Este trabalho foi apoiado pelos Institutos canadenses de Pesquisa em Saúde e Ciências Naturais.

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+relationship+between+hippocampal+place+units+and+the+EEG+theta+rhythm.">Phase relationship between hippocampal place units and the EEG theta rhythm.</a> Hippocampus. 3 (3), 317-330 (1993).</li><li>Winson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+hippocampal+theta+rhythm+results+in+spatial+memory+deficit+in+the+rat.">Loss of hippocampal theta rhythm results in spatial memory deficit in the rat.</a> Science. 201 (4351), 160-163 (1978).</li><li>M'Harzi, M., Jarrard, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategy+selection+in+a+task+with+spatial+and+nonspatial+components:+effects+of+fimbria-fornix+lesions+in+rats.">Strategy selection in a task with spatial and nonspatial components: effects of fimbria-fornix lesions in rats.</a> Behav Neural Biol. 58 (3), 171-179 (1992).</li><li>Osipova, D., 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=Theta+and+gamma+oscillations+predict+encoding+and+retrieval+of+declarative+memory.">Theta and gamma oscillations predict encoding and retrieval of declarative memory.</a> J Neurosci. 26 (28), 7523-7531 (2006).</li><li>Stumpf, C., Petsche, H., Gogolak, 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+significance+of+the+rabbit's+septum+as+a+relay+station+between+the+midbrain+and+the+hippocampus.+II.+The+differential+influence+of+drugs+upon+both+the+septal+cell+firing+pattern+and+the+hippocampus+theta+activity.">The significance of the rabbit's septum as a relay station between the midbrain and the hippocampus. II. The differential influence of drugs upon both the septal cell firing pattern and the hippocampus theta activity.</a> Electroencephalogr Clin Neurophysiol. 14, 212-219 (1962).</li><li>Mitchell, S. J., Ranck, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+theta+rhythm+in+medial+entorhinal+cortex+of+freely+moving+rats.">Generation of theta rhythm in medial entorhinal cortex of freely moving rats.</a> Brain Res. 189 (1), 49-66 (1980).</li><li>Alonso, A., Garcia-Austt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+sources+of+theta+rhythm+in+the+entorhinal+cortex+of+the+rat.+I.+Laminar+distribution+of+theta+field+potentials.">Neuronal sources of theta rhythm in the entorhinal cortex of the rat. I. Laminar distribution of theta field potentials.</a> Exp Brain Res. 67 (3), 493-501 (1987).</li><li>Vertes, R. P., Kocsis, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brainstem-diencephalo-septohippocampal+systems+controlling+the+theta+rhythm+of+the+hippocampus.">Brainstem-diencephalo-septohippocampal systems controlling the theta rhythm of the hippocampus.</a> Neuroscience. 81 (4), 893-926 (1997).</li><li>Bland, B. H., Colom, L. V., Konopacki, J., Roth, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+records+of+carbachol-induced+theta+rhythm+in+hippocampal+slices.">Intracellular records of carbachol-induced theta rhythm in hippocampal slices.</a> Brain Res. 447 (2), 364-368 (1988).</li><li>Cobb, S. R., Buhl, E. H., Halasy, K., Paulsen, O., Somogyi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+neuronal+activity+in+hippocampus+by+individual+GABAergic+interneurons.">Synchronization of neuronal activity in hippocampus by individual GABAergic interneurons.</a> Nature. 378 (6552), 75-78 (1995).</li><li>Williams, J. H., Kauer, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+carbachol-induced+oscillatory+activity+in+rat+hippocampus.">Properties of carbachol-induced oscillatory activity in rat hippocampus.</a> J Neurophysiol. 78 (5), 2631-2640 (1997).</li><li>Chapman, C. A., Lacaille, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+induction+of+theta-frequency+oscillations+in+hippocampal+inhibitory+interneurons+and+pacing+of+pyramidal+cell+firing.">Cholinergic induction of theta-frequency oscillations in hippocampal inhibitory interneurons and pacing of pyramidal cell firing.</a> J Neurosci. 19 (19), 8637-8645 (1999).</li><li>Strata, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+oscillations+in+CA3+hippocampal+pyramids:+physiological+relevance+to+theta+rhythm+generation.">Intrinsic oscillations in CA3 hippocampal pyramids: physiological relevance to theta rhythm generation.</a> Hippocampus. 8 (6), 666-679 (1998).