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

<ol>
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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. 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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 enquanto atravessam camadas individuais. A integridade do hipocampo é preservada juntamente com conectividade funcional e propriedades das populações neuronais locais. Isso fornece uma ferramenta complexa e poderosa para investigar pequenas e grandes oscilações de rede no hipocampo. Por exemplo, a combinação de circuitos pré-conectados e as propriedades específicas do tipo celular na rede produzem ondas tete e gamma rítmicas geradas intrinsecamente e espontaneamente que imitam características importantes da hipocaminaAtividade mpal in vivo . Usando os métodos apresentados neste protocolo, o hipocampo pode ser extraído rapidamente do cérebro do roedor e permanecer viável por várias horas em um ambiente aCSF de fluxo rápido. As técnicas eletrofisiológicas básicas são facilmente aplicadas para investigar como a atividade síncrona e a função sináptica de tipos neuronais específicos influenciam a dinâmica do hipocampo. O nosso procedimento proporcionou a primeira oportunidade de explorar sistematicamente a dinâmica das oscilações de potencial de campo local em todo o eixo septo-temporal (dorso-ventral) do hipocampo in vitro (ver Refs 22 , 23 , 24 , 25 , 26 , 27 , 28 ). Na rede e nos níveis fisiológicos, a preparação intacta preserva: 1) O sistema operacional synaInterações entre as células necessárias para suportar de forma confiável as oscilações da rede intrínseca com a faixa de freqüência e o perfil das ondas de theta in vivo , 2) A mudança acentuada na fase relativa da oscilação de theta ao nível do estrato pyramidale, 3) A distribuição espacial e a coerência da theta Oscilação no hipocampo e sua interação com os ritmos locais de freqüência gama, 4) a relação amplitude-fase das ondas theta / gamma e 5) a associação temporal específica da célula principal e internuron com as oscilações theta do potencial de campo. Enquanto a ausência de entrada externa ao hipocampo pode parecer uma limitação da técnica, também permite o estudo da dinâmica interna do hipocampo de maneira que não pode ser realizada in vivo . Além disso, as entradas externas podem ser imitadas pela estimulação optogenética de terminais de fibra específicos e vias aferentes. Os preparativos intactos oferecem novas possibilidades de estudoComo as atividades da rede se propagam e interagem em redes de grande escala que envolvem o hipocampo e outras estruturas conectadas. Como tal, uma aplicação principal da técnica de preparação intacta é a investigação da conectividade de rede funcional dentro ou entre regiões cerebrais. O uso do hipocampo intacto in vitro deve, portanto, não só fornecer mais informações sobre propriedades celulares e de rede do hipocampo, mas também esclarecer sobre como elas interagem para moldar processamento, integração e geração de informações dentro do cérebro. As oscilações da rede rítmica que envolvem a sinalização eletroquímica coordenada de grandes conjuntos neuronais atuam como um mecanismo central para codificar, armazenar e transferir informações dentro e entre áreas cerebrais. As oscilações do hipocampo são consideradas cruciais para o processamento de informações, codificações e memória espaciotemporais. Por outro lado, pensa-se que a disfunção do hipocampo está subjacente a distúrbiosH como doença de Alzheimer e esquizofrenia, que estão associados a padrões de oscilação alterados 29 . Como tal, o estudo das oscilações do hipocampo autogeradas utilizando preparações intactas do hipocampo tornou-se um meio renovado para elucidar as propriedades básicas da função e disfunção do sistema nervoso.

