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

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

Tutti i protocolli sperimentali sono stati approvati dal Comitato di Cura e uso istituzionale degli animali della University of Arkansas per le scienze mediche (numero protocollo # 3593) ed erano in accordo con le linee guida del National Institutes of Salute per la cura e l&#39;uso di animali da laboratorio. 1. Preparazione del liquido cerebrospinale standard-artificiale (aCSF) <ol><li> Preparazione della soluzione Stock A <ol><li> Aggiungere 700 ml di acqua distillata ad una pulita 1 L bicchiere prima di aggiungere sostanze chimiche. </li><li> Mentre agitando continuamente un volume di 500 ml, aggiungere 136.75 g di NaCl, 6,99 g di KCl, 2,89 g di MgSO 4, e 2,83 g di NaH 2 PO 4. </li><li> Aggiungere acqua distillata per raggiungere un volume finale di 1 L. Dopo la diluizione, la concentrazione finale di ogni composto è 117 mM NaCl, 4,69 mM KCl, 1,2 mM MgSO 4, e 1,18 mM NaH 2 PO 4. </li><li> Conservare in frigorifero a 4 ° C per un massimo di 2 settimane. </li></ol></li><li> Preparazione della soluzione della B <ol><li> Aggiungere 700 ml di acqua distillata in un becher da 1L pulito prima di aggiungere sostanze chimiche. </li><li> Mentre agitando continuamente un volume di 500 ml, aggiungere 41,45 g di D-glucosio e 41.84 g di NaHCO 3. </li><li> Aggiungere acqua distillata per raggiungere un volume finale di 1 L. Dopo la diluizione, la concentrazione finale di ogni composto è 11,5 mM D-glucosio e 24,9 mM NaHCO 3. </li><li> Conservare in frigorifero a 4 ° C per un massimo di 2 settimane. </li><li> Prima di ogni mix esperimento 50 ml di brodo A con 50 ml di B e portare a 1 L con acqua distillata per ottenere una soluzione definitiva concentrazione aCSF e lasciare a temperatura ambiente, mentre continua ossigenante con CarboGen (95% O 2 - 5% di CO 2 mix) per almeno 30 min. Preparare aCSF concentrazione finale solo all&#39;inizio di ogni esperimento e scartare alla fine della giornata. </li></ol></li></ol> 2. Preparazione di liquido cerebrospinale saccarosio-artificiale(Saccarosio-aCSF) <ol><li> Preparazione della soluzione della C <ol><li> Aggiungere 700 ml di acqua distillata ad una pulita 1 L bicchiere prima di aggiungere sostanze chimiche. </li><li> Mentre continuo agitazione 500 ml di acqua distillata, aggiungere 240 g di saccarosio, 6,55 g di NaHCO 3, 0,671 g di KCl, 4,88 g di MgCl 2, 0,22 g di CaCl 2 e 0,21 g di acido ascorbico. </li><li> Aggiungere acqua distillata per raggiungere un volume finale di 1 L. Dopo la diluizione, la concentrazione finale di ogni composto è 701.1 mM di saccarosio, 78 mM NaHCO 3, 9 mM KCl, 24 mM MgCl 2, 1.5 mM CaCl 2 e 1,2 mM di acido ascorbico . </li><li> Mantenere Soluzione C a 4 ° C per un massimo di una settimana. </li><li> Prima di ogni miscela esperimento 300 ml di 50 ml di brodo C con 600 ml di acqua distillata per ottenere la soluzione di saccarosio-aCSF alla concentrazione finale e lasciare a temperatura ambiente mentre continuamente ossigenante con CarboGen (95% O 2 - 5% CO 2 mix) per almeno 30 min. </li></ol&gt; </li></ol> 3. Preparazione Slice <ol><li> Posizionare un bicchiere pulito con 100 ml di soluzione di saccarosio-aCSF in ghiaccio mentre ossigenante con CarboGen e fissare il pH a 7,4 con un pH-metro mentre l&#39;aggiunta di qualche goccia di soluzione 0,1 M di NaOH (quando il pH &lt;7,4) o 0,1 soluzione M HCl (quando pH&gt; 7.4). </li><li> Riempire una camera di taglio di un vibratome con saccarosio aCSF e ossigenare esso. Accendere il sistema di refrigerazione glicerolo basato accoppiato alla camera di taglio e attendere 15 min per permettere raffreddare 0 - 4 ° C. </li><li> Anestetizzare cuccioli di ratto (dai 9 ai 12 da adulti al momento giusto in gravidanza ratti Sprague-Dawley) con ketamina (70 mg / kg, ip; utilizzando &lt;50 ml di volume finale). Quando cucciolo è calmo, doppio controllo che la coda pizzico riflesso è assente. </li><li> Decapitare cuccioli. <ol><li> Tagliare la pelle testa longitudinalmente dalla parte anteriore alla parte posteriore con un bisturi in acciaio al carbonio, e tirare la pelle ai lati con pinze. Tagliare l&#39;osso che copre il cervello in movimento scissors lateralmente e totalmente rimuoverlo per esporre il cervello. </li><li> Quindi, rimuovere rapidamente il cervello utilizzando una spatola (inizialmente posto sotto il bulbo olfattivo per spingere delicatamente il cervello dal più rostrale verso le zone più caudali). Spingere delicatamente il cervello in ossigenato liquido cerebrospinale di saccarosio-ghiaccio artificiale raffreddato (saccarosio-aCSF). </li></ol></li><li> Effettuare un taglio parasagittale nell&#39;emisfero destro (rimuovendo circa un terzo della semisfera), ed incollare la parte tagliata del cervello su un disco metallico che sarà magneticamente fissato alla camera di taglio di un vibroslicer tagliare sagittali 400 sezioni micron contenenti la pedunculopontine nucleo (PPN). Mantenere fette PPN a temperatura ambiente per 45 minuti prima di tutto-cell patch-clamp. </li></ol> 4. Oscillazioni Recordings banda gamma a PPN Fette <ol><li> Preparazione di potassio gluconato soluzione intracellulare (High-K + Solution) <ol><li> Posizionare inghiaccio un bicchiere