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).

Todos os protocolos experimentais foram aprovados pelo Comitê de Cuidado e Uso Institucional Animal da Universidade de Arkansas para Ciências Médicas (número Protocolo # 3593) e estavam de acordo com os Institutos Nacionais de Saúde diretrizes para o cuidado e uso de animais de laboratório. 1. Preparação do líquido cefalorraquidiano Standard-artificial (aCSF) <ol><li> Preparação da Solução A de <ol><li> Adicionar 700 ml de água destilada para uma proveta de 1 L limpo antes da adição de produtos químicos. </li><li> Enquanto agitando continuamente um volume de 500 ml, adicionar 136,75 g de NaCl, 6,99 g de KCl, 2,89 g de MgSO4, e 2,83 g de NaH 2 PO 4. </li><li> Adicionar mais água destilada para atingir um volume final de 1 L. Depois da diluição, a concentração final de cada composto é de NaCl a 117 mM, KCl 4,69 mM, 1,2 mM de MgSO4, e 1,18 mM de NaH 2 PO 4. </li><li> Manter refrigerado a 4 ° C durante até 2 semanas. </li></ol></li><li> Preparação de Solução Stock B <ol><li> Adicionar 700 ml de água destilada para uma proveta de 1 L limpo antes da adição de produtos químicos. </li><li> Enquanto agitando continuamente um volume de 500 ml, adicionar 41,45 g de D-glucose e 41,84 g de NaHCO3. </li><li> Adicionar mais água destilada para atingir um volume final de 1 L. Depois da diluição, a concentração final de cada composto é de 11,5 mM de D-glucose 24,9 mM e NaHCO 3. </li><li> Manter refrigerado a 4 ° C durante até 2 semanas. </li><li> Antes de cada mix experimento 50 ml de estoque A com 50 ml estoque B e trazer para 1 L com água destilada para obter solução final concentração aCSF e deixar à temperatura ambiente, enquanto oxigenando-o continuamente com carbogênio (95% O 2 - 5% de CO 2 mix) durante pelo menos 30 min. Prepare aCSF concentração final apenas no início de cada experiência e descartar no final do dia. </li></ol></li></ol> 2. Preparação de líquido cefalorraquidiano sacarose-artificial(Sacarose-aCSF) <ol><li> Preparação de Solução Stock C <ol><li> Adicionar 700 ml de água destilada para uma proveta de 1 L limpo antes da adição de produtos químicos. </li><li> Enquanto agitando continuamente a 500 ml de água destilada, adicionar 240 g de sacarose, 6,55 g de NaHCO3, 0,671 g de KCl, 4,88 g de MgCl2, 0,22 g de CaCl 2 e 0,21 g de ácido ascórbico. </li><li> Adicionar mais água destilada para atingir um volume final de 1 L. Depois da diluição, a concentração final de cada composto é sacarose 701.1 mM, 78 mM de NaHCO 3, 9 mM de KCl, 24 mM de MgCl 2, CaCl 1,5 mM ácido ascórbico 1,2 mM e 2 . </li><li> Mantenha a solução estoque C a 4 ° C durante até uma semana. </li><li> Antes de cada mistura experimental 300 ml de 50 ml de estoque de C com 600 ml de água destilada para se obter a solução de sacarose-aCSF na concentração final e deixar à temperatura ambiente durante a oxigenação continuamente com carbogénio (95% O2 - 5% CO 2 mix) durante pelo menos 30 min. </li></ol&gt; </li></ol> 3. Preparação Slice <ol><li> Coloque um copo limpo com 100 ml de solução de sacarose-aCSF em gelo enquanto oxigenar com carbogénio e fixar o pH em 7,4 usando um medidor de pH durante