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).

Tüm deneysel protokoller Arkansas Tıbbi Bilimler Üniversitesi (Protokol numarası # 3593) Kurumsal Hayvan Bakım ve Kullanım Komitesi tarafından onaylanmış ve bakım ve laboratuvar hayvanlarının kullanımına ilişkin Sağlık kılavuzların National Institutes ile anlaşma idi. Standart yapay beyin omurilik sıvısının hazırlanması 1. (CSF) <ol><li> Stok Çözeltisi A Hazırlanması <ol><li> kimyasal ilave edilmeden önce, temiz bir 1 L&#39;lik bir kap için distile su, 700 ml ilave edilir. </li><li> Sürekli 500 ml bir hacme karıştırılırken, NaCI 136,75 g KCI 6.99 g, MgSO 4 2.89 g, ve NaH 2 PO 4 2.83 g ekleyin. </li><li> Seyreltildikten sonra 1 L bir son hacme ulaşmak için daha fazla damıtılmış su ekleyin, her bir bileşiğin nihai konsantrasyon 117 mM NaCI, 4.69 mM KCI olduğu, 1.2 ila 4 mm MgSO, ve 1.18 mM NaH 2 PO 4. </li><li> 2 hafta boyunca 4 ° C&#39;de buzdolabında tutun. </li></ol></li><li> Stok Çözeltisi B&#39;nin Hazırlanması <ol><li> kimyasal ilave edilmeden önce, temiz bir 1 L çanağa damıtılmış 700 ml su ekleyin. </li><li> Sürekli 500 ml bir hacme karıştırılırken, D-glukoz 41.45 g NaHCO 3 41.84 g ekleyin. </li><li> Seyreltildikten sonra 1 L bir son hacme ulaşmak için daha fazla damıtılmış su ekleyin, her bir bileşiğin nihai konsantrasyon 11.5 mM D-glikoz ve 24.9 mM NaHCO 3&#39;tür. </li><li> 2 hafta boyunca 4 ° C&#39;de buzdolabında tutun. </li><li> Her deney karışımı önce 50 50 ml stok B ile stok ml sürekli karbojen (-% 5 CO2 karışımı% 95 O 2) ile oksijene ederken nihai konsantrasyon aCSF solüsyon elde etmek ve oda sıcaklığında bırakın damıtılmış su ile 1 L&#39;ye getir en az 30 dakika karıştırıldı. Sadece her bir deney başında nihai konsantrasyon aCSF hazırlayın ve günün sonunda atın. </li></ol></li></ol> Sakkaroz-yapay beyin omurilik sıvısının 2. Hazırlık(Sakroz-CSF) <ol><li> Stok Çözeltisi C hazırlanması <ol><li> kimyasal ilave edilmeden önce, temiz bir 1 L&#39;lik bir kap için distile su, 700 ml ilave edilir. </li><li> Sürekli damıtık su 500 mi karıştırılırken, sükroz, 240 g, NaHCO 3 6.55 g, KCI 0.671 g, MgCl2 4.88 g, CaCI2 0.22 g askorbik asit, 0.21 g ekleyin. </li><li> Seyreltildikten sonra 1 L bir son hacme ulaşmak için daha fazla damıtılmış su ekleyin, her bir bileşiğin nihai konsantrasyon 701,1 mM sakaroz, 78 mM NaHCO 3, 9 mM KCI, 24 mM MgCl2, 1.5 mM CaCl2 ve 1.2 mM askorbik asit . </li><li> en fazla bir hafta boyunca 4 ° C&#39;de stok çözeltisi C tutun. </li><li> Her deney karışımı önce sürekli bir karbojen (% 95 O 2-5% CO2 karışımı) ile oksijene ise son konsantrasyonda sukroz-CSF çözelti elde etmek ve oda sıcaklığında bırakın distile su, 600 ml stok C, 50 ml, 300 mi en az 30 dakika karıştırıldı. </li></ol&gt; </li></ol> 3. Dilim Hazırlık <ol><li> karbojen ile oksijene, buz içinde 100 mi sükroz aCSF çözeltisi ile temiz bir beher yerleştirilir ve 0.1 M NaOH solüsyonu birkaç damla eklenirken bir pH metre kullanılarak 7.4 pH düzeltme (zaman pH &lt;7.4) ya da 0.1 M HCI çözeltisi ( pH&gt; 7.4). </li><li> sakaroz-aCSF ile bir vibratome bir kesim odasına kadar doldurun ve oksijen. Kesme odasına bağlanmış gliserol tabanlı soğutma sistemi açın ve 0 kadar soğuması için 15 dakika bekleyin - 4 ° C. </li><li> (; &lt;50 ul son hacim ile 70 mg / kg, ip), Ketamin ile (yetişkin Zamanlı gebe Sprague-Dawley fareleri ile 12 yaş 9) yavrular anestezisi. yavru sakin olduğunda, çift o kuyruk tutam refleks yoktur edin. </li><li> yavrular başını kesmek. <ol><li> Bir karbonlu çelik neşter bıçak kullanarak geri ve forseps kullanarak yanlara cilt çekin önden uzunlamasına kafa cildi kesin. sc hareketli beyini çevreleyen kemik kesmekyanal issors ve tamamen beyin açığa çıkarın. </li><li> Sonra hızla (başlangıçta hafifçe en kaudal alanlara doğru en rostral beyni dışarı itmek için koku ampul altına) bir spatula kullanarak beyin kaldırmak. Yavaşça buzla soğutulmuş oksijenli sükroz yapay beyin omurilik sıvısı (sükroz-CSF) içine beyin itin. </li></ol></li><li> (Yarımkürede yaklaşık üçte birini öldürmesi) sağ hemisfer bir parasagital kesim olun ve manyetik içeren sagital 400 mikron bölümleri kesmek için bir vibroslicer kesme odasına sabit olacak bir metalik diske beynin kesilmiş tarafını tutkal pedunculopontine çekirdeği (PPN). bütün hücre patch-clamp kayıtları önce 45 dakika boyunca oda sıcaklığında PPN dilimleri tutun. </li></ol> PPN Dilimleri 4. Kayıtlar Gamma-bant Salınımlılığı <ol><li> Potasyum glukonat hücre içi Çözüm hazırlanması (High-K + Çözümü) <ol><li> Yerleştirmekdistile su 10 ml ile temiz bir beher buz. K + -gluconate, 90,68 mg phosphocreatine di Tris tuz, 47.66 mg HEPES, 1.5 mg EGTA, 40.58 mg Mg 2+ ATP ve 4 mg Na + 2 -GTP: Sürekli eklemek karıştırırken. </li><li> KOH 7.3 (damıtılmış su içinde 100 mM), pH ayarlama. 