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 border="0" cellpadding="0" cellspacing="0" width="820">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Reagents</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S9625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S9378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaH2PO4 - sodium phosphate monobasic</td>
			<td>Sigma Aldrich</td>
			<td>S8282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium sulfate</td>
			<td>Sigma Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-(+)-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>C5080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Ascorbate</td>
			<td>Sigma Aldrich</td>
			<td>A7631-25G</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:319px;">Name</td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard Dissecting Scissors</td>
			<td>Fisher Scientific</td>
			<td>08-951-25</td>
			<td>brain extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel Handle #4, 14cm</td>
			<td>WPI</td>
			<td>500237</td>
			<td>brain extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter forceps, flat jaws, straight (11cm)</td>
			<td>WPI</td>
			<td>500456</td>
			<td>brain extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paragon Stainless Steel Scalpel Blades #20</td>
			<td>Ultident</td>
			<td>02-90010-20</td>
			<td>brain extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine Point Curved Dissecting Scissors</td>
			<td>Thermo Fisher Scientific</td>
			<td>711999</td>
			<td>brain extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Teflon (PTFE) -coated thin spatula</td>
			<td>VWR</td>
			<td>82027-534</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hayman Style Microspatula</td>
			<td>Fisher Scientific</td>
			<td>21-401-25A</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lab spoon</td>
			<td>Fisher Scientific</td>
			<td>14-375-20</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate Glass Pasteur Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20A</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Droper</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor blades Single edged</td>
			<td>VWR</td>
			<td>55411-055</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lens paper (4X6 inch)</td>
			<td>VWR</td>
			<td>52846-001</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass petri dishes (100 x 20 mm)</td>
			<td>VWR</td>
			<td>25354-080</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Single Inline Solution Heater</td>
			<td>Warner Instruments</td>
			<td>SH-27B</td>
			<td>perfusion system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aquarium air stones for bubbling</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>perfusion system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Electric Skillet</td>
			<td>Black &#38; Decker</td>
			<td>n.a.</td>
			<td>perfusion system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">95% O2/5% CO2 gas mixture (carbogen)&#160;</td>
			<td>Vitalaire</td>
			<td>SG466204A</td>
			<td>perfusion system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass bottles/flasks (4 x 1 L)</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>perfusion system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Submerged recording Chamber</td>
			<td>custom design (FM)</td>
			<td>n.a.</td>
			<td style="width:247px;">Commercial alternative may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass pipettes (1.5 / 0.84 OD/ID (mm) )</td>
			<td>WPI</td>
			<td>1B150F-4</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hum Bug 50/60 Hz Noise Eliminator</td>
			<td>Quest Scientific</td>
			<td>Q-Humbug</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multiclamp 700B patch-clamp amplifier</td>
			<td>Molecular devices</td>
			<td>MULTICLAMP</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multiclamp 700B Commander Program</td>
			<td>Molecular devices</td>
			<td>MULTICLAMP</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital/Analogue converter</td>
			<td>Molecular devices</td>
			<td>DDI440</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCLAMP10</td>
			<td>Molecular devices</td>
			<td>PCLAMP10</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibration isolation table&#160;</td>
			<td>Newport</td>
			<td>n.a.</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulators (manually operated )</td>
			<td>Siskiyou&#160;</td>
			<td>MX130</td>
			<td>electrophysiology (LFP)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulators (automated)</td>
			<td>Siskiyou&#160;</td>
			<td>MC1000e</td>
			<td>electrophysiology (patch)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Audio monitor&#160;</td>
			<td>A-M Systems</td>
			<td>Model 3300</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette/Patch pipette puller</td>
			<td>Sutter</td>
			<td>P-97</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Custom-built upright fluorescence microscope</td>
			<td>Siskiyou</td>
			<td>n.a.</td>
			<td>Imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Analogue video camera</td>
			<td>COHU</td>
			<td>4912-2000/0000</td>
			<td>Imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital frame grabber with imaging software</td>
			<td>EPIX, Inc</td>
			<td>PIXCI-SV7</td>
			<td>Imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus 2.5x objective</td>
			<td>Olympus</td>
			<td>MPLFLN</td>
			<td>Imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus 40x water immersion objective</td>