a adição de algumas gotas de solução de NaOH 0,1 M (quando o pH &lt;7,4) ou uma solução 0,1 M de HCl (quando pH&gt; 7,4). </li><li> Encha-se de uma câmara de corte de um vibratome com sacarose-aCSF e oxigenar-lo. Ligue o sistema de refrigeração à base de glicerol acoplado à câmara de corte e esperar 15 minutos para permitir que ele esfriar, 0 - 4 ° C. </li><li> Anestesiar ratos jovens (com idade de 9 a 12 a partir de adulto cronometrado-grávidas de ratos Sprague-Dawley) com cetamina (70 mg / kg, ip; usando &lt;50 uL de volume final). Quando filhote é calmo, verifique que reflex cauda pitada está ausente. </li><li> Decapitar filhotes. <ol><li> Cortar a cabeça pele longitudinalmente a partir da frente para trás usando uma lâmina de bisturi de aço carbono e puxar a pele para os lados usando uma pinça. Cortar o osso que cobre o cérebro movendo o SCissors lateralmente e removê-lo totalmente para expor o cérebro. </li><li> Em seguida, remove-se rapidamente o cérebro utilizando uma espátula (inicialmente colocado sob o bolbo olfactivo, a fim de empurrar suavemente para fora do cérebro do mais rostral para as áreas mais caudal). Com cuidado, empurre o cérebro em fluido gelada oxigenado sacarose-artificial cerebrospinal (sacarose-aCSF). </li></ol></li><li> Fazer um corte parassagital no hemisfério direito (remoção de aproximadamente um terço do hemisfério) e colar o lado cortado do cérebro para um disco metálico que irá ser magneticamente fixado à câmara de corte de um vibroslicer para reduzir sagitais 400 uM secções contendo o núcleo pedunculopontino (PPN). Manter fatias PPN à TA durante 45 min antes de célula inteira gravações de patch-clamp. </li></ol> 4. Oscilações Recordings Gamma-banda em PPN Slices <ol><li> Preparação de Solução de potássio intracelular-gluconato (alta-K + Solução) <ol><li> Colocar emgelo num copo limpo com 10 ml de água destilada. Enquanto continuamente agitando acrescentar: K + -gluconate, 90,68 mg phosphocreatine di sal Tris, 47,66 mg HEPES, 1,5 mg EGTA, 40,58 mg Mg 2+ -ATP e 4 mg Na + 2 -GTP. </li><li> Ajustar o pH para 7,3 com KOH (100 mM em água destilada). Adiciona-se água destilada para atingir um volume final de 20 mL. Se necessário, ajustar a osmolaridade com sacarose (100 mM em água destilada) a ser 280-290 mOsm. Após diluição, a concentração final de cada composto é K + 124 mM -gluconate, fosfocreatina sal di Tris 10 mM, HEPES 10 mM, EGTA 0,2 mM, 4 mM de Mg2 + -ATP, e 0,3 mM de Na + 2 -GTP. </li><li> soluções alíquota intracelulares em 1 ml tubos e congelar a -20 ° C. Utilize uma aliquota por dia e manter a 4 ° C durante as experiências. </li></ol></li><li> Whole-cell Grampo patch Recordings <ol><li> Coloque as fatias de uma câmara de imersão e perfundir-los (1,5 ml / min) com oxigenado(95% de O2 - 5% CO 2) aCSF contendo os seguintes: antagonistas de receptor de antagonista de receptor de ácido 2-amino-5-phosphonovaleric selectivo de NMDA (APV, 40 uM), competitiva AMPA / cainato antagonista do receptor de glutamato de 6-ciano-7- nitroquinoxalina-2.3-diona (CNQX, 10 uM), antagonista do receptor de glicina estricnina (STR, 10 | iM), o GABA específica gabazine Um antagonista do