20 mL&#39;lik bir son hacme ulaşmak için saf su ekle. 290 mOsm - gerekli olduğunda, 280 olarak sakaroz (damıtılmış su içinde 100 mM) ile, ozmolarite ayarlayın. Seyreltildikten sonra, her bir bileşiğin nihai konsantrasyon 124 mM K + -gluconate olan, 10 mM fosfokreatin di Tris tuzu, 10 mM HEPES, 0.2 mM EGTA, 4 mM Mg2 + -ATP ve 0.3 mM, Na + 2 -GTP. </li><li> Kısım hücre 1 ml tüpler çözeltiler ve dondurularak -20 ° C&#39;de ilave edildi. günde bir kısım kullanın ve deneyler süresince 4 ° C&#39;de tutun. </li></ol></li><li> Tüm hücre yama Kelepçe Kayıtlar <ol><li> bir daldırma bölmesi dilimleri yerleştirin ve oksijenli ile (1.5 mL / dakika) ile perfüze(% 95 O2 - CO2,% 5), aşağıdaki reseptör antagonistlerini içeren CSF: Selektif NMDA reseptör antagonisti, bir 2-amino-5-phosphonovaleric asit (APV, 40 uM), rekabetçi AMP A / kainat bir glutamat reseptör antagonisti 6-siyano-7- nitrokuinoksalin-2,3-dion (CNQX, 10 uM), glisin reseptör antagonisti striknin (STR, 10 uM), belirli bir GABA A reseptör antagonisti gabazine (GBZ, 10 uM) ve sodyum kanal bloke tetrodoksin (TTX 3μM) NOT: sonuçlar ve rakamlar, bu antagonistleri toplu olarak sinaptik bloker veya SBs adlandırılır. </li><li> Kayıt yama pipetler dolgu, bir ticari olarak elde edilebilen parça pipet dolgu maddeleri kullanılarak hücre içi yüksek K + çözeltisi (Direnç 2-7 MQ 1,0 mm dış çapı ve 0.6 mm iç çap normal, ticari olarak temin edilebilir kalın duvarlı borosilikat cam kılcal yapılmış) çözelti filtre. onun amplifikatörün tutucu pipet yerleştirin. Küçük bir pos uygulaBir üç yollu vanaya bağlı bir silikon tüp kullanılarak pipet sahibine bağlı 1 ml&#39;lik şırınga ile gösterilmiş pozitif basıncı. Bir yama kelepçe amplifikatör pipet tutucu arka bağlayın. </li><li> yakın kızılötesi diferansiyel girişim kontrast optik kombine bir 4X objektif kullanılarak PPN çekirdeğe yakın bir mekanik mikromanipülatör kullanarak kayıt pipet hareket ettirin. NOT: PPN çekirdeği superior serebellar sapı ile dilimler dorsal görülebilir (SCP; akson kalın bir paket olarak gözlemlenebilir). Kayıt pipetler PPN&#39;nin yerleşmişti sapı arka ucuna dorsal hemen bulunduğu compactadaki, pars. </li><li> 40X suya daldırma lens kullanarak görüntülenmiştir iken PPN nöron ile temas halinde kayıt pipet getirin ve hızla hücre ile bir mühür formu negatif emiş uygulanır. </li><li> Üreticinin protokolü kullanılarak negatif emme sırasında pipet direnci izlemek için gerilim kelepçe mühür yazılımını kullanın. <ol><li> Ne zaman olumsuz suction yavaş yavaş artar ve pipet ucu yama-kelepçe monitör tarafından okuma direnç değerleri 80 ulaşır - hızla mV -50 ve negatif basınç serbest tutma potansiyeli değiştirmek, 100 Mohm. Sürekli nöronun membran ve elektrik ile erişim tam hücre konfigürasyonunda elde edilir yırtılma girene kadar, emme uygulamak başlayın. </li><li> gerilim kelepçe mühür yazılımı ile ölçülen erişim direnç değerleri 10 MOhm veya daha yüksek ise, o zaman küçük miktarlarda negatif emme uygulayarak devam edin. </li></ol></li><li> Kapasitans telafi gerilim kelepçe modunda ve seri direnç (yani, yavaş ve hızlı geçici hücrenin zarı kırılması sonra gözlenen). Köprü değerleri telafi hızla geçerli kelepçe kayıt moduna geçmek ve (örneğin, amplifikatör düğmesini taşımak veya computer&#39;s fareyi kullanarak otomatik kompanzasyon menüsünü tıklayın). <ol><li> PPN nöronun sürekli dinlenme monitörü zar potansiyeli u kaydediliyorüreticinin protokolünü şarkı. depolarizan veya hiperpolarizan değerlere doğru membran potansiyeli kayması istirahat, daha sonra optimum -50 mV nihai değerini tutmak için doğru akım (100 pA kadar) küçük miktarlarda kullanın. NOT: -48 mV istikrarlı istirahat membran potansiyeli (RMP) veya daha fazla hiperpolarize (yani, RMP &lt;-48 mV) ile sadece PPN nöronlar tutun. </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 nöronlar onları uyanık ya da REM uykusu sırasında değil, yavaş dalga uykusu 2,3,5,13-17 sırasında olan hayvanlardan elde in vivo kayıtları sırasında beta / gama bandı frekanslarında aksiyon potansiyelleri ateş izin içsel özelliklere sahiptir. PPN EEG kayıtları sırasında gama frekansları azaltılmış dışında yazarlar daha ön seviyelerinde olduğu beyin sapı terimleri göstermiştir. Beyin sapı lezyonları bu çekirdek bulunduğu yere posterior Ancak, PPN&#39;nin doğr...