			<td>Olympus</td>
			<td>UIS2 LUMPLFLN</td>
			<td>Imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Custom-made light-emitting diode (LED) system&#160;</td>
			<td>custom</td>
			<td>n.a.</td>
			<td>optogenetic stimulation (Amhilon et al., 2015)</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:319px;">Name</td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">PV::Cre (KI) mice</td>
			<td style="width:135px;">Jackson Laboratory</td>
			<td style="width:119px;">stock number 008069</td>
			<td style="width:247px;">Allow&#160; Cre-directed gene expression in PV interneurons</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Constitutive-conditional Ai9 mice (R26-lox-stop-lox-tdTomato (KI))</td>
			<td style="width:135px;">Jackson Laboratory</td>
			<td style="width:119px;">stock number 007905</td>
			<td style="width:247px;">Express TdTomato following Cre-mediated recombination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Ai32 mice (R26-lox-stop-lox-ChR2(H134R)-EYFP</td>
			<td style="width:135px;">Jackson Laboratory</td>
			<td style="width:119px;">stock 
			number 012569</td>
			<td style="width:247px;">Express the improved channelrhodopsin-2/EYFP fusion protein following exposure to Cre recombinase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PVChY mice</td>
			<td>In house breeding</td>
			<td>n.a.</td>
			<td style="width:247px;">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 sincronizadas para populações neuronais em diferentes regiões e camadas do hipocampo isolado ( Figura 2 ). Os resultados representativos da grampeamento simultâneo de grampos de células inteiras e gravações extracelulares são apresentados para caracterizar as propriedades de disparo e sináptica de tipos celulares específicos durante oscilações espontâneas do hipocampo ( Figura 3 ), bem como durante a manipulação optogenética da atividade rítmica (G &quot;&gt; Figura 4). <img alt="figura 1" class="xfigimg" src="/files/ftp_upload/55851/55851fig1.jpg" /> Figura 1: Procedimento de dissecação para a preparação isolada de hipocampo intacto. (A) Vista geral da configuração dissecção. Topo direito: balão de solução de sacarose gelatinizada gelatinada (1); Inferior esquerda: bandeja de plástico cheia de gelo (2) segurando o prato de dissecação coberto com papel de lentes (3); A câmara de retenção a frio contendo solução de sacarose (4); E um conjunto de ferramentas cirúrgicas (5). ( B ) Vista do cérebro do mouse antes da hemisecão no prato de dissecação. ( C ) Recuperação do cérebro hemisecado na câmara de retenção a frio e vista ampliada (inserção) do hemisfério cerebral esquerdo antes de inserir a extremidade pequena da espátula revestida sob o septo. ( D ) Espátula revestida colocada sob o hipocampo isolado, ao longo da região CA1 / SUB, com o cérebro restanteO tecido é puxado para fora por baixo. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55851/55851fig1large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" class="xfigimg" src="/files/ftp_upload/55851/55851fig2.jpg" /> Figura 2: Configuração da Configuração para Gravação de Ontas Theta in vitro da Preparação Intacta do Hipocampo na Câmara de Gravação Submersa. (A) O hipocampo é mostrado isolado com um esquema de regiões do hipocampo e múltiplos eléctrodos colocados em quatro locais de gravação diferentes distribuídos septotemporally (indicado por asteriscos branco). Na vista da plataforma da câmara de gravação mostrada acima (inserção i), a entrada e a saída para fluxo de perfusão rápida são indicadas pelos números (1, 2). Na imagem ampliada do hipocampo mostrado abaixo (inserção ii), um único eletrodo é colocado no meioCA1 sepetotécnico e as fibras dos alvéolos são facilmente visíveis, correndo diagonalmente em direção ao subículo. S: septal, T: temporal, f / fx: fimbria-fornix. ( B ) Representação esquemática da organização de camadas CA1 com traços LFP representativos gravados simultaneamente de stratum oriens (cinza) e stratum radiatum (preto). Observe a fase invertida de sinais entre as duas camadas. Alv: stratum alveus, PR: stratum pyramidale, SR: stratum radiatum. SLM: Stratum Lacunosum Moleculare. ( C ) Exemplo de Rastreamento de LFP que mostra oscilação teta espontânea gravada a partir da área CA1 / SUB (segmento de 20 segundos) e segmentos expandidos de 2 segundos (abaixo) de sinal não filtrado; Passagem de banda filtrada para freqüências theta (0,5 - 12 Hz); Gama lenta (25 - 55 Hz); E gama rápida (125 - 250 Hz). <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55851/55851fig2large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a> Figura 3: Atividade específica do tipo celular da célula piramidal e PV Interneuron durante as oscilações da teta espontânea na preparação intacta do hipocampo a partir de um mouse PV-TOM. (A) actividade sináptica gravado a partir de uma célula piramidal durante teta oscilações. Os traços do braço atual (painel esquerdo) mostram disparo espontâneo, mas não rítmico em repouso, e potenciais pós-sinápticos inibitórios (iPSPs) que não estavam claramente sincronizados com a oscilação do LFP lentamente emergente. As gravações de tensão (painel direito) mostram que as correspondentes correntes inibitórias inibitórias (iPSCs) têm seu potencial de reversão em torno de -70 mV. ( B ) Atividade sináptica registrada a partir de um interneurônio PV fluorescente de rápida expansão durante as oscilações de theta. No braçadeira atual (esquerda), esta célula disparou espontaneamente em repouso e foi fortemente conduzida por poções excitatórias rítmicasPotenciais stsinápticos (ePSPs) que foram bloqueados em fase com a oscilação estável do LFP. Nas gravações de tensão-braçadeira (direita), o potencial excitatório de reversão da corrente pós-sináptica foi de aproximadamente 0 mV. Os desenhos esquemáticos em cima mostram a configuração experimental juntamente com a colocação de eléctrodos de parche e campo nas imagens de ampliação CA1 / SUB e alta (40X) da célula piramidal registrada e internurônico PV fluorescente. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55851/55851fig3large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 4" class="xfigimg" src="/files/ftp_upload/55851/55851fig4.jpg" /> Figura 4: Potenciais de campo local e gravações simultâneas de pinças de cliques a partir de neurônios piramidais e PV VPN únicos CA1 / SUB durante as oscilações de Theta espontâneas e optogeneticamente no Hipocampo isolado. ( Um </stRong&gt;) Diagrama mostrando os locais de gravação de eletrodos de LFP e patch com colocação do guia de fibra leve acima da região CA1 / SUB que expressa um opsin sensível à luz azul (ChETA acoplado com o eYFP de fluoróforo). ( B ) Caracterização de um neurônio piramidal que mostra as propriedades típicas do Spike regular (RS) (superior) e alta ampliação (40X) da célula gravada (inferior). ( C ) Gravação de corrente de corrente da amostra da mesma célula em potencial de membrana despolarizada (preto) juntamente com o sinal LFP (cinza) e mostrando IPSPs rítmicos sincronizados durante a oscilação espontânea (esquerda) e durante a estimulação de luz de frequência de onda (6 Hz) ( certo). O padrão de estimulação de luz (sombreamento azul) é representado em cima dos traços de tensão. ( D ) Spectrograma e espectro de potência da forma de onda LFP antes, durante e após uma simulação de luz de 6 Hz (linha de base, estimulação, pós). ( E ) Imagens de campo brilhante e de fluorescência de baixa ampliação do isolatPreparação de hipocampo mostrando fluorescência de eYFP (em verde) localizada na região CA1 / SUB. ( F ) Caracterização de etapas atuais mostrando o comportamento de Fast-Spiking (FS) de um interneurônio PV-TOM gravado (imagem de fluorescência 40X abaixo). ( G ) Gravação de potencial de membrana mostrando grandes EPSPs e disparo rítmico da célula fotográfica gravada sincronizada com o sinal LFP durante a oscilação espontânea do campo (esquerda) e durante a estimulação de luz (direita) a 3 Hz. ( H ) Média de Disparo de Campo (FTA) de picos de células PV sobre o sinal CA1 / SUB LFP. A descarga da célula fotovoltaica em múltiplos ensaios (centrada nos picos da LFP) foi convertida em FTA de picos registrados durante a oscilação espontânea (linha de base) e durante a estimulação de luz (stim). Gráficos médios e inferiores mostram as parcelas de espetadas e histogramas de probabilidade de pico (a probabilidade média é mostrada em vermelho). Os gráficos mais altos mostram gráficos do sinal LFP médio que aumentou em potência durante o ligHt estimulação em paralelo com disparo altamente sincronizado da célula fotovoltaica fase-bloqueada para o pico da oscilação LFP. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55851/55851fig4large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="4"> SOLUÇÃO DE SUCROSE (1X) PARA HIPPOCAMPUS ISOLADO </td></tr><tr><td colspan="2"> (Solução de estoque) </td><td></td><td></td></tr><tr><td> Composto </td><td> MW </td><td> Final Conc. (milímetros) </td><td> Quantidade para 1 L (g) </td></tr><tr><td> Sacarose </td><td> 342.3 </td><td> 252 </td><td> 86,26 g </td></tr><tr><td> NaHCO 3 </td><td> 84.01 </td><td> 24 </td><td> 2,020 g </td></tr><tr><td> glicose </td><td> 180,2 </td><td> 10 </td><td&gt; 1.800 g </td></tr><tr><td> KCl </td><td> 74,55 </td><td> 3 </td><td> 0,223 g </td></tr><tr><td> MgSO 4 </td><td> 120,4 </td><td> 2 </td><td> 0,241 g </td></tr><tr><td> NaH 2 PO 4 </td><td> 120 </td><td> 1,25 </td><td> 0,150 g </td></tr><tr><td> CaCl 2 .2H2O [1 M] stock </td><td> 147 </td><td> 1.2 </td><td> 120 μL / 0,1 L * </td></tr><tr><td colspan="4"> * Adicionar 360 uL de CaCl2 [1 M] durante 0,3 L de solução de sacarose oxigenado </td></tr><tr><td></td><td></td><td colspan="2"> PH = 7,4 quando oxigenado, Osm 310 - 320 </td></tr></tbody></table> Tabela 1. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="4"> SOLUÇÃO DE ACSF STANDARD (5X) PARA PERFUSÃO </td></tr><tr><td colspan="2"> (Solução de estoque) </td><td></td><td></td></tr><tr><td> Composto </td><td> MW </td><td> Final Conc. (milímetros) </td><td> Quantidade para 2 L 5X </td></tr><tr><td> NaCl </td><td> 58,44 </td><td> 126 </td><td> 73.6 </td></tr><tr><td> NaHCO 3 </td><td> 84.01 </td><td> 24 </td><td> 20.2 </td></tr><tr><td> glicose </td><td> 180,2 </td><td> 10 </td><td> 18 </td></tr><tr><td> KCl </td><td> 74,55 </td><td> ♦ 4.5 </td><td> 3.355 </td></tr><tr><td> MgSO 4 </td><td> 120,4 </td><td> 2 </td><td> 2.41 </td></tr><tr><td> NaH 2 PO 4 </td><td> 120 </td><td> 1,25 </td><td> 1,5 </td></tr><tr><td> Ascorbato </td><td> 176.1 </td><td> 0,4 </td><td> 0,705 </td></tr><tr><td> CaCl 2 .2H2O [1 M] stock </td><td> 147 </td><td> 2 </td><td> 2 mL / L *</td></tr><tr><td colspan="4"> * Adicionar 2 ml de CaCl 2 [1 M] durante 1 L aCSF (1x) solução oxigenada </td></tr><tr><td></td><td></td><td colspan="2"> PH = 7,4 quando oxigenado, Osm 310 - 320 </td></tr><tr><td colspan="4"> ♦ Um [K + ] o ligeiramente elevado é usado para esta solução de aCSF (em comparação com o KCl normal aCSF 2,5 mM) para aumentar a excitabilidade das redes do hipocampo e facilitar o surgimento das oscilações theta. </td></tr></tbody></table> Mesa 2.