receptor (GBZ, 10 uM), e tetrodotoxina bloqueador do canal de sódio (TTX, 3 um) NOTA: Nos resultados e números, estes antagonistas são referidos coletivamente bloqueadores como sinápticas ou SBS. </li><li> Encha as pipetas de patch de gravação (Resistência 2-7 mohms; feita a partir de capilares regulares, disponíveis comercialmente espessura da parede de vidro de borosilicato de diâmetro exterior 1,0 mm e diâmetro interno 0,6 mm) com solução intracelular high-K + usando enchimentos patch-pipetas disponíveis comercialmente com um Filtro de solução. Insira a pipeta no suporte de seu amplificador. Aplique uma pequena postivo de pressão usando uma seringa de 1 ml ligada ao suporte da pipeta usando um tubo de silicone ligado a uma válvula de três vias. Ligue a volta do suporte da pipeta para um amplificador de patch clamp. </li><li> Mover a pipeta de gravação usando um micromanipulador mecânica perto do núcleo PPN usando uma objetiva de 4X combinados para infravermelho próximo de interferência diferencial óptica de contraste. NOTA: PPN núcleo pode ser observada em fatias dorsal para o pedúnculo cerebelar superior (SCP; observável como um feixe de espessura dos axónios). Pipetas de registo foram localizados na PPN pars compacta, que está localizado imediatamente dorsal para a extremidade posterior do pedúnculo. </li><li> Traga a pipeta de gravação em contacto com um neurónio PPN enquanto visualizadas utilizando uma lente de imersão em água 40X, e aplicar rapidamente sucção negativa para formar uma vedação com a célula. </li><li> Utilize software selo de tensão-clamp para monitorar a resistência da pipeta durante a aspiração negativa usando o protocolo do fabricante. <ol><li> Quando s negativosução é aumentada lentamente e valores de resistência de leitura pelo monitor de patch-clamp na ponta da pipeta atingir 80-100 MOhm, mudar rapidamente o potencial de manutenção de -50 mV e aliviar a pressão negativa. Comece a aplicar continuamente sucção negativa até ruptura de membrana e o acesso eléctrico do neurónio é conseguido na configuração de célula inteira. </li><li> Se os valores de resistência de acesso medidos pelo software selo de tensão-clamp são 10 MOhm ou superior, em seguida, continuar a aplicar sucção negativa em quantidades menores. </li></ol></li><li> Compensar capacitância (ou seja, transientes rápidos e lentos observada após rotura da membrana da célula) e resistência em série em modo de tensão-grampo. Mudar o modo de gravação a pinça de corrente, e rapidamente compensar valores ponte (por exemplo, mover o botão amplificador ou clique no menu automático de compensação usando o mouse computer&#39;s). <ol><li> Monitorar continuamente potencial de repouso da membrana da PPN neurônio sendo gravado ucantar o protocolo do fabricante. Se membrana em repouso mudança potencial para valores despolarização ou hiperpolarizantes, em seguida, usar pequenas quantidades de corrente contínua (até 100 Pa) para manter o valor final mV óptima -50. NOTA: Mantenha apenas os neurônios PPN com um potencial estável de membrana em repouso (RMP) de -48 mV ou mais hyperpolarized (ie, RMP &lt;-48 mV). </li></ol></li></ol></li></ol>