PPN çekirdeği anatomik kaudal Mezensefalik tegmentumdaki dahildir. PPN RAS 1 önemli bir bileşenidir. PPN (uyanma, REM uykusu yani) 2 davranışsal aktif devletlerin bakım katılır. Sıçan bilateral PPN lezyonların azalır veya REM uykusunu 4 elimine ederken, kortikal EEG 3 - (40 Hz 20) in vivo PPN&#39;nin elektrik stimülasyonu hızlı salınım kaynaklı. PPN nöronların çoğunluğu beta / gama-bant frekansı aksiyon potansiyelleri ateş ederken (20-80 Hz), bazı nöronlar spontan ateşleme düşük oranlar sundu (&lt;10 Hz) 5. Ayrıca, PPN gibi motivasyon ve dikkat 6 gibi davranış diğer yönleri dahil olmak görünüyor. Doğrudan yüksek frekans (40 - 60 Hz) deserebre hayvanlarda PPN çekirdeğinin 7 elektrik stimülasyonu hareketlilik teşvik edebilir. Son yıllarda, derin beyin stimülasyonu (DBS) PPN&#39;nin sağa sola şikayet eden hastaları tedavi etmek için kullanılır olmuşturParkinson hastalığı (PH) 8 olarak yürüyüş açıkları kapsayan m bozukluklar. Önceki raporlar kare akım darbeleri 9 kullanılarak depolarize zaman neredeyse tüm PPN nöronlar gamma bant frekansı aksiyon potansiyelleri ateş göstermiştir. Çünkü mV kadar veya -25 altında kare bakliyat depolarizasyonlarından sırasında voltaj bağımlı potasyum kanallarının şiddetli aktivasyon. Bunun bir sonucu olarak, herhangi bir güçlü gama salınımlar Tetrodotoksin 10 kullanılarak aksiyon potansiyelleri nesil engelleme sonra gözlenmiştir. Böyle bir sorun atlamak için bir çaba, 1 - uzun cari rampaları depolarizan 2 sn kullanıldı. Kısmen voltaj bağımlı potasyum kanallarını inaktive ederken Rampaları yavaş yavaş, 0 mV kadar değerleri istirahat membran potansiyeli depolarize. Net gama bandı membran salınımlar 10 (-25 mV ve -0 mV arasında, yani) yüksek eşik kalsiyum kanallarının voltaj bağımlılığı penceresi içinde belirgindi. Sonuç olarak, gama bandı olduğu faaliyetlerty PPN nöronlar 9 gözlendi ve her iki P / Q ve N-tipi voltaj bağımlı kalsiyum kanalları PPN 10 gama bant salınımları oluşturmak amacıyla aktive edilmesi gerekir. bir dizi çalışma PPN nöronlarda yüksek eşik kalsiyum kanallarının yerini tespit edilmiştir. Boyaların kombinasyonunu enjekte oranlı metrik floresan görüntüleme geçerli rampalar 11 ile depolarize zaman farklı dendritler aktive edilir voltaj kapılı kalsiyum kanalları aracılığıyla geçici kalsiyum gösterdi. PPN nöronların içsel özellikleri dolayısıyla RAS ve talamokortikal döngüler arasında yüksek frekanslı titreşimli nöronal aktiviteyi uyaran, uyanma ve REM uykusu sırasında bu hücrelerin simultane aktivasyonu izin öne sürülmüştür. Bu tür uzun erimli bir etkileşim güvenilir bir şekilde sürekli olarak 12 etrafımızdaki dünya değerlendirebilecek bir beyin durumunu desteklemek için kabul edilir. Burada, biz denemeyi tarifGerekli al koşulları oluşturmak ve in vitro PPN hücrelerinde gama bandı salınımını korumak için. Bu protokol daha önce tarif edilmemiştir, ve diğer beyin alanlarında gama bandı aktivitesini aracılık içsel membran özelliklerini incelemek için grupların bir dizi yardımcı olacaktır. Ayrıca, mevcut adım y bandı aktivitesi bu hücrelerde üretilen edilemez hatalı sonuca yol açabilir.

