activity+in+the+pedunculopontine+nucleus+(PPN).">Mechanisms behind gamma band activity in the pedunculopontine nucleus (PPN).</a> Abstr Soc Neurosci. 38, 257.20 (2014).</li><li>Luster, B., D'Onofrio, S., Urbano, F. J., Garcia-Rill, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-threshold+Ca2++channels+behind+gamma+band+activity+in+the+pedunculopontine+nucleus+(PPN).">High-threshold Ca2+ channels behind gamma band activity in the pedunculopontine nucleus (PPN).</a> Physiol. Rep. 3 (6), e12431 (2015).</li></ol>

The authors declare that they have no competing financial interests.

PPN神经元有内在属性，使他们在从动物醒着REM睡眠过程中，而不是在慢波睡眠2,3,5,13-17 体内录音开火β/γ频段频率动作电位。其他作者发现，脑干横断在更前水平在脑电图记录比减少PPN伽玛频率。然而，当脑干病变后路到这个核心所在，PPN的直接刺激所允许的EEG 2,3,5,18-21皮质伽玛活动的表现。伽马带神经活动已经报道在小鼠PPN 体外 22，在大鼠REM睡眠诱导区域...

PPN核被解剖列入尾脑被盖。该PPN是RAS 1的重要组成部分。该PPN参与维护行为激活状态（ 即清醒，REM睡眠）2。 体内 PPN电刺激诱 ​​发的快速振荡（20 - 40赫兹）在皮层脑电图3，而双侧病变PPN在大鼠中减少或消除REM睡眠4。虽然大多数PPN神经元开火β/γ波段频率动作电位（20 - 80赫兹），一些神经元呈现自发放电率较低（&lt;10赫兹）5。此外，PPN似乎参与的行为其他方面，如动机和关注6。直接高频（40 - 60赫兹）在去大脑动物PPN核7电刺激可以促进运动。近年来，深部脑刺激（DBS）PPN的已被用于治疗来回患者涉及步态缺陷米障碍，例如帕金森氏病（PD）8。 以前的报告显示，几乎所有的神经元PPN可以用平方电流脉冲去极化9时在伽马射线波段频率发射动作电位。因为电压 - 门控钾通道的过程中方形脉冲去极化到或在-25毫伏的急剧的活化。其结果是，阻止使用河豚毒素10动作电位产生之后没有观察到鲁棒伽马振荡。在努力绕过这样的问题，1 - 2秒长的去极化电流斜坡中使用。坡道从休息值高达0毫伏逐渐去极化膜电位，而部分失活的电压 - 门控钾通道。清除伽玛带膜振荡高门槛钙通道的电压依赖性窗口内是明显的（ 即 -25 mV至-0之间毫伏）10。总之，γ-带活性状况TY在PPN神经元9观察到的，和两个P / Q-和N型电压门控钙通道需要，以便产生在PPN 10伽玛波段振荡被激活。 一系列的研究确定了PPN神经元的高门槛钙通道的位置。注射染料的组合， 比例荧光成像通过在不同的树突激活使用电流斜11去极化时的电压门控钙通道表明钙瞬变。 PPN神经元的固有特性已建议允许唤醒和REM睡眠，从而诱发RAS和丘脑皮层环路之间高频振荡神经元活动中这些细胞的同时激活。这种长期深远的互动被认为是支持能够在连续的基础上12可靠地评估我们周围的世界的大脑状态。这里，我们描述了实验必要人条件，以产生和维持伽马频带振荡PPN细胞在体外 。该协议还没有被先前所描述，并有助于若干组研究固有膜性能在其他脑区介导伽马带活性。此外，目前的步骤可能会导致错误的结论伽马射线波段活动不能在这些细胞中产生。