<ol><li>Profice, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurophysiological+evaluation+of+the+pedunculopontine+nucleus+in+humans%5BTitle%5D)+AND+%22J.+Neural.+Transm+%28Vienna%29%22%5BJournal%5D)">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/pubmed?term=((Neuronal+activities+in+brain-stem+cholinergic+nuclei+related+to+tonic+activation+processes+in+thalamocortical+systems%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Fast+oscillations+%2820-40+Hz%29+in+thalamocortical+systems+and+their+potentiation+by+mesopontine+cholinergic+nuclei+in+the+cat%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">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/pubmed?term=((Lesions+of+the+pedunculopontine+tegmental+nucleus+reduce+paradoxical+sleep+%28PS%29+propensity%3A+evidence+from+a+short-term+PS+deprivation+study+in+rats%5BTitle%5D)+AND+%22Eur.+J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Different+cellular+types+in+mesopontine+cholinergic+nuclei+related+to+ponto-geniculo-occipital+waves%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((The+pedunculopontine+tegmental+nucleus%3A+a+role+in+cognitive+processes%3F%5BTitle%5D)+AND+%22Brain+Res.+Brain+Res.+Rev%22%5BJournal%5D)">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/pubmed?term=((The+pedunculopontine+tegmental+nucleus%3A+from+basic+neuroscience+to+neurosurgical+applications%3A+arousal+from+slices+to+humans%3A+implications+for+DBS%5BTitle%5D)+AND+%22J.+Neural.+Transm%22%5BJournal%5D)">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/pubmed?term=((Implantation+of+human+pedunculopontine+nucleus%3A+a+safe+and+clinically+relevant+target+in+Parkinson%27s+disease%5BTitle%5D)+AND+%22Neuroreport%22%5BJournal%5D)">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/pubmed?term=((Gamma+band+unit+activity+and+population+responses+in+the+pedunculopontine+nucleus%5BTitle%5D)+AND+%22J.+Neurophysiol%22%5BJournal%5D)">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/pubmed?term=((Mechanism+behind+gamma+band+activity+in+pedunculopontine+nucleus+%28PPN%29%5BTitle%5D)+AND+%22Eur.+J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Spatiotemporal+properties+of+high+speed+calcium+oscillations+in+the+pedunculopontine+nucleus%5BTitle%5D)+AND+%22J.+Appl.+Physiol+%281985%29%22%5BJournal%5D)">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/pubmed?term=((Temporal+binding+via+cortical+coincidence+detection+of+specific+and+nonspecific+thalamocortical+inputs%3A+a+voltage-dependent+dye-imaging+study+in+mouse+brain+slices%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">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/pubmed?term=((Discharge+profiles+across+the+sleep-waking+cycle+of+identified+cholinergic%2C+gabaergic%2C+and+glutamatergic+neurons+in+the+pontomesencephalic+tegmentum+of+the+rat%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Single+cell+activity+patterns+of+pedunculopontine+tegmentum+neurons+across+the+sleep-wake+cycle+in+the+freely+moving+rats%5BTitle%5D)+AND+%22J.+Neurosci.+Res%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">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/pubmed?term=((Firing+of+%27possibly%27+cholinergic+neurons+in+the+rat+laterodorsal+tegmental+nucleus+during+sleep+and+wakefulness%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">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/pubmed?term=((Inhibition+by+carbachol+microinjections+of+presumptive+cholinergic+PGO-on+neurons+in+freely+moving+cats%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">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/pubmed?term=((Effect+upon+the+EEG+of+acute+injury+to+the+brainstem+activating+system%5BTitle%5D)+AND+%22Electroenceph.+Clin.+Neurophysiol%22%5BJournal%5D)">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/pubmed?term=((The+sleep-waking+cycle%5BTitle%5D)+AND+%22Ergeb.+Physiol%22%5BJournal%5D)">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/pubmed?term=((Brain+stem+reticular+formation+and+activation+of+the+EEG%5BTitle%5D)+AND+%22Electroenceph.+Clin.+Neurophysiol%22%5BJournal%5D)">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/pubmed?term=((Correlations+between+alterations+of+the+cortical+transaminase+activity+and+EEG+patterns+of+sleep+and+wakefulness+induced+by+brainstem+transections%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">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/pubmed?term=((Orexin+receptor+activation+generates+gamma+band+input+to+cholinergic+and+serotonergic+arousal+system+neurons+and+drives+an+intrinsic+Ca2%2B+-dependent+resonance+in+LDT+and+PPT+cholinergic+neurons%5BTitle%5D)+AND+%22Frontiers+Neurol%22%5BJournal%5D)">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/pubmed?term=((Electrophysiological+characterization+of+neurons+in+the+dorsolateral+pontine+REM+sleep+induction+zone+of+the+rat%3A+intrinsic+membrane+properties+and+responses+to+carbachol+and+orexins%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">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/pubmed?term=((On+the+role+of+the+pedunculopontine+nucleus+and+mesencephalic+reticular+formation+in+locomotion+in+non-human+primates%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Pedunculopontine+nucleus+area+oscillations+during+stance%2C+stepping+and+freezing+in+Parkinson%27s+disease%5BTitle%5D)+AND+%22PLOS+ONE%22%5BJournal%5D)">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/pubmed?term=((Mechanisms+behind+gamma+band+activity+in+the+pedunculopontine+nucleus+%28PPN%29%5BTitle%5D)+AND+%22Abstr+Soc+Neurosci%22%5BJournal%5D)">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/pubmed?term=((High-threshold+Ca2%2B+channels+behind+gamma+band+activity+in+the+pedunculopontine+nucleus+%28PPN%29%5BTitle%5D)+AND+%22Physiol.+Rep%22%5BJournal%5D)">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.