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recording gamma band oscillations in pedunculopontine nucleus neurons

(Cı / PG çekirdeği, örneğin, centrolateral / parafascicular) PPN sinaptik efferentler birçok intralaminar talamik bölgelerin nöronal aktivitenin modüle bilinmektedir. PPN veya in vivo Cı / PG çekirdekleri ya aktivasyonu hayvanın uyarılma ve kortikal elektroansefalogram (EEG) gama bandı aktivitesinde bir artış uyarılması için tarif edilmiştir. Retiküler Aktive Sistemde gama bandı salınımları üretimi için hücresel mekanizmalar (RAS) nöronlar diğer beyinleri çekirdeklerde gama bandı salınımlarını oluşturmak için bulunanlarla aynıdır. (9 dan parasagital dilim - 25 gün eski sıçan) PPN nöronlarının akım kelepçe kayıtları sırasında, kare adımlar depolarizan kullanımı hızla -25 mV ötesinde depolarize olmaktan PPN nöronlar engelledi voltaj bağımlı potasyum kanallarını aktive. 1 enjekte - uzun cari rampaları depolarizan 2 sn yavaş yavaş PPN zar potansiyeli res depolarizeting 0 mV doğru değerleri. Ancak, enjekte depolarizan kare bakliyat rampalar tarafından oluşturulan salınımlar kıyasla genlik küçük olması gösterdi membran potansiyelinin gama-bant salınımlar oluşturdu. Bütün deneyler, voltaj bağımlı sodyum kanalı ve hızlı sinaptik reseptörleri bloke edicilerin mevcudiyetinde gerçekleştirilmiştir. Yüksek eşikli gerilime-bağlı kalsiyum kanallarının aktivasyonu PPN nöronlarda y-bant salınımlı aktivitesi altında yatan gösterilmiştir. Spesifik metodolojik ve farmakolojik müdahalelerin neden ve in vitro PPN eşikaltı gama bandı salınımını sürdürmek için gerekli araçları sağlayarak, burada açıklanmıştır.