<table><tbody><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S8501</td><td>C&lt;sub&gt;12&lt;/sub&gt;H&lt;sub&gt;22&lt;/sub&gt;O&lt;sub&gt;11&lt;/sub&gt;, molecular weight = 342.30</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma-Aldrich</td><td>S6014</td><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;, molecular weight = 84.01</td></tr><tr><td>Potassium Chloride</td><td>Sigma-Aldrich</td><td>P3911</td><td>KCl, molecular weight = 74.55</td></tr><tr><td>Magnesium Chloride Hexahydrate</td><td>Sigma-Aldrich</td><td>M9272</td><td>MgCl&lt;sub&gt;2&lt;/sub&gt; &amp;middot; 6H&lt;sub&gt;2&lt;/sub&gt;O, molecular weight =&amp;nbsp; 203.30</td></tr><tr><td>Calcium Chloride Dihydrate</td><td>Sigma-Aldrich</td><td>C3881</td><td>CaCl&lt;sub&gt;2&lt;/sub&gt; &amp;middot; 2H&lt;sub&gt;2&lt;/sub&gt;O, molecular weight =147.02</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G5767</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;12&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;, molecular weight = 180.16</td></tr><tr><td>L-Ascorbic Acid</td><td>Sigma-Aldrich</td><td>A5960</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;8&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;, molecular weight =176.12</td></tr><tr><td>Sodium Chloride</td><td>Acros Organics</td><td>327300025</td><td>NaCl, molecular weight =&amp;nbsp; 58.44</td></tr><tr><td>Potassium Gluconate</td><td>Sigma-Aldrich</td><td>G4500</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;11&lt;/sub&gt;KO&lt;sub&gt;7&lt;/sub&gt;, molecular weight =&amp;nbsp; 234.25</td></tr><tr><td>Phosphocreatine di(tris) salt</td><td>Sigma-Aldrich</td><td>P1937</td><td>C&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;10&lt;/sub&gt;N&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;5&lt;/sub&gt;P &amp;middot; 2C&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;11&lt;/sub&gt;NO&lt;sub&gt;3&lt;/sub&gt;, molecular weight =&amp;nbsp; 453.38</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td>C&lt;sub&gt;8&lt;/sub&gt;H&lt;sub&gt;18&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt;S, molecular weight = 238.30</td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E0396</td><td>[-CH&lt;sub&gt;2&lt;/sub&gt;OCH&lt;sub&gt;2&lt;/sub&gt;CH&lt;sub&gt;2&lt;/sub&gt;N(CH&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;2&lt;/sub&gt;H)&lt;sub&gt;2&lt;/sub&gt;]&lt;sub&gt;2&lt;/sub&gt;, molecular weight = 380.40</td></tr><tr><td>Adenosine 5&#039;-triphosphate magnesium salt</td><td>Sigma-Aldrich</td><td>A9187</td><td>&amp;nbsp;C&lt;sub&gt;10&lt;/sub&gt;H&lt;sub&gt;16&lt;/sub&gt;N&lt;sub&gt;5&lt;/sub&gt;O&lt;sub&gt;13&lt;/sub&gt;P&lt;sub&gt;3&lt;/sub&gt; &amp;middot; xMg2+, molecular weight = 507.18</td></tr><tr><td>Guanosine 5&#039;-triphosphate sodium salt hydrate</td><td>Sigma-Aldrich</td><td>G8877</td><td>C&lt;sub&gt;10&lt;/sub&gt;H&lt;sub&gt;16&lt;/sub&gt;N&lt;sub&gt;5&lt;/sub&gt;O&lt;sub&gt;14&lt;/sub&gt;P&lt;sub&gt;3&lt;/sub&gt; &amp;middot; xNa+, molecular weight = 523.18</td></tr><tr><td>Tetrodotoxin citrate</td><td>Alomone Labs</td><td>T-550</td><td>C&lt;sub&gt;11&lt;/sub&gt;H&lt;sub&gt;17&lt;/sub&gt;N&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;8&lt;/sub&gt;, molecular weight = 319.27</td></tr><tr><td>&amp;nbsp;DL-2-Amino-5-Phosphonovaleric Acid</td><td>Sigma-Aldrich</td><td>A5282</td><td>&amp;nbsp;C&lt;sub&gt;5&lt;/sub&gt;H&lt;sub&gt;12&lt;/sub&gt;NO&lt;sub&gt;5&lt;/sub&gt;P, molecular weight = 197.13</td></tr><tr><td>CNQX disodium salt hydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C239</td><td>C&lt;sub&gt;9&lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;N&lt;sub&gt;4&lt;/sub&gt;Na&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt; &amp;middot; xH2O, molecular weight = 276.12</td></tr><tr><td>Strychnine</td><td>Sigma-Aldrich</td><td>S0532</td><td>C&lt;sub&gt;21&lt;/sub&gt;H&lt;sub&gt;22&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;, molecular weight = 334.41</td></tr><tr><td>Mecamylamine hydrochloride</td><td>Sigma-Aldrich</td><td>M9020</td><td>&amp;nbsp;C&lt;sub&gt;11&lt;/sub&gt;H&lt;sub&gt;21&lt;/sub&gt;N &amp;middot; HCl, molecular weight = 203.75</td></tr><tr><td>Gabazine (SR-95531)</td><td>Sigma-Aldrich</td><td>S106</td><td>C&lt;sub&gt;15&lt;/sub&gt;H&lt;sub&gt;18&lt;/sub&gt;BrN&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt;, molecular weight = 368.23</td></tr><tr><td>Ketamine hydrochloride</td><td>Mylan</td><td>67457-001-00</td><td></td></tr><tr><td>Microscope</td><td>Nikon</td><td>Eclipse E600FN</td><td></td></tr><tr><td>Micromanipulator</td><td>Sutter Instruments</td><td>ROE-200</td><td></td></tr><tr><td>Micromanipulator</td><td>Sutter Instruments</td><td>MPC-200</td><td></td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>Multiclamp 700B</td><td></td></tr><tr><td>A/D converter</td><td>Molecular Devices</td><td>Digidata 1440A</td><td></td></tr><tr><td>Heater</td><td>Warner Instruments</td><td>TC-324B</td><td></td></tr><tr><td>Pump</td><td>Cole-Parmer</td><td>Masterflex L/S 7519-20</td><td></td></tr><tr><td>Pump cartridge</td><td>Cole-Parmer</td><td>Masterflex 7519-85</td><td></td></tr><tr><td>Pipette puller</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr><tr><td>Camera</td><td>Q-Imaging</td><td>RET-200R-F-M-12-C</td><td></td></tr><tr><td>Vibratome</td><td>Leica Biosystems</td><td>&amp;nbsp;Leica VT1200 S</td><td></td></tr><tr><td>Refrigeration system</td><td>Vibratome Instruments</td><td>900R</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>microscope</td><td>Nikon</td><td>Eclipse E600FN</td><td></td></tr><tr><td>micromanipulator</td><td>Sutter Instruments</td><td>ROE-200</td><td></td></tr><tr><td>micromanipulator</td><td>Sutter Instruments</td><td>MPC-200</td><td></td></tr><tr><td>amplifier</td><td>Molecular Devices</td><td>Multiclamp 700B</td><td></td></tr><tr><td>A/D converter</td><td>Molecular Devices</td><td>Digidata 1440A</td><td></td></tr><tr><td>heater</td><td>Warner Instruments</td><td>TC-324B</td><td></td></tr><tr><td>pump</td><td>Cole-Parmer</td><td>Masterflex L/S 7519-20</td><td></td></tr><tr><td>pump cartridge</td><td>Cole-Parmer</td><td>Masterflex 7519-85</td><td></td></tr><tr><td>pipette puller</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr><tr><td>camera</td><td>Q-Imaging</td><td>RET-200R-F-M-12-C</td><td></td></tr></tbody></table>