Neurônios PPN têm propriedades intrínsecas que lhes permitem disparar potenciais de ação em frequências da banda beta / gama durante em gravações vivo a partir de animais que são acordado ou durante o sono REM, mas não durante o sono de ondas lentas 2,3,5,13-17. Outros autores mostraram que os transectos do tronco cerebral em mais níveis anteriores de PPN reduzida frequências gama durante as gravações de EEG. No entanto, quando as lesões no tronco cerebral posterior ao núcleo, onde es...

PPN núcleo está anatomicamente incluídos no tegmento mesencefálico caudal. O PPN é um componente chave da RAS 1. O PPN participa na manutenção de estados ativados comportamentais (ou seja, de vigília, o sono REM) 2. A estimulação elétrica do PPN in vivo induzida oscilação rápida (20 - 40 Hz) no EEG cortical 3, enquanto lesões bilaterais PPN no rato reduzidos ou eliminados sono REM 4. Enquanto a maioria dos neurônios PPN disparar potenciais de ação em frequência beta / gama-band (20 - 80 Hz), alguns neurônios apresentou baixas taxas de queima espontânea (&lt;10 Hz) 5. Além disso, o PPN parece estar envolvido em outros aspectos do comportamento, tais como motivação e atenção 6. Alta freqüência direta (40 - 60 Hz) 7 estimulação elétrica do núcleo PPN em animais decerebrate pode promover a locomoção. Nos últimos anos, a estimulação cerebral profunda (DBS) de PPN tem sido utilizado para tratar pacientes que sofrem frodistúrbios m envolvendo défices da marcha, tais como a doença de Parkinson (DP) 8. Relatórios anteriores demonstraram que quase todos os neurônios PPN pode disparar potenciais de ação na frequência da banda de gama quando despolarizado usando pulsos de corrente quadrados 9. Devido à activação drástica dos canais de potássio dependentes de voltagem durante pulsos quadrados despolarizações até ou menos de -25 mV. Como consequência, há oscilações gama robustas foram observados após o bloqueio geração de potenciais de ação usando tetrodotoxina 10. Em um esforço para contornar tal problema, 1 - foram utilizados 2 seg longo despolarização rampas atuais. Rampas gradualmente despolarizado o potencial de membrana a partir de valores de repouso até 0 mV, enquanto inativar parcialmente canais de potássio dependentes de voltagem. Oscilações da membrana banda gama claras foram evidentes dentro da janela dependência da voltagem dos canais de cálcio limiar elevado (isto é, entre -25 mV e -0 mV) 10. Em conclusão, a banda gama activiTy foi observada em neurónios PPN 9, e ambos P / Q e canais de cálcio dependentes da voltagem de tipo N precisa de ser activado, a fim de gerar oscilações da banda gama na PPN 10. Uma série de estudos determinou-se a localização dos canais de cálcio limiar elevado em neurónios PPN. Injetando a combinação de corantes, imagens de fluorescência ratiometric mostrou transientes de cálcio através de canais de cálcio dependentes da voltagem que são ativados em diferentes dendrites quando despolarizado usando rampas atuais 11. propriedades intrínsecas de neurónios PPN têm sido sugeridos para permitir a activação simultânea dessas células durante a vigília e sono REM, induzindo, assim, a actividade neuronal oscilatória de alta frequência entre o RAS e lacetes tálamo. Tal interação de longo alcance é considerado para apoiar um estado cerebral capaz de avaliar de forma confiável o mundo em torno de nós em uma base contínua 12. Aqui, descrevemos o experimentoal condições necessárias para gerar e manter a gama da banda de oscilação em células in vitro PPN. Este protocolo não foi descrito anteriormente, e ajudaria um número de grupos para estudar as propriedades de membrana intrínsecas que medeiam a actividade banda gama a outras áreas do cérebro. Além disso, os passos atuais poderia levar à conclusão errônea de que a atividade banda gama não pode ser gerada nestas células.