</li><li>Kocsis, B., Bragin, A., Buzsaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interdependence+of+multiple+theta+generators+in+the+hippocampus:+a+partial+coherence+analysis.">Interdependence of multiple theta generators in the hippocampus: a partial coherence analysis.</a> J Neurosci. 19 (14), 6200-6212 (1999).</li><li>Fellous, J. M., Sejnowski, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+induction+of+oscillations+in+the+hippocampal+slice+in+the+slow+(0.5-2+Hz),+theta+(5-12+Hz),+and+gamma+(35-70+Hz)+bands.">Cholinergic induction of oscillations in the hippocampal slice in the slow (0.5-2 Hz), theta (5-12 Hz), and gamma (35-70 Hz) bands.</a> Hippocampus. 10 (2), 187-197 (2000).</li><li>Gillies, M. J., 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=A+model+of+atropine-resistant+theta+oscillations+in+rat+hippocampal+area+CA1.">A model of atropine-resistant theta oscillations in rat hippocampal area CA1.</a> J Physiol. 543 (Pt 3), 779-793 (2002).</li><li>Gloveli, T., 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=Orthogonal+arrangement+of+rhythm-generating+microcircuits+in+the+hippocampus.">Orthogonal arrangement of rhythm-generating microcircuits in the hippocampus.</a> Proc Natl Acad Sci U S A. 102 (37), 13295-13300 (2005).</li><li>Konopacki, J., Eckersdorf, B., Kowalczyk, T., Golebiewski, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Firing+cell+repertoire+during+carbachol-induced+theta+rhythm+in+rat+hippocampal+formation+slices.">Firing cell repertoire during carbachol-induced theta rhythm in rat hippocampal formation slices.</a> Eur J Neurosci. 23 (7), 1811-1818 (2006).</li><li>Hajos, N., 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=Maintaining+network+activity+in+submerged+hippocampal+slices:+importance+of+oxygen+supply.">Maintaining network activity in submerged hippocampal slices: importance of oxygen supply.</a> Eur J Neurosci. 29 (2), 319-327 (2009).</li><li>Manseau, F., Goutagny, R., Danik, M., Williams, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hippocamposeptal+pathway+generates+rhythmic+firing+of+GABAergic+neurons+in+the+medial+septum+and+diagonal+bands:+an+investigation+using+a+complete+septohippocampal+preparation+in+vitro.">The hippocamposeptal pathway generates rhythmic firing of GABAergic neurons in the medial septum and diagonal bands: an investigation using a complete septohippocampal preparation in vitro.</a> J Neurosci. 28 (15), 4096-4107 (2008).</li><li>Goutagny, R., Jackson, J., Williams, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-generated+theta+oscillations+in+the+hippocampus.">Self-generated theta oscillations in the hippocampus.</a> Nat Neurosci. 12 (12), 1491-1493 (2009).</li><li>Jackson, J., Goutagny, R., Williams, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+slow+gamma+rhythms+are+intrinsically+and+independently+generated+in+the+subiculum.">Fast and slow gamma rhythms are intrinsically and independently generated in the subiculum.</a> J Neurosci. 31 (34), 12104-12117 (2011).</li><li>Amilhon, B., 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=Parvalbumin+Interneurons+of+Hippocampus+Tune+Population+Activity+at+Theta+Frequency.">Parvalbumin Interneurons of Hippocampus Tune Population Activity at Theta Frequency.</a> Neuron. 86 (5), 1277-1289 (2015).</li><li>Huh, C. Y., 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=Excitatory+Inputs+Determine+Phase-Locking+Strength+and+Spike-Timing+of+CA1+Stratum+Oriens/Alveus+Parvalbumin+and+Somatostatin+Interneurons+during+Intrinsically+Generated+Hippocampal+Theta+Rhythm.">Excitatory Inputs Determine Phase-Locking Strength and Spike-Timing of CA1 Stratum Oriens/Alveus Parvalbumin and Somatostatin Interneurons during Intrinsically Generated Hippocampal Theta Rhythm.</a> J Neurosci. 36 (25), 6605-6622 (2016).</li><li>Gu, N., 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=NMDA-dependent+phase+synchronization+between+septal+and+temporal+CA3+hippocampal+networks.">NMDA-dependent phase synchronization between septal and temporal CA3 hippocampal networks.</a> J Neurosci. 33 (19), 8276-8287 (2013).</li><li>Jackson, J., 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=Reversal+of+theta+rhythm+flow+through+intact+hippocampal+circuits.">Reversal of theta rhythm flow through intact hippocampal circuits.</a> Nat Neurosci. 17 (10), 1362-1370 (2014).</li><li>Gonzalez-Burgos, G., Lewis, D. 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Os autores declaram que não há interesses comerciais ou financeiros concorrentes.