pulito con 10 ml di acqua distillata. Mentre continua a mescolare aggiungere: K + -gluconate, 90.68 mg phosphocreatine di Tris sale, 47.66 mg HEPES, 1,5 mg EGTA, 40.58 mg Mg 2+ -ATP, e 4 mg Na + 2 -GTP. </li><li> Regolare il pH a 7,3 con KOH (100 mm di acqua distillata). Aggiungere acqua distillata per raggiungere un volume finale di 20 ml. Se necessario, regolare osmolarità con saccarosio (100 mm di acqua distillata) ad essere 280-290 mOsm. Dopo la diluizione, la concentrazione finale di ogni composto è 124 mm K + -gluconate, 10 mM phosphocreatine di Tris di sale, 10 mM HEPES, 0,2 mM EGTA, 4 mM Mg 2+ -ATP, e 0,3 mm Na + 2 -GTP. </li><li> Le soluzioni Aliquota intracellulari in 1 ml tubi e congelare a -20 ° C. Utilizzare un&#39;aliquota al giorno e mantenere a 4 ° C durante gli esperimenti. </li></ol></li><li> Cellula intera Bloccare Registrazioni patch <ol><li> Mettere le fette in una camera di immersione e li (1,5 ml / min) profumato con ossigenato(95% O 2 - 5% di CO 2) aCSF contenente i seguenti antagonisti del recettore NMDA: antagonista dei recettori dell&#39;acido 2-amino-5-phosphonovaleric selettivo (APV, 40 micron), competitivo AMPA / kainato dei recettori del glutammato antagonista del 6-ciano-7- nitroquinoxaline-2,3-dione (CNQX, 10 micron), del recettore della glicina antagonista del stricnina (STR, 10 micron), il GABA specifica un antagonista del recettore gabazine (GBZ, 10 micron), e sodio-antagonista tetrodotossina (TTX, 3μM) NOTA: Nei risultati e le cifre, questi antagonisti sono indicate collettivamente bloccanti come Synaptic o SB. </li><li> Riempire le pipette di patch di registrazione (Resistenza 2-7 MW, costituito da regolari, capillari in commercio di spessore parete di vetro borosilicato di 1,0 mm di diametro esterno e diametro interno 0,6 millimetri) con la soluzione intracellulare alta K + utilizzando disponibili in commercio filler patch-pipetta con filtro soluzione. Inserire la pipetta nel supporto del suo amplificatore. Applicare una piccola pospressione itive utilizzando una siringa da 1 ml collegato al supporto pipetta utilizzando un tubo in silicone collegato ad una valvola a tre vie. Collegare il retro del supporto pipetta ad un amplificatore di patch clamp. </li><li> Spostare la pipetta registrazione utilizzando un micromanipolatore meccanico vicino al nucleo PPN utilizzando un obiettivo 4X combinati per l&#39;ottica di contrasto di interferenza differenziale ad infrarossi. NOTA: PPN nucleo può essere osservato in fette dorsale al peduncolo cerebellare superiore (SCP; osservabile come uno spesso fascio di assoni). Pipette di registrazione erano situati nella PPN pars compacta, che si trova immediatamente dorsale alla estremità posteriore del peduncolo. </li><li> Portare la pipetta di registrazione a contatto con un neurone PPN mentre visualizzati utilizzando un obiettivo ad immersione 40X acqua, e applicare rapidamente aspirazione negativo per formare una tenuta con la cella. </li><li> Utilizzare il software di tenuta di tensione-clamp per monitorare la resistenza pipetta durante l&#39;aspirazione negativo utilizzando il protocollo del produttore. <ol><li> Quando s negativiuzione è lentamente aumentata e valori di resistenza di lettura dal monitor patch-clamp sulla punta della pipetta raggiungere 80-100 MOhm, cambiare rapidamente il potenziale tenendo a -50 mV e rilasciare la pressione negativa. Avviare l&#39;applicazione in continuo di aspirazione negativa fino a quando la rottura della membrana ed elettrica accesso del neurone è realizzato nella cellula intera configurazione. </li><li> Se i valori di resistenza accesso rilevati dal software di tenuta di tensione-clamp sono 10 MOhm o superiore, quindi continuare ad applicare l&#39;aspirazione negativa in piccole quantità. </li></ol></li><li> Compensare capacità (cioè, transitori lenti e veloci osservati dopo rottura della membrana della cellula) e resistenza serie in modo tensione-clamp. Passare modalità di registrazione a pinza di corrente, e rapidamente compensare valori ponte (ad esempio, spostare la manopola amplificatore o fare clic sul menu compensazione automatica utilizzando il mouse del computer utilizzato). <ol><li> Monitorare costantemente riposo potenziale di membrana del neurone PPN in fase di registrazione ucantare protocollo del produttore. Se riposo potenziale di membrana spostamento verso valori depolarizzanti o iperpolarizzanti, quindi utilizzare una piccola quantità di corrente continua (fino a 100 Pa) per mantenere il valore finale mV ottimale -50. NOTA: Conservare soltanto neuroni PPN con un potenziale di membrana a riposo stabile (RMP) di -48 mV o più hyperpolarized (vale a dire, RMP &lt;-48 mV). </li></ol></li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Neuroni PPN hanno proprietà intrinseche che permettono loro di fuoco potenziali d&#39;azione a frequenze in banda beta / gamma durante le registrazioni in vivo da animali che sono sveglio o durante il sonno REM, ma non durante il sonno ad onde lente 2,3,5,13-17. Altri autori hanno mostrato che transections tronco cerebrale a più livelli anteriori di PPN ha ridotto le frequenze di gamma durante le registrazioni EEG. Tuttavia, quando le lesioni del tronco cerebrale posteriore al punto in cui si trova...