recording gamma band oscillations in pedunculopontine nucleus neurons

从PPN突触传出已知的几种调节丘脑板内核区域的神经元活动（ 例如 ，centrolateral /束旁;氯/ PF核）。无论是PPN 或体内氯/ Pf的核的活化已经描述以诱 ​​导动物的觉醒和伽玛频带活性皮质脑电图（EEG）的增量。在网状活化系统伽马频带的振荡的产生的细胞机制（RAS）的神经元的那些相同发现生成其他大脑原子核伽玛波段振荡。期间PPN神经元的电流钳记录（从9矢状切片 - 40日龄鼠），使用去极化平方步骤的快速激活电压依赖性钾通道，阻止PPN神经元被去极化超出-25毫伏。 注射1 - 2秒长的去极化电流逐渐逐渐去极化PPN膜电位资源婷对0毫伏值。然而，注射去极化方形脉冲产生的膜电位的伽玛波段振荡即表明要与由斜面所产生的振动的振幅变小。所有实验都在电压 - 门控钠通道和快速突触受体阻断剂的存在下进行。它已经表明，高阈值电压依赖性钙通道的活化背后伽马波段振荡活性PPN神经元。具体的方法和药物干预在这里描述的，提供了必要的工具来诱导和维持PPN阈下伽玛波段振荡体外 。

最初，伽马振荡采用方形电流脉冲引起的。突触阻滞剂和TTX的存在PPN神经元的电流钳记录连续监测，以确保静息膜电位在约-50 mV的（ 图1A）保持稳定。两个第二长方形的电流脉冲是由通过记录吸管膜片钳放大器细胞内注射，增加其幅度从200 pA的600 PA（ 图1A）。膜电位去极化电压等级接近-20 mV的时候600 pA的，造成小幅度的伽马振荡。伽玛振荡的权力...