Watch this Scientific Journal Video about Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit at JoVE.com

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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Nanyang Technological University

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JoVE Journal

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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

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

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

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

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

Labeling fluorescentes retrógrada Permite alvejado Extracelular Gravação Single-unidade dos neurônios identificados<em&gt; In vivo</em

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

Neurociência

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

Investigações sobre alterações da função Circuito Hippocampal Após lesão cerebral traumática leve

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Dois registros eletrofisiológicos de sinapticamente evocadas Respostas astroglial e neuronal em fatias hipocampais agudas

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Multi-unidade Métodos de gravação para caracterizar a atividade neural no Locust (<em&gt; Schistocerca Americana</em&gt;) Circuitos olfatórias

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Entrega intraventricular direta de medicamentos para o Sistema Nervoso Central Rodent

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

Um<em&gt; In-vitro</em&gt; Preparação de neurônios entéricos isolados e células gliais do plexo mioentérico do Rato Adulto

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

Utilizando metodologias combinadas para definir o papel do Plasma Membrane entrega durante Axon Ramificação e Neuronal morfogênese

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the centrolateral/parafascicular; Cl/Pf nucleus). The activation of either the PPN or Cl/Pf nuclei in vivo has been described to induce the arousal of the animal and an increment in gamma band activity in the cortical electroencephalogram (EEG). The cellular mechanisms for the generation of gamma band oscillations in Reticular Activating System (RAS) neurons are the same as those found to generate gamma band oscillations in other brains nuclei. During current-clamp recordings of PPN neurons (from parasagittal slices from 9 - 25 day-old rats), the use of depolarizing square steps rapidly activated voltage-dependent potassium channels that prevented PPN neurons from being depolarized beyond -25 mV.
Injecting 1 - 2 sec long depolarizing current ramps gradually depolarized PPN membrane potential resting values towards 0 mV. However, injecting depolarizing square pulses generated gamma-band oscillations of membrane potential that showed to be smaller in amplitude compared to the oscillations generated by ramps. All experiments were performed in the presence of voltage-gated sodium channels and fast synaptic receptors blockers. It has been shown that the activation of high-threshold voltage-dependent calcium channels underlie gamma-band oscillatory activity in PPN neurons. Specific methodological and pharmacological interventions are described here, providing the necessary tools to induce and sustain PPN subthreshold gamma band oscillation in vitro.

The pedunculopontine nucleus (PPN) is located in the brainstem and its neurons are maximally activated during waking and rapid eye movement (REM) sleep brain states. This work describes the experimental approach to record in vitro gamma band subthreshold membrane oscillation in PPN neurons.

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 ...

Personalized Needles for Microinjections in the Rodent Brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

We describe here a protocol for microinjection in the rodent brain that uses quartz needles. These needles do not produce detectable tissue damage and ensure reliable delivery even in deep regions. Moreover, they can be adapted to research needs by personalized designs and can be re-used.

Agulhas personalizadas para alevinos do cérebro de roedor

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Gravação de plasticidade sináptica em fatias Hippocampal agudas, mantidas em um pequeno volume reciclagem - perfusão- e sistema de câmara de submersão-tipo

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...