Embora as gravações eletrofisiológicas de fatias agudas do hipocampo constituam uma técnica in vitro padrão, os métodos aqui apresentados diferem substancialmente da abordagem clássica. Ao contrário das preparações finas em fatias onde as camadas celulares específicas são visíveis na superfície e podem ser examinadas diretamente, as preparações intactas do hipocampo são mais parecidas com as configurações in vivo onde os eletrodos são abaixados em regiões cerebrais direcionadas enq...

As oscilações do hipocampo (4 - 12 Hz) estão entre as formas mais predominantes de atividade rítmica no cérebro de mamíferos e acreditam desempenhar papéis fundamentais nas funções cognitivas, como o processamento da informação espaciotemporal e a formação de memórias episódicas 1 , 2 , 3 . Enquanto vários estudos in vivo que realçam a relação das células do quadrado modificadas com estudos espaciais de navegação e lesão, além de evidências clínicas, sustentam a visão de que as oscilações do theta do hipocampo estão envolvidas na formação da memória 4 , 5 , 6 , os mecanismos associados Com a geração das oscilações do theta do hipocampo ainda não são totalmente compreendidas. As primeiras investigações in vivo sugeriram que a atividade theta dependia principalmente de osciladores extrínsecos, em particular a entrada rítmicaA partir de estruturas cerebrais aferentes, como o septo e o córtex entorrinal 7 , 8 , 9 , 10 . Um papel para fatores intrínsecos - conectividade interna das redes neurais do hipocampo juntamente com as propriedades dos neurônios do hipocampo - também foi postulado com base em observações in vitro 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 . No entanto, além de alguns estudos históricos 19 , 20 , 21 , dificuldades no desenvolvimento de abordagens que poderiam replicar atividades de população fisiologicamente realistas em simples preparações de fatia in vitroS têm, durante muito tempo, um exame experimental mais detalhado das habilidades intrínsecas do hipocampo e áreas relacionadas para auto-gerar oscilações theta. Uma desvantagem importante da configuração experimental padrão de fatias finas in vitro é que a organização celular e sináptica 3D das estruturas cerebrais geralmente está comprometida. Isso significa que muitas formas de atividades de rede concertada baseadas em conjuntos de células espacialmente distribuídos, que vão desde grupos localizados (≤ 1 mm de raio) até populações de neurônios espalhados por uma ou mais áreas cerebrais (&gt; 1 mm), não podem ser suportadas. Diante dessas considerações, era necessário um tipo diferente de abordagem para estudar como as oscilações theta emergem no hipocampo e se propagam para estruturas de saída corticais e subcorticais relacionadas. Nos últimos anos, o desenvolvimento inicial da preparação &quot;septo-hipocampo completo&quot; para examinar a interação bidirecionalAs citações das duas estruturas 22 e a evolução subseqüente da preparação do &quot;hipocampo isolado&quot; revelaram que as oscilações teta intrínsecas ocorrem espontaneamente no hipocampo que não possui entrada rítmica externa 23 . O valor dessas abordagens reside na visão inicial de que toda a estrutura funcional dessas regiões teve que ser preservada para funcionar como um gerador de ritmo theta in vitro 22 .

