This work was funded by the Whitehall Foundation and the NIH. We thank Clifford Dax (University of Oregon Technical Support Administration) for his help and expertise in designing a circuit for light delivery.

NOTA: Il seguente protocollo è conforme con il National Institutes of linee guida per la salute, come approvato dalla University of Oregon Animal Care and Use Committee. 1. Chirurgia acuta <ol><li> Anestetizzare l&#39;animale con un cocktail di ketamina-medetomidina, via intraperitoneale (ip) iniezione (Tabella 1). NOTA: I topi utilizzati in questi esperimenti sono generati attraversando un cre-dipendente ChR2-EYFP transgenico line10 di interneuroni linee conducente (Pvalb-iCre11, PV +; Sst-iCre12, SOM +; Cr-iCre12, CR +). Consegna virale di ChR2 o opsine correlati dovrebbe funzionare altrettanto bene, assumendo livelli di espressione simili si ottengono. </li><li> Prima di iniziare l&#39;intervento, in modo che le esibizioni di animali non risposta ad un pizzico punta dolce. Ri-somministrare anestetici durante l&#39;esperimento come necessario per mantenere questa profondità dell&#39;anestesia. Se si utilizza anestetici iniettabili, eventualmente impiantare un catetere ip per preparazioni iniettabili di manutenzione. </li><li> Mantenere l&#39;animale idratato con soluzione salina o di Ringer lattato durante l&#39;esperimento (circa 3 ml / kg / ora), per esempio usando un cocktail anestetico opportunamente diluito per preparazioni iniettabili manutenzione (Tabella 1). </li><li> Posto l&#39;animale in un apparato porta-teste stereotassico o altro. Assicurarsi che il cranio è ben protetto,. Ciò è essenziale per mantenere registrazioni monocellulari stabili. </li><li> Applicare una pomata opthalamic agli occhi per prevenire la secchezza. Mantenere la temperatura corporea a 36,5-37 ° C. </li><li> Eseguire uno scarico della cisterna per la stabilità ulteriore registrazione. Usando un bisturi, rimuovere il tessuto dalla faccia posteriore del cranio per esporre la cisterna magna. Fare un piccolo nick nella dura per drenare il liquido cerebrospinale. Utilizzare solo la punta della lama, ed evitare qualsiasi contatto il gambo cervelletto o del cervello. </li><li> Utilizzando il bisturi, eseguire una piccola craniotomia (~ 2 mm 2) sopra la zona corticale di interesse (per l&#39;acusticacorteccia, circa -2.3 mm posteriormente di bregma, 4,5 millimetri laterale della linea mediana). </li><li> Rimuovere la dura e coprire la corteccia esposta con uno strato di agarosio calda 0,5 - spessore di 1 mm (1,5% agarosio in soluzione salina, 0,9% di NaCl; applicare a ~ 37 ° C). Mantenere l&#39;umido agarosio tutto l&#39;esperimento applicando periodicamente alcune gocce di soluzione salina. </li></ol> 2. Registrazione Set-up <ol><li> Preparare un elettrodo di tungsteno (7-14 MW, 127 micron di diametro, 12 ° punta affusolata, rivestimento epossidico). Superglue l&#39;elettrodo di un tubo capillare di vetro e aggiungere termorestringente per una presa (figura 1). </li><li> Montare l&#39;elettrodo su un micromanipolatore motorizzato (o idraulico), impostare viaggiare ortogonale alla superficie corticale. Far scorrere un filo di terra sotto la pelle, contro il cranio. Evitare il contatto con i muscoli sul lato della testa e la parte posteriore del collo, in quanto possono generare artefatti elettromiografici. </li><li> Amplificazione del segnale elettrico utilizzando un amplificatore extracellulareli fi catore adatto per la registrazione singola unità, preferibilmente dotata di una modalità di controllo dell&#39;impedenza. Monitorare l&#39;attività chiodare in corso (banda passante di 300 - 5.000 Hz) con due oscilloscopi e un set di altoparlanti amplificati. Qui, i dati registrati vengono digitalizzate continuo ad una frequenza di campionamento di 10 kHz; picchi vengono estratti in linea. </li><li> Avanzare l&#39;elettrodo attraverso l&#39;agarosio finché la punta raggiunge la superficie della corteccia. Osservare questo passaggio attraverso un microscopio. &quot;Zero&quot; questa posizione sul micromanipolatore. </li><li> Per meglio approssimare la profondità della registrazione, confermare visivamente che la punta dell&#39;elettrodo esce dalla superficie corticale ad una profondità di zero quando ritirare l&#39;elettrodo al termine di una penetrazione. NOTA: Una discrepanza tra la lettura manipolatore e la posizione dell&#39;elettrodo osservato indica che è spostata rispetto al tessuto. Questo può essere causato da instabilità del cervello o animale, o manipolatore deriva. <ol><li> Come un ulteriore controllo per garantire chequesto set-punto