PPN nucleo è anatomicamente incluso nel tegmento mesencefalica caudale. Il PPN è un componente chiave di RAS 1. Il PPN partecipa alla manutenzione degli stati attivati ​​comportamentali (ad esempio, la veglia, il sonno REM) 2. La stimolazione elettrica del PPN in vivo indotta oscillazione veloce (20 - 40 Hz) nella EEG corticale 3, mentre le lesioni bilaterali PPN nel ratto ridotti o eliminati sonno REM 4. Mentre la maggioranza dei neuroni PPN fuoco potenziali d&#39;azione a frequenza beta / gamma-banda (20 - 80 Hz), alcuni neuroni hanno presentato bassi tassi di scarica spontanea (&lt;10 Hz) 5. Inoltre, la PPN sembra essere coinvolti in altri aspetti del comportamento come la motivazione e l&#39;attenzione 6. Ad alta frequenza diretta (40 - 60 Hz) 7 stimolazione elettrica del nucleo PPN negli animali decerebrate può promuovere la locomozione. Negli ultimi anni, la stimolazione cerebrale profonda (DBS) del PPN è stato usato per trattare pazienti affetti frodisturbi m coinvolgono deficit della deambulazione come il morbo di Parkinson (PD) 8. Studi precedenti hanno dimostrato che quasi tutti i neuroni PPN possono sparare potenziali d&#39;azione a frequenza di banda di gamma quando depolarizzato utilizzando impulsi di corrente quadrati 9. A causa della drastica attivazione dei canali del potassio voltaggio-dipendenti durante impulsi depolarizzazioni quadrati pari o sotto -25 mV. Di conseguenza, non sono state osservate robusti oscillazioni gamma dopo il blocco generazione potenziali d&#39;azione usando tetrodotoxin 10. Nel tentativo di aggirare tale problema, 1 - sono stati utilizzati 2 sec depolarizzante lungo le rampe di corrente. Rampe gradualmente depolarizzati il ​​potenziale di membrana dai valori fino a 0 mV a riposo, mentre in parte l&#39;inattivazione canali del potassio voltaggio-dipendenti. Oscillazioni della membrana banda gamma Cancella erano evidenti all&#39;interno della finestra di dipendenza di tensione dei canali del calcio soglia alta (cioè tra -25 mV e -0 mV) 10. In conclusione, banda gamma Activity stata osservata nei neuroni PPN 9, ed entrambi P / Q e canali del calcio voltaggio-dipendenti di tipo N deve essere attivato al fine di generare oscillazioni banda gamma nella PPN 10. Una serie di studi ha determinato la posizione dei canali del calcio ad alta soglia nei neuroni PPN. Iniettando la combinazione di coloranti, imaging di fluorescenza raziometrico mostrato transienti di calcio attraverso i canali del calcio voltaggio-dipendenti che si attivano in diversi dendriti quando depolarizzato utilizzando le rampe di corrente 11. proprietà intrinseche dei neuroni PPN sono state proposte per permettere l&#39;attivazione simultanea di queste cellule durante la veglia e il sonno REM, inducendo così oscillatoria ad alta frequenza attività neuronale tra la RAS e loop talamocorticali. Tale interazione lunga portata è considerata supportare uno stato del cervello in grado di valutare attendibilmente il mondo intorno noi su base continua 12. Qui, descriviamo l&#39;esperimentoAl condizioni necessarie per generare e mantenere gamma banda di oscillazione in cellule in vitro PPN. Questo protocollo non è stato descritto in precedenza, e avrebbe aiutato un certo numero di gruppi per studiare le proprietà intrinseche della membrana che mediano l&#39;attività banda gamma in altre aree cerebrali. Inoltre, a pochi passi attuali potrebbero portare alla conclusione erronea che l&#39;attività banda gamma non può essere generata in queste cellule.