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma Aldrich</td><td>S9625</td><td></td></tr><tr><td>Sucrose</td><td>Sigma Aldrich</td><td>S9378</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma Aldrich</td><td>S5761</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; - sodium phosphate monobasic</td><td>Sigma Aldrich</td><td>S8282</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Sigma Aldrich</td><td>M7506</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma Aldrich</td><td>P3911</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma Aldrich</td><td>G7528</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma Aldrich</td><td>C5080</td><td></td></tr><tr><td>Sodium Ascorbate</td><td>Sigma Aldrich</td><td>A7631-25G</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Standard Dissecting Scissors</td><td>Fisher Scientific</td><td>08-951-25</td><td>brain extraction</td></tr><tr><td>Scalpel Handle #4, 14 cm</td><td>WPI</td><td>500237</td><td>brain extraction</td></tr><tr><td>Filter forceps, flat jaws, straight (11 cm)</td><td>WPI</td><td>500456</td><td>brain extraction</td></tr><tr><td>Paragon Stainless Steel Scalpel Blades #20</td><td>Ultident</td><td>02-90010-20</td><td>brain extraction</td></tr><tr><td>Fine Point Curved Dissecting Scissors</td><td>Thermo Fisher Scientific</td><td>711999</td><td>brain extraction</td></tr><tr><td>Teflon (PTFE) -coated thin spatula</td><td>VWR</td><td>82027-534</td><td>hippocampal preparation</td></tr><tr><td>Hayman Style Microspatula</td><td>Fisher Scientific</td><td>21-401-25A</td><td>hippocampal preparation</td></tr><tr><td>Lab spoon</td><td>Fisher Scientific</td><td>14-375-20</td><td>hippocampal preparation</td></tr><tr><td>Borosilicate Glass Pasteur Pipets</td><td>Fisher Scientific</td><td>13-678-20A</td><td>hippocampal preparation</td></tr><tr><td>Droper</td><td>Fisher Scientific</td><td></td><td>hippocampal preparation</td></tr><tr><td>Razor blades Single edged</td><td>VWR</td><td>55411-055</td><td>hippocampal preparation</td></tr><tr><td>Lens paper (4 x 6&quot;)</td><td>VWR</td><td>52846-001</td><td>hippocampal preparation</td></tr><tr><td>Glass petri dishes (100 x 20 mm)</td><td>VWR</td><td>25354-080</td><td>hippocampal preparation</td></tr><tr><td>Plastic tray for ice; size 30 x 20 x 5 cm</td><td>n.a.</td><td>n.a.</td><td>hippocampal preparation</td></tr><tr><td>Single Inline Solution Heater</td><td>Warner Instruments</td><td>SH-27B</td><td>perfusion system</td></tr><tr><td>Aquarium air stones for bubbling</td><td>n.a.</td><td>n.a.</td><td>perfusion system</td></tr><tr><td>Tygon E-3603 tubing (ID 1/16 OD 1/8)</td><td>Fisherbrand</td><td>14-171-129</td><td>perfusion system</td></tr><tr><td>Electric Skillet</td><td>Black &amp;amp; Decker</td><td>n.a.</td><td>perfusion system</td></tr><tr><td>95% O&lt;sub&gt;2&lt;/sub&gt;/5% CO&lt;sub&gt;2&lt;/sub&gt; gas mixture (carbogen)&amp;nbsp;</td><td>Vitalaire</td><td>SG466204A</td><td>perfusion system</td></tr><tr><td>Glass bottles/flasks (4 x 1 L)</td><td>n.a.