<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

Eferentes sinápticos de PPN são conhecidas por modular a actividade neuronal das várias regiões talâmicos intralaminares (por exemplo, o centrolateral / parafascicular; Cl / Pf núcleo). A activação quer do PPN ou núcleos Cl / pf in vivo tem sido descrito para induzir a estimulação do animal e um aumento na actividade de banda na gama cortical electroencefalograma (EEG). Os mecanismos celulares para a geração de oscilações da banda gama de sistema ativando reticular (RAS) neurónios são os mesmos que aqueles encontrados para gerar oscilações banda gama de outros núcleos cérebros. Durante gravações corrente-clamp de neurónios PPN (a partir de fatias de parassagitais 9 - ratos 25 dias de idade), a utilização de medidas de despolarização quadrados activado rapidamente os canais de potássio dependentes da voltagem que impediram neurónios PPN seja despolarizada além de -25 mV. Injectar 1 - 2 seg longo despolarização rampas atuais gradualmente despolarizado PPN potencial de membrana resting valores na direção de 0 mV. No entanto, injetáveis ​​despolarizantes pulsos quadrados gerado oscilações gama-banda de potencial de membrana que se mostraram menores em amplitude em comparação com as oscilações geradas por rampas. Todas as experiências foram realizadas na presença de canais de sódio dependentes da voltagem e rápidos bloqueadores dos receptores sinápticos. Tem sido demonstrado que a activação dos canais de cálcio dependentes da voltagem de limiar elevado subjacentes actividade oscilatório gama-banda em neurónios PPN. Intervenções metodológicas e farmacológicos específicos são descritos aqui, fornecendo as ferramentas necessárias para induzir e manter PPN subliminares oscilação banda gama in vitro.

Inicialmente, as oscilações gama foram evocados usando impulsos de corrente quadrados. Actual de gravação de braçadeira de neurónios PPN na presença de agentes bloqueadores sinápticas e TTX foi monitorizada continuamente para assegurar que o potencial de repouso da membrana foi mantida estável a ~ -50 mV (Figura 1A). Dois impulsos de corrente quadrados longo segundo foram injectados intracelularmente pelo amplificador de patch clamp através da pipeta de gravaç...

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

O núcleo pedunculopontino (PPN) está localizado no tronco cerebral e seus neurônios são maximamente ativado durante a vigília e movimento rápido dos olhos (REM) do cérebro sono estados. Este trabalho descreve a abordagem experimental para gravar em oscilação vitro membrana subliminares banda gama em neurônios PPN.

Gravação Gama Oscilações banda em pedunculopontino Núcleo Neurônios

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

8.6K visualizações.  University of Buenos Aires.  O núcleo pedunculopontino (PPN) está localizado no tronco cerebral e seus neurônios são maximamente ativado durante a vigília e movimento rápido dos olhos (REM) do cérebro sono estados. Este trabalho descreve a abordagem experimental para gravar em oscilação vitro membrana subliminares banda gama em neurônios PPN. 

Vídeo: Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

Caracterização fisiológica, morfológica e neuroquímicos de Neurônios Modulada por Movimento

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Neurociência

Automatic Detection of Highly Organized Theta Oscillations in the Murine EEG

Theta activity is generated in the septohippocampal system and can be recorded using deep intrahippocampal electrodes and implantable electroencephalography (EEG) radiotelemetry or tether system approaches. Pharmacologically, hippocampal theta is heterogeneous (see dualistic theory) and can be differentiated into type I and type II theta. These individual EEG subtypes are related to specific cognitive and behavioral states, such as arousal, exploration, learning and memory, higher integrative functions, etc. In neurodegenerative diseases such as Alzheimer&#39;s, structural and functional alterations of the septohippocampal system can result in impaired theta activity/oscillations. A standard quantitative analysis of the hippocampal EEG includes a Fast-Fourier-Transformation (FFT)-based frequency analysis. However, this procedure does not provide details about theta activity in general and highly-organized theta oscillations in particular. In order to obtain detailed information on highly-organized theta oscillations in the hippocampus, we have developed a new analytical approach. This approach allows for time- and cost-effective quantification of the duration of highly-organized theta oscillations and their frequency characteristics.