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

recording gamma band oscillations in pedunculopontine nucleus neurons

Efferenze Synaptic dal PPN sono noti per modulare l&#39;attività neuronale di diverse regioni del talamo intralaminari (ad esempio, la centrolateral / parafascicular; Cl / Pf nucleo). L&#39;attivazione di una PPN o nuclei Cl / Pf in vivo è stata descritta per indurre l&#39;eccitazione dell&#39;animale e un incremento dell&#39;attività banda gamma nel elettroencefalogramma corticale (EEG). I meccanismi cellulari per la generazione di oscillazioni banda gamma di Attivazione Reticolare System (RAS) neuroni sono gli stessi di quelli trovati per generare oscillazioni banda gamma di altri nuclei cervello. Durante registrazioni current-clamp su neuroni PPN (da fette parasagittali da 9 - 25 giorni-ratti), l&#39;uso di depolarizzante punti quadrato rapidamente attivato canali del potassio voltaggio-dipendenti che impedivano neuroni PPN venga depolarizzato oltre -25 mV. Iniezione 1 - 2 sec depolarizzante lungo rampe attuali gradualmente depolarizzata PPN potenziale di membrana resting Valori verso 0 mV. Tuttavia, parenterale depolarizzanti impulsi quadrati generati oscillazioni gamma-banda di potenziale di membrana che mostravano ad essere più piccole in ampiezza rispetto alle oscillazioni generate da rampe. Tutti gli esperimenti sono stati eseguiti in presenza di canali del sodio voltaggio-dipendenti e veloci recettori sinaptici bloccanti. E &#39;stato dimostrato che l&#39;attivazione dei canali del calcio voltaggio-dipendenti soglia alta sottendono attività oscillatoria banda gamma nei neuroni PPN. Interventi metodologici e farmacologici sono descritte qui, fornendo gli strumenti necessari per indurre e sostenere PPN sottosoglia oscillazione banda gamma in vitro.