</td><td>n.a.</td><td>perfusion system</td></tr><tr><td>Submerged recording Chamber</td><td>custom design (FM)</td><td>n.a.</td><td>Commercial alternative may be used</td></tr><tr><td>Glass pipettes (1.5/0.84 OD/ID (mm) )</td><td>WPI</td><td>1B150F-4</td><td>electrophysiology</td></tr><tr><td>Hum Bug 50/60 Hz Noise Eliminator</td><td>Quest Scientific</td><td>Q-Humbug</td><td>electrophysiology</td></tr><tr><td>Multiclamp 700B patch-clamp amplifier</td><td>Molecular devices</td><td>MULTICLAMP</td><td>electrophysiology</td></tr><tr><td>Multiclamp 700B Commander Program</td><td>Molecular devices</td><td>MULTICLAMP</td><td>electrophysiology</td></tr><tr><td>Digital/Analogue converter</td><td>Molecular devices</td><td>DDI440</td><td>electrophysiology</td></tr><tr><td>PCLAMP10</td><td>Molecular devices</td><td>PCLAMP10</td><td>electrophysiology</td></tr><tr><td>Vibration isolation table&amp;nbsp;</td><td>Newport</td><td>n.a.</td><td>electrophysiology</td></tr><tr><td>Micromanipulators (manually operated )</td><td>Siskiyou&amp;nbsp;</td><td>MX130</td><td>electrophysiology (LFP)</td></tr><tr><td>Micromanipulators (automated)</td><td>Siskiyou&amp;nbsp;</td><td>MC1000e</td><td>electrophysiology (patch)</td></tr><tr><td>Audio monitor&amp;nbsp;</td><td>A-M Systems</td><td>Model 3300</td><td>electrophysiology</td></tr><tr><td>Micropipette/Patch pipette puller</td><td>Sutter</td><td>P-97</td><td>electrophysiology</td></tr><tr><td>Custom-built upright fluorescence microscope</td><td>Siskiyou</td><td>n.a.</td><td>Imaging</td></tr><tr><td>Analogue video camera</td><td>COHU</td><td>4912-2000/0000</td><td>Imaging</td></tr><tr><td>Digital frame grabber with imaging software</td><td>EPIX, Inc</td><td>PIXCI-SV7</td><td>Imaging</td></tr><tr><td>Olympus 2.5X objective</td><td>Olympus</td><td>MPLFLN</td><td>Imaging</td></tr><tr><td>Olympus 40X water immersion objective</td><td>Olympus</td><td>UIS2 LUMPLFLN</td><td>Imaging</td></tr><tr><td>Custom-made Light-Emitting Diode (LED) system&amp;nbsp;</td><td>custom</td><td>n.a.</td><td>optogenetic stimulation (Amhilon et al., 2015)</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Animals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PV::Cre (KI) mice</td><td>Jackson Laboratory</td><td>stock number 008069</td><td>Allow&amp;nbsp; Cre-directed gene expression in PV interneurons</td></tr><tr><td>Constitutive-conditional Ai9 mice (R26-lox-stop-lox-tdTomato (KI))</td><td>Jackson Laboratory</td><td>stock number 007905</td><td>Express TdTomato following Cre-mediated recombination</td></tr><tr><td>Ai32 mice (R26-lox-stop-lox-ChR2(H134R)-EYFP</td><td>Jackson Laboratory</td><td>stock&lt;br/&gt; number 012569</td><td>Express the improved channelrhodopsin-2/EYFP fusion protein following exposure to Cre recombinase</td></tr><tr><td>PVChY mice</td><td>In house breeding</td><td>n.a.</td><td>Offspring obtained from cross-breeding the PV-Cre line with Ai32 mice (R26-lox-stop-lox-ChR2(H134R)-EYFP</td></tr></tbody></table>