Theta activity in the hippocampus is related to specific cognitive and behavioral stages. Here, we describe an analytical method to detect highly-organized theta oscillations within the hippocampus using a time-frequency (i.e., wavelet analysis)-based approach.

Detecção automática de altamente organizada Oscilações Theta no Murino EEG

Theta activity is generated in the septohippocampal system and can be recorded using deep intrahippocampal electrodes and implantable ...

Comportamento

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.

Fabricação e utilização Piggy-back Multibarrel Eletrodos para<em&gt; Em vivo</em&gt; As manipulações farmacológicas de respostas neurais

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

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.

Geração de locais Oscilações CA1 γ por Tetânica Estimulação

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.

Tuning na banda Theta do hipocampo<em&gt; In Vitro</em&gt;: Metodologias para gravação a partir do Circuito de Septohippocampal de Roedores Isolado

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.

Agudo<em&gt; In Vivo</em&gt; Gravações eletrofisiológicas de Potenciais de campo locais e atividade de múltiplas unidades a partir do caminho hiperdiretivo em ratos anestesiados

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

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.

Optogenetic de arrastamento de Theta Hippocampal oscilações em comportar-se ratos

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

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.

Gravação espacialmente restrita oscilações no hipocampo de ratos de comportar-se

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

Electrophysiological Investigations of Retinogeniculate and Corticogeniculate Synapse Function

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ganglion cells and project it to the visual cortex. In addition, relay neurons receive top-down excitation from the cortex. The two main excitatory inputs to the relay neurons differ in several aspects. Each relay neuron receives input from only a few retinogeniculate synapses, which are large terminals with many release sites. This is reflected by the comparably strong excitation, the relay neurons receive, from retinal ganglion cells. Corticogeniculate synapses, in contrast, are simpler with few release sites and weaker synaptic strength. The two synapses also differ in their synaptic short-term plasticity. Retinogeniculate synapses have a high release probability and consequently display a short-term depression. In contrast, corticogeniculate synapses have a low release probability. Corticogeniculate fibers traverse the reticular thalamic nuclei before entering the lateral geniculate nucleus. The different locations of the reticular thalamic nucleus (rostrally from the lateral geniculate nucleus) and optic tract (ventro-laterally from the lateral geniculate nucleus) allow stimulating corticogeniculate or retinogeniculate synapses separately with extracellular stimulation electrodes. This makes the lateral geniculate nucleus an ideal brain area where two excitatory synapses with very different properties impinging onto the same cell type, can be studied simultaneously. Here, we describe a method to investigate the recording from relay neurons and to perform detailed analysis of the retinogeniculate and corticogeniculate synapse function in acute brain slices. The article contains a step-by-step protocol for the generation of acute brain slices of the lateral geniculate nucleus and steps for recording activity from relay neurons by stimulating the optic tract and corticogeniculate fibers separately.

Here, we present protocols for the preparation of acute brain slices containing the lateral geniculate nucleus and the electrophysiological investigation of retinogeniculate and corticogeniculate synapses function. This protocol provides an efficient way to study synapses with the high-release and low-release probability in the same acute brain slices.

Investigações eletrofisiológicas de Retinogeniculate e função do Synapse de Corticogeniculate

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ...

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

Source: Urbano, F. J., et al. <a href="https://appjove.unicartagenaproxy.elogim.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.

Registrando oscilações de banda gama de neurônios do núcleo pedunculopontino em uma fatia de cérebro

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

Gravação Gama Oscilações banda em pedunculopontino Núcleo Neurônios

Resumo

Explore mais vídeos