Inizialmente, oscillazioni gamma sono stati evocati utilizzando impulsi di corrente quadrati. Clamp attuale dei neuroni PPN in presenza di bloccanti sinaptiche e TTX è stata continuamente monitorata per assicurare che riposa potenziale di membrana è stato mantenuto stabile a ~ -50 mV (Figura 1A). Due impulsi di corrente quadrati lunghi secondi sono stati iniettati intracellulare dall&#39;amplificatore patch clamp attraverso la pipetta registrazione, aumentando la loro ...

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

Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

Il nucleo pedunculopontine (PPN) si trova nel tronco cerebrale e le sue neuroni vengono attivati ​​al massimo durante gli stati di veglia e movimento rapido degli occhi (REM) del cervello sonno. Questo lavoro descrive l&#39;approccio sperimentale di registrare in banda gamma di oscillazione membrana sottosoglia vitro in neuroni PPN.

Registrazione Gamma banda oscillazioni Pedunculopontine Nucleo neuroni

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

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

Research

JoVE Journal

Neuroscience

8.6K Views.  University of Buenos Aires.  Il nucleo pedunculopontine (PPN) si trova nel tronco cerebrale e le sue neuroni vengono attivati ​​al massimo durante gli stati di veglia e movimento rapido degli occhi (REM) del cervello sonno. Questo lavoro descrive l'approccio sperimentale di registrare in banda gamma di oscillazione membrana sottosoglia vitro in neuroni PPN. 

Video: Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

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

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

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

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

Produzione e utilizzo di Piggy-back Multibarrel Elettrodi per<em&gt; In vivo</em&gt; Manipolazioni farmacologiche di risposte neurali

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

Ricerca

Neuroscienze

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

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

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

Generazione di locali oscillazioni CA1 γ di Tetanic Stimolazione

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

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

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

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

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

Tuning nella Banda Theta Ippocampale<em&gt; In vitro</em&gt;: Metodologie per la registrazione dal circuito isolante di roditore Septohippocampal

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

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

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

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

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

acuto<em&gt; In Vivo</em&gt; Registrazioni elettrofisiologiche dei potenziali del campo locale e dell&#39;attività multi-unità dal percorso iperdirezionale nei ratti anestetizzati

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

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

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

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

Registrazione simultanea di co-localizzato elettroencefalografia e potenziali di campo locale nel roditore

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

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

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

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

Registrazione e modulazione di attività Epileptiform in fettine di cervello del roditore accoppiate a matrici di microelettrodi

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.

Registrazione spazialmente limitate oscillazioni nell'ippocampo dei topi di comportarsi

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

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

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

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

Imaging ottico basato su fotodiodi per la registrazione di dinamiche di rete con risoluzione a singolo neurone in invertebrati non transgenici

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

Biologia

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

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

Registrazione delle oscillazioni della banda gamma dai neuroni del nucleo peduncolopontino in una fetta di cervello

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

Neurophysiology

Electrophysiology Techniques

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

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

Registrazione Gamma banda oscillazioni Pedunculopontine Nucleo neuroni

Riepilogo

Esplora Altri Video

Capitoli in questo video

Produzione e utilizzo di Piggy-back Multibarrel Elettrodi per

Generazione di locali oscillazioni CA1 γ di Tetanic Stimolazione

Tuning nella Banda Theta Ippocampale

acuto

Registrazione simultanea di co-localizzato elettroencefalografia e potenziali di campo locale nel roditore

Registrazione e modulazione di attività Epileptiform in fettine di cervello del roditore accoppiate a matrici di microelettrodi

Registrazione spazialmente limitate oscillazioni nell'ippocampo dei topi di comportarsi

Imaging ottico basato su fotodiodi per la registrazione di dinamiche di rete con risoluzione a singolo neurone in invertebrati non transgenici

Registrazione delle oscillazioni della banda gamma dai neuroni del nucleo peduncolopontino in una fetta di cervello

Registrazioni elettrofisiologiche in vivo di attività cerebrali da più siti nei ratti