Watch this Scientific Journal Video about Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons at JoVE.com

Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

脚桥核（PPN）位于脑干和神经元在清醒和快速眼动（REM）睡眠大脑状态最大限度地激活。这部作品描述了实验方法在 PPN神经元体外伽玛带阈下膜振荡记录。

录音伽玛乐队在振荡脚桥核神经元

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the ...

recording-gamma-band-oscillations-in-pedunculopontine-nucleus-neurons

Research

JoVE Journal

Neuroscience

8.6K 次观看.  University of Buenos Aires. 脚桥核（PPN）位于脑干和神经元在清醒和快速眼动（REM）睡眠大脑状态最大限度地激活。这部作品描述了实验方法在 PPN神经元体外伽玛带阈下膜振荡记录。 

视频: Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

In vivo recordings from single neurons allow an investigator to examine the firing properties of neurons, for example in response to sensory stimuli. Neurons typically receive multiple excitatory and inhibitory afferent and/or efferent inputs that integrate with each other, and the ultimate measured response properties of the neuron are driven by the neural integrations of these inputs. To study information processing in neural systems, it is necessary to understand the various inputs to a neuron or neural system, and the specific properties of these inputs. A powerful and technically relatively simple method to assess the functional role of certain inputs that a given neuron is receiving is to dynamically and reversibly suppress or eliminate these inputs, and measure the changes in the neuron's output caused by this manipulation. This can be accomplished by pharmacologically altering the neuron's immediate environment with piggy-back multibarrel electrodes. These electrodes consist of a single barrel recording electrode and a multibarrel drug electrode that can carry up to 4 different synaptic agonists or antagonists. The pharmacological agents can be applied iontophoretically at desired times during the experiment, allowing for time-controlled delivery and reversible reconfiguration of synaptic inputs. As such, pharmacological manipulation of the microenvironment represents a powerful and unparalleled method to test specific hypotheses about neural circuit function.
Here we describe how piggy-back electrodes are manufactured, and how they are used during in vivo experiments. The piggy-back system allows an investigator to combine a single barrel recording electrode of any arbitrary property (resistance, tip size, shape etc) with a multibarrel drug electrode. This is a major advantage over standard multi-electrodes, where all barrels have more or less similar shapes and properties. Multibarrel electrodes were first introduced over 40 years ago 1-3, and have undergone a number of design improvements 2,3 until the piggy-back type was introduced in the 1980s 4,5. Here we present a set of important improvements in the laboratory production of piggy-back electrodes that allow for deep brain penetration in intact in vivo animal preparations due to a relatively thin electrode shaft that causes minimal damage. Furthermore these electrodes are characterized by low noise recordings, and have low resistance drug barrels for very effective iontophoresis of the desired pharmacological agents.

Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

Iontophoresis of neural agonists and antagonists during extracellular in vivo recordings is a powerful way to manipulate a neuron&#x2019;s microenvironment. These manipulations can most easily be done via piggy-back multibarrel electrodes. Here we describe how to manufacture them and use them during auditory recordings.

生产和使用背驮式多管电极<em在体内</em&gt;药理手法的神经反应

In vivo recordings from single neurons allow an investigator to examine the firing properties of neurons, for example in response to sensory stimuli. ...

科研

神经科学

Generation of Local CA1 &#947; Oscillations by Tetanic Stimulation

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A protocol is provided to generate a model for studying CA1 &#947; oscillations (20 - 80 Hz). These &#947; oscillations are stable for at least 30 min&#160;and depend upon excitatory and inhibitory synaptic activity in addition to activation of pacemaker currents. Tetanically stimulated oscillations have a number of reproducible and easily quantifiable characteristics including spike count, oscillation duration, latency and frequency that report upon the network state. The advantages of the electrically stimulated oscillations include stability, reproducibility and episodic acquisition enabling robust characterization of network function. This model of CA1 &#947; oscillations can be used to study cellular mechanisms and to systematically investigate how neuronal network activity is altered in disease and by drugs. Disease state pharmacology can be readily incorporated by the use of brain slices from genetically modified or interventional animal models to enable selection of drugs that specifically target disease mechanisms.