zero corrisponde alla superficie della corteccia, monitorare potenziali di campo evocati stimolo mentre avanza attraverso le prime diverse centinaia di micrometri di tessuto. Durante il monitoraggio potenziali di campo, abbassare il più basso filtro passa-banda di taglio dell&#39;amplificatore extracellulare a 10 Hz. Verificare che la polarità del potenziale campo locale inverte intorno una profondità di circa 100 micron, in prossimità dello strato di confine I / II 13. Questo è un punto di riferimento affidabile per la corteccia uditiva, ma può essere diverso per le altre aree corticali. </li></ol></li><li> Montare una fibra ottica accoppiata ad una sorgente di luce blu (figura 2) su un micromanipolatore manuale. Utilizzando il microscopio, posizionare la punta della fibra più vicino alla superficie del agarosio possibile. Centrare il fascio in cui l&#39;elettrodo si entra il tessuto. </li><li> Regolare l&#39;uscita della sorgente di luce con una unità di controllo in grado di erogare un TTL (transistor-transistor logic) treno di impulsi di larghezza e duratio specificaton- esempio, un Arduino (figura 3), una scheda PC I / O (come mostrato), o un generatore di impulsi disponibile in commercio. <ol><li> Monitorare il segnale su entrambi oscilloscopi. </li></ol></li><li> Misurare potenza luminosa totale (mW) sulla punta della fibra ottica utilizzando un misuratore di potenza. Utilizzare questo valore per calcolare irradianza (mW / mm 2) dividendo per l&#39;area della sezione trasversale del nucleo della fibra. Iniziare con un valore di intensità nell&#39;intervallo 10 - 15 mW / mm 2 e regolare verso il basso se necessario, fino artefatti sono eliminati. </li><li> Verificare la presenza di manufatti leggeri con l&#39;elettrodo nella agarosio, pronta a entrare nel tessuto. Avviare la ricerca treno di impulsi (ad esempio, 30 impulsi di luce msec, 500 msec intervallo interstimulus (ISI)). <ol><li> Eliminare manufatti leggeri transitori (Figura 4) per riposizionare la fibra ottica rispetto all&#39;elettrodo per cambiare l&#39;angolo della luce incidente. Se artefatti persistono, provare a diminuire la potenza luminosa. In the caso raro che gli artefatti non possono essere completamente eliminati (solo ridotto al minimo), estendere la durata dell&#39;impulso di ricerca. Questo può aiutare a identificare i veri picchi neuronali. </li></ol></li></ol> 3. Dritto PINP-in &#39; <ol><li> Avanzare l&#39;elettrodo lentamente attraverso il cervello ad una velocità di circa 1 micron / sec. Utilizzare un oscilloscopio per monitorare l&#39;attività in corso. Dall&#39;altro, scatenare gli impulsi laser. </li><li> Attendere il &#39;hash&#39; deboli di picchi luce evocata sul monitor audio, che indica che l&#39;elettrodo si avvicina una cella ChR2 + e spesso può essere percepito ben prima del segnale elettrico risulta sull&#39;oscilloscopio. Rallentare la velocità di avanzamento. NOTA: Se la cella si trova direttamente nel percorso dell&#39;elettrodo, l&#39;attività di luce evocata crescerà più grande (e più forte). Come l&#39;elettrodo si avvicina un singolo interneuroni, l&#39;hash risolverà in piccoli ma ben definiti picchi di forma e dimensione uniformi. </li><li> Appena lipicchi GHT-evocato sono grandi abbastanza per innescare della singolarmente sull&#39;oscilloscopio, cominciano a farlo. Regolare la scala sull&#39;asse orizzontale per osservare la forma esatta della forma d&#39;onda di picco. </li><li> Fermarsi e attendere per il tessuto di stabilirsi. Resistete alla tentazione di spostare l&#39;elettrodo più vicino alla cella. Questo passaggio è fondamentale per una registrazione stabile. Se dopo alcuni minuti il ​​rapporto segnale-rumore non è migliorata - cioè, il tessuto è costante, ma non ha portato la cella più vicino alla punta dell&#39;elettrodo - avanzamento 5 micron ulteriormente e attendere. <ol><li> Ripetere questo processo fino a quando la punta o la depressione del potenziale d&#39;azione possono essere catturati in modo affidabile con una soglia di tensione impostata ben al di sopra del rumore di fondo (ad esempio, +/- 300 mV o superiore). NOTA: C&#39;è poco rischio di falsi positivi o ambiguità quando PINPing interneuroni inibitori. La maggior parte delle cellule in grado di seguire facilmente un treno di impulsi di durata di 30 msec e inter-pulIntervallo SE 500 msec, o più veloce, con una certa latenza primo picco di 2-5 msec (Figura 5). La maggior parte delle cellule fotovoltaiche +, in particolare, può sostenere cottura per un totale di 1 sec (Figura 6). Perché questi sono neuroni inibitori, disynaptic attivazione (indiretto) non è una preoccupazione importante. </li></ol></li><li> Durante la registrazione, monitorare l&#39;attività in corso sul primo oscilloscopio. Tieni d&#39;occhio le dimensioni e la forma del picchi utilizzando il secondo oscilloscopio. Si noti la qualità del loro suono sull&#39;altoparlante. <ol><li> Ascoltate con attenzione per i cambiamenti improvvisi, che indicano che la cella è sia volge troppo vicino all&#39;elettrodo (dove rischia di essere impalato o danneggiato), o è alla deriva. Se le punte si allargano e distorta, tornare indietro; se crescono più piccolo, far avanzare l&#39;elettrodo. Si muovono lentamente in 2 micron passi. </li><li> Verificare la qualità della registrazione post-hoc sovrapponendo tutte le forme d&#39;onda di picco, allineati a picco o depressione. Variare le THRES tensionetenere. Attraverso una vasta gamma di valori di soglia, ci dovrebbe essere solo una forma coerente picco di altezza uniforme (figura 5a). </li></ol></li><li> Se in qualsiasi momento il segnale venga contaminato con punte da un neurone vicino, andare avanti. Avanza lentamente, sulla remota possibilità che l&#39;elettrodo passerà fuori dalla portata della cella vicina, ma rimanere nel raggio d&#39;azione del neurone bersaglio. (Questo è di solito senza successo.) </li><li> Spostare l&#39;elettrodo attraverso l&#39;intera profondità della corteccia (900 + micron) in ogni penetrazione. Se non ci sono i neuroni di luce-sensibili si incontrano dopo molti penetrazioni o, al contrario, le registrazioni sono regolarmente contaminati con punte di cellule ChR2- vicini, provare un nuovo elettrodo. NOTA: Anche all&#39;interno di una stessa partita, la geometria impedenza e punta di singoli elettrodi può variare considerevolmente. Entrambi i fattori contribuiscono alla effettiva &quot;ascolto raggio&quot; dell&#39;elettrodo, che deve essere sufficientemente grande per rilevare i picchi di luce evocati whricerca ile, ma limitato sufficiente per permettere la registrazione di un singolo interneuroni in isolamento. </li><li> Testare l&#39;impedenza degli elettrodi lo desideri. Per evitare di danneggiare il tessuto, farlo con la punta dell&#39;elettrodo nella parte superiore dello strato di agarosio, ben al di fuori del cervello. All&#39;interno della gamma di 7-14 MW, l&#39;impedenza esatta non è un sicuro indicatore delle prestazioni, o cedere, per questa applicazione. Detto questo, si tratta di un proxy ragionevole per l&#39;ascolto radio: elettrodi più bassa impedenza raccogliere più unità; elevata impedenza, meno. </li><li> Alla fine dell&#39;esperimento, eutanasia dell&#39;animale mediante istituzionalmente approvati, quali overdose di anestetico, o dislocazione cervicale sotto un profondo piano di anestesia. </li></ol>

<ol>
	<li>Lima, S. Q., Hromadka, T., Znamenskiy, P., Zador, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINP:+a+new+method+of+tagging+neuronal+populations+for+identification+during+in+vivo+electrophysiological+recording.">PINP: a new method of tagging neuronal populations for identification during in vivo electrophysiological recording.</a> PLoS One. 4, (2009).</li><li>Moore, A. K., Wehr, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parvalbumin-expressing+inhibitory+interneurons+in+auditory+cortex+are+well-tuned+for+frequency.">Parvalbumin-expressing inhibitory interneurons in auditory cortex are well-tuned for frequency.</a> J Neurosci. 33, 13713-13723 (2013).</li><li>Merchant, H., de Lafuente, V., Pena-Ortega, F., Larriva-Sahd, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+impact+of+interneuronal+inhibition+in+the+cerebral+cortex+of+behaving+animals.">Functional impact of interneuronal inhibition in the cerebral cortex of behaving animals.</a> Prog Neurobiol. 99, 163-178 (2012).</li><li>Markram, H., 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=Interneurons+of+the+neocortical+inhibitory+system.">Interneurons of the neocortical inhibitory system.</a> Nat Rev Neurosci. 5, 793-807 (2004).</li><li>Atallah, B. V., Bruns, W., Carandini, M., Scanziani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parvalbumin-expressing+interneurons+linearly+transform+cortical+responses+to+visual+stimuli.">Parvalbumin-expressing interneurons linearly transform cortical responses to visual stimuli.</a> Neuron. 73, 159-170 (2012).</li><li>Wilson, N. R., Runyan, C. A., Wang, F. L., Sur, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Division+and+subtraction+by+distinct+cortical+inhibitory+networks+in+vivo.">Division and subtraction by distinct cortical inhibitory networks in vivo.</a> Nature. 