tuning in the hippocampal theta band in vitro: methodologies for recording from the isolated rodent septohippocampal circuit

Este protocolo descreve os procedimentos para a preparação e gravação do hipocampo inteiro isolado, de WT e camundongos transgênicos, além de melhorias recentes em metodologias e aplicações para o estudo das oscilações theta. Uma caracterização simples da preparação isolada do hipocampo é apresentada pelo qual a relação entre os osciladores internamente da teta do hipocampo é examinada em conjunto com a atividade das células piramidais e interneurônios GABAérgicos, nas áreas cornu ammonis-1 (CA1) e subículo (SUB). No geral, mostramos que o hipocampo isolado é capaz de gerar oscilações theta intrínsecas in vitro e que a ritmicidade gerada no hipocampo pode ser manipulada com precisão pela estimulação optogenética de interneurônios com parvalbumina positiva (PV). A preparação de hipocampo isolado in vitro oferece uma oportunidade única de usar gravações simultâneas de grampos e grampos intracelulares de neu visualmente identificadoRons para entender melhor os mecanismos subjacentes à geração de ritmo theta.

<html> Esta seção ilustra exemplos de resultados que podem ser obtidos estudando oscilações theta na preparação de hipocampo isolado no mouse in vitro . O procedimento de dissecção para extração do hipocampo isolado está ilustrado na Figura 1 . Usando esta preparação, as oscilações teta intrínsecas podem ser examinadas durante a colocação de eletrodos de campo múltiplos, registrando atividade geral e entradas sinápticas sincroniza...

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

Aqui, apresentamos um protocolo para a gravação da teta de rede neuronal rítmica e das oscilações gama de uma preparação isolada do hipocampo inteiro. Descrevemos os passos experimentais da extração do hipocampo para detalhes de gravações de grampos de campo, unidade e toda célula, bem como a estimulação optogenética do ritmo de theta.

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 ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

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

9.7K visualizações.  McGill University.  Aqui, apresentamos um protocolo para a gravação da teta de rede neuronal rítmica e das oscilações gama de uma preparação isolada do hipocampo inteiro. Descrevemos os passos experimentais da extração do hipocampo para detalhes de gravações de grampos de campo, unidade e toda célula, bem como a estimulação optogenética do ritmo de theta. 

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

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

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.

Em larga escala de gravação de Neurônios por sondas de silicone móveis em Comportando Roedores

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

Neurociência

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Whole-cell Recordings patch-clamp de Morphologically- e identificados Neuroquimicamente hipocampo Interneurônios

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

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

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

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

Gravação e modulação da atividade epileptiforme em fatias de cérebro de roedor acoplado para matrizes de microeletrodos

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

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

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

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

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 ...

Selective Manipulation of Theta Oscillations in a Mouse Model

Source: Bender, F. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/57349">Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice.</a> J. Vis. Exp (2018)
This video demonstrates a protocol to modulate theta oscillations in a genetically engineered mouse model with light-sensitive ion channels in its GABAergic medial septum neurons. By delivering light pulses to the hippocampus, the protocol aims to synchronize neuronal activity and generate theta oscillations, which are recorded as local field potentials.

Manipulação seletiva de oscilações em um modelo de camundongo

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

JoVE Encyclopedia of Experiments

Neurophysiology

Stimulation and Modulation Techniques

Implanting an Electrode Array and Optic Fiber into the Mouse Hippocampus

Source: Bender, F., et. al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/57349">Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice.</a> J. Vis. Exp. (2018)
This video demonstrates the stereotactic implantation of a wire electrode array and an optic fiber in the hippocampus of a mouse model.

Implantando um feixe de eletrodos e fibra óptica no hipocampo do camundongo

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

Optogenetic Control of Hippocampal Network Dynamics

Source: Manseau, F., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55851">Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit.</a> J. Vis. Exp. (2017)
This video demonstrates the method to use optogenetics to modulate hippocampal network dynamics by activating genetically modified light-sensitive interneurons in a mouse brain slice, enhancing theta oscillation synchronization and studying neural circuit behavior.

Controle optogenético da dinâmica da rede hipocampal

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

	Optogenetic control in the ...

In Vitro Recording of Theta Oscillations in the Mouse Hippocampus

Source: Manseau, F., et.al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55851">Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit.</a> J. Vis. Exp. (2017).
This video demonstrates the recording of rhythmic neuronal network activity, also known as theta oscillations, in an isolated hippocampus. Electrodes are inserted into various layers and spatial positions to detect and analyze theta oscillations. This shows the unique neuronal compositions and the connectivity of the neuronal network across different layers of the hippocampus.

Tuning na banda Theta do hipocampo

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