Başlangıçta, gama salınımlar kare akım darbeleri kullanarak uyarılmış bulundu. Sinaptik bloker ve TTX huzurunda PPN nöronların Güncel kelepçe kayıt sürekli istirahat membran potansiyeli ~ -50 mV (Şekil 1A) istikrarlı tutuldu sağlamak için izlenmiştir. İki saniyelik kare akım darbeleri 200 Pa ila 600 Pa (Şekil 1A) genliği artan kayıt pipet ile yama kelepçe amplifikatör tarafından hücre enjekte edilmiştir. Membran potansiyeli ...

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

pedunculopontine çekirdeği (PPN) beyin sapı bulunur ve nöronlar maksimum uyanma ve hızlı göz hareketleri (REM) uykusu beyin devletler sırasında aktive edilir. Bu eser PPN nöronlar in vitro gama bandı eşik altı membran salınım kayıt için deneysel bir yaklaşım anlatılmaktadır.

Pedunculopontine Nucleus Nöronlar Kayıt Gama Bant Salınımlılığı

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 Görüntüleme.  University of Buenos Aires.  pedunculopontine çekirdeği (PPN) beyin sapı bulunur ve nöronlar maksimum uyanma ve hızlı göz hareketleri (REM) uykusu beyin devletler sırasında aktive edilir. Bu eser PPN nöronlar in vitro gama bandı eşik altı membran salınım kayıt için deneysel bir yaklaşım anlatılmaktadır. 

Video: Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

Dilim hazırlanması, Organotipik Doku ekimi ve suprakiazmatik nükleus Saat Gen Faaliyet Lusiferaz Kayıt

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

Nörobilim

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

Kemirgenler davranmak yılında Hareketli Silikon Problar tarafından Nöronlar Büyük ölçekli Kaydı

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Retrograd Floresan Etiketleme Belirlenen Nöronlar gelen Hedefli Hücre dışı Tek ünitesi Kayıt izin verir<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

Transgenik Balık Sağlam Beyin Tespit Edilen nöronlar Elektrik Aktivitesi Kayıt

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

Hücre içi Kaydı, Duyu Alan Haritalama ve Sülük de Kültüre Belirlenen Nöronlar Hirudo medicinalis</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Eşzamanlı Elektrofizyolojik Kayıt ve Suprakiazmatik Nucleus Nöronlar Kalsiyum Görüntüleme

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Bozulmamış neuroepithelium Fare Koku Duyu Nöronlar Delikli Patch-kelepçe Kayıt: Bir Belirlenen Odorant Reseptör ifade Nöronlar Fonksiyonel Analiz

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

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.

Hipokampal Theta Bandında Tuning<em&gt; In vitro</em&gt;: İzole edilmiş Kemirgen Protokokal Circüse Kayıt için Metodolojiler

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

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Hipokampal teta salınımlarını fareler davranışlar içinde Optogenetic sürüklenme

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

Pedunculopontine Nucleus Nöronlar Kayıt Gama Bant Salınımlılığı

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Dilim hazırlanması, Organotipik Doku ekimi ve suprakiazmatik nükleus Saat Gen Faaliyet Lusiferaz Kayıt

Kemirgenler davranmak yılında Hareketli Silikon Problar tarafından Nöronlar Büyük ölçekli Kaydı

Retrograd Floresan Etiketleme Belirlenen Nöronlar gelen Hedefli Hücre dışı Tek ünitesi Kayıt izin verir

Transgenik Balık Sağlam Beyin Tespit Edilen nöronlar Elektrik Aktivitesi Kayıt

Hücre içi Kaydı, Duyu Alan Haritalama ve Sülük de Kültüre Belirlenen Nöronlar Hirudo medicinalis

Eşzamanlı Elektrofizyolojik Kayıt ve Suprakiazmatik Nucleus Nöronlar Kalsiyum Görüntüleme

Bozulmamış neuroepithelium Fare Koku Duyu Nöronlar Delikli Patch-kelepçe Kayıt: Bir Belirlenen Odorant Reseptör ifade Nöronlar Fonksiyonel Analiz

Hipokampal Theta Bandında Tuning

Hipokampal teta salınımlarını fareler davranışlar içinde Optogenetic sürüklenme

Dopamin D1-Alfa reseptör kemirgen Nucleus Accumbens bir yenilikçi RNA Situ içinde hafiye teknoloji tarafından içinde tanımlaması