488, 343-348 (2012).</li><li>Letzkus, J. 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+disinhibitory+microcircuit+for+associative+fear+learning+in+the+auditory+cortex.">A disinhibitory microcircuit for associative fear learning in the auditory cortex.</a> Nature. 480, 331-335 (2011).</li><li>Pi, H. 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=Cortical+interneurons+that+specialize+in+disinhibitory+control.">Cortical interneurons that specialize in disinhibitory control.</a> Nature. 503, 521-524 (2013).</li><li>Adesnik, H., Bruns, W., Taniguchi, H., Huang, Z. J., Scanziani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neural+circuit+for+spatial+summation+in+visual+cortex.">A neural circuit for spatial summation in visual cortex.</a> Nature. 490, 226-231 (2012).</li><li>Madisen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+toolbox+of+Cre-dependent+optogenetic+transgenic+mice+for+light-induced+activation+and+silencing.">A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing.</a> Nat Neurosci. 15, 793-802 (2012).</li><li>Hippenmeyer, S., 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+developmental+switch+in+the+response+of+DRG+neurons+to+ETS+transcription+factor+signaling.">A developmental switch in the response of DRG neurons to ETS transcription factor signaling.</a> PLoS Biol. 3, 159 (2005).</li><li>Taniguchi, H., 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+resource+of+Cre+driver+lines+for+genetic+targeting+of+GABAergic+neurons+in+cerebral+cortex.">A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex.</a> Neuron. 71, 995-1013 (2011).</li><li>Christianson, G. 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The authors have no competing financial interests.

Anche se PINP è concettualmente semplice, può essere difficile in pratica. Un fattore determinante del successo è la scelta dell&#39;elettrodo. Il raggio di ascolto elettrica è il parametro critico. Deve essere sufficientemente grande per rilevare i picchi di luce evocata quando la punta è ancora lontano da una cella ChR2 +, in modo che si può regolare la velocità di avanzamento di conseguenza. Allo stesso tempo, deve essere limitato sufficiente a consentire un buon isolamento singola unità. Cioè, l&#39;elettro...

Characterizing the myriad cell types that comprise the mammalian brain has been a central, but long-elusive goal of neurophysiology. For instance, the properties and function of different inhibitory cell types in the cerebral cortex are topics of great interest but are still relatively unknown. This is in part because conventional blind in vivo recording techniques are limited in their ability to distinguish between different cell types. Extracellular spike width can be used to separate putative parvalbumin-positive inhibitory neurons from excitatory pyramidal cells, but this method is subject to both type I and type II errors2,3. Alternatively, recorded neurons can be filled, recovered, and stained to later confirm their morphological and molecular identity, but this is a pain-staking and time-consuming process. Recently, genetically identified populations of inhibitory interneurons have become accessible by means of calcium imaging or visually guided patch recordings. In these approaches, viral or transgenic expression of a calcium reporter (such as GCaMP) or fluorescent protein (such as GFP) allows identification and characterization of cell types defined by promoter expression. These approaches use 2-photon microscopy, which requires expensive equipment, and are also limited to superficial cortical layers due to the light scattering properties of brain tissue. 
Recently, Lima and colleagues1 developed a novel application of optogenetics to target electrophysiological recordings to genetically identified neuronal types in vivo, termed &#8220;PINP&#8221; &#8211; or Photostimulation-assisted Identification of Neuronal Populations. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. Unlike many other optogenetic applications, the goal is not to manipulate circuit function but simply to identify neurons belonging to a genetically-defined class, which can then be characterized during normal brain function. The technique can be implemented with standard extracellular recording equipment and can therefore serve as an accessible and inexpensive alternative to calcium imaging or visually-guided patching. Here we describe an approach to PINPing specific cell types in the anesthetized auditory cortex, with the expectation that the more general points can be usefully applied in other preparations and brain regions.