Oscillations are fundamental network properties and are modulated by disease and drugs. Studying brain-slice oscillations allows characterization of isolated networks under controlled conditions. Protocols are provided for the preparation of acute brain slices for evoking CA1 &#947; oscillations.

本地CA1γ振荡通过强直刺激生成

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A ...

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent improvements in methodologies and applications for the study of theta oscillations. A simple characterization of the isolated hippocampal preparation is presented whereby the relationship between internal hippocampal theta oscillators is examined together with the activity of pyramidal cells, and GABAergic interneurons, of the cornu ammonis-1 (CA1) and subiculum (SUB) areas. Overall, we show that the isolated hippocampus is capable of generating intrinsic theta oscillations in vitro and that rhythmicity generated within the hippocampus can be precisely manipulated by optogenetic stimulation of parvalbumin-positive (PV) interneurons. The in vitro isolated hippocampal preparation offers a unique opportunity to use simultaneous field and intracellular patch-clamp recordings from visually-identified neurons to better understand the mechanisms underlying theta rhythm generation.

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

Here, we present a protocol for recording rhythmic neuronal network theta and gamma oscillations from an isolated whole hippocampal preparation. We describe the experimental steps from extraction of the hippocampus to details of field, unitary and whole-cell patch clamp recordings as well as optogenetic pacing of the theta rhythm.

调整在海马Theta乐队<em&gt;体外</em&gt;:从孤立的啮齿动物皮下注射电路记录的方法

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

急性<em&gt;体内</em&gt;麻醉大鼠超导途径局部场电位和多单位活动的电生理记录

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Concurrent Recording of Co-localized Electroencephalography and Local Field Potential in Rodent

Although electroencephalography (EEG) is widely used as a non-invasive technique for recording neural activities of the brain, our understanding of the neurogenesis of EEG is still very limited. Local field potentials (LFPs) recorded via a multi-laminar microelectrode can provide a more detailed account of simultaneous neural activity across different cortical layers in the neocortex, but the technique is invasive. Combining EEG and LFP measurements in a pre-clinical model can greatly enhance understanding of the neural mechanisms involved in the generation of EEG signals, and facilitate the derivation of a more realistic and biologically accurate mathematical model of EEG. A simple procedure for acquiring concurrent and co-localized EEG and multi-laminar LFP signals in the anesthetized rodent is presented here. We also investigated whether EEG signals were significantly affected by a burr hole drilled in the skull for the insertion of a microelectrode. Our results suggest that the burr hole has a negligible impact on EEG recordings.

This protocol describes a simple method for concurrent recording of co-localized electroencephalography (EEG) and multi-laminar local field potential in an anesthetized rat. A burr hole drilled in the skull for the insertion of a microelectrode is shown to produce negligible distortion of the EEG signal.

啮齿动物联合局部脑电图和局部电位的同步记录

Although electroencephalography (EEG) is widely used as a non-invasive technique for recording neural activities of the brain, our understanding of the ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

鼠脑切片偶联微电极阵列癫痫活性的记录与调控

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

行为小鼠海马空间受限振荡的记录

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

基于光二极管的光学成像，用于记录非转基因无脊椎动物的单神经元分辨率的网络动态

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...

生物学

Recording Gamma Band Oscillations From Pedunculopontine Nucleus Neurons in a Brain Slice

Source: Urbano, F. J., et al. <a href="https://app.jove.com/v/54685">Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons.</a> J. Vis. Exp. (2016).
The video demonstrates the method to record gamma-band oscillations in PPN neurons. Using a sagittal brain section with synaptic blockers in aCSF, it employs positive current pulses via a patch pipette to open calcium channels, inducing crucial subthreshold activity for wakefulness.

记录脑切片中带蒂核神经元的伽马波段振荡

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

In Vivo Electrophysiological Recordings of Brain Activities from Multiple Sites in Rats

Source: Haumesser, J. K., et al. <a href="https://app.jove.com/v/55940">Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats.</a> J. Vis. Exp.(2017).
This video demonstrates the detailed procedure for preparing an anesthetized rat for electrophysiological recordings of brain activity from multiple sites. The scalp is dissected to access the skull, and holes are drilled at precise locations to install and secure electrodes. Tungsten microwire electrodes are then inserted into the targeted location to record the data.

录音伽玛乐队在振荡脚桥核神经元

摘要

探索更多视频

此视频中的章节

生产和使用背驮式多管电极