In cortex, PINP holds particular promise for investigating the in vivo response properties of inhibitory interneurons. GABAergic interneurons comprise a small, heterogeneous subset of cortical neurons4. Different subtypes, marked by the expression of particular molecular markers, have recently been shown to perform different computational roles in cortical circuits5-9. As genetic tools improve it may eventually be possible to distinguish morphologically- and physiologically-separable types that fall within these broad classes. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. This strategy was developed specifically for targeting parvalbumin-positive (PV+) cells, but we have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons. Although PINPing is conceptually straightforward, it can be surprisingly unyielding in practice. We learned a number of tips and tricks through trial-and-error that may be useful to others attempting the method.

<table border="0" cellpadding="0" cellspacing="0" width="1482">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Name of Material/Equipment</td>
			<td style="width:171px;">Company</td>
			<td style="width:341px;">Product/Stock Number</td>
			<td style="width:708px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ChR2-EYFP Line</td>
			<td style="width:171px;">Jackson Colonies</td>
			<td style="width:341px;">12569</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pvalb-iCre (PV) Line</td>
			<td style="width:171px;">Jackson Colonies</td>
			<td style="width:341px;">8069</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sst-iCre (SOM) Line</td>
			<td style="width:171px;">Jackson Colonies</td>
			<td style="width:341px;">13044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cr-iCre (CR) Line</td>
			<td style="width:171px;">Jackson Colonies</td>
			<td>10774</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Agarose</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:341px;">A9793</td>
			<td>Type III-A, High EEO</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Micro Point (dural hook)</td>
			<td style="width:171px;">FST</td>
			<td style="width:341px;">10066-15</td>
			<td style="width:708px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Surgical Scissors</td>
			<td style="width:171px;">FST</td>
			<td>14084-09</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Scalpel</td>
			<td style="width:171px;">FST</td>
			<td>10003-12 (handle), 10011-00 (blades)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Puralube Ophthalmic Ointment</td>
			<td style="width:171px;">Foster &#38; Smith</td>
			<td style="width:341px;">9N-76855</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Homeothermic Blanket</td>
			<td style="width:171px;">Harvard Apparatus</td>
			<td style="width:341px;"><a name="RANGE!C11">507220F</a></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tungsten Microelectrodes</td>
			<td style="width:171px;">A-M Systems</td>
			<td>577200</td>
			<td>12 M&#937; AC resistance, 127 &#956;m diameter, 12&#176; tapered tip, epoxy-coated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Capillary Glass Tubing</td>
			<td style="width:171px;">Warner Instruments</td>
			<td style="width:341px;">G150TF-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Heat Shrink Tubing</td>
			<td style="width:171px;">DigiKey</td>
			<td>A332B-4-ND</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Zapit Accelerator</td>
			<td style="width:171px;">DVA</td>
			<td style="width:341px;">SKU ZA/ZAA</td>
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a guide to in vivo single-unit recording from optogenetically identified cortical inhibitory interneurons

Una sfida importante in neurofisiologia è stato quello di caratterizzare le proprietà di risposta e la funzione dei numerosi tipi di cellule inibitoria nella corteccia cerebrale. Siamo qui condividiamo la nostra strategia per l&#39;ottenimento di stabili, ben isolate-single-unit registrazioni da interneuroni inibitori identificati nel topo corticale anestetizzato utilizzando un metodo sviluppato da Lima e colleghi 1. Le registrazioni vengono eseguite in topi che esprimono channelrhodopsin-2 (ChR2) in specifiche sottopopolazioni neuronali. I membri della popolazione sono identificati dal loro risposta ad un breve lampo di luce blu. Questa tecnica - chiamata &quot;PINP&quot;, o Identificazione Fotostimolazione-assistita di popolazioni neuronali - possono essere implementati con equipaggiamento di serie di registrazione extracellulare. Può servire come un&#39;alternativa economica e accessibile per l&#39;imaging calcio o patch visivamente guidato, al fine di indirizzare registrazioni extracellulari alle cellule geneticamente identificati. Here mettiamo a disposizione una serie di linee guida per l&#39;ottimizzazione del metodo nella pratica di tutti i giorni. Abbiamo raffinato la nostra strategia specifica per il targeting (PV +), le cellule parvalbumin-positivo, ma abbiamo trovato che funziona per altri tipi di interneuroni, nonché, come ad esempio (CR +) interneuroni-calretinina esprimono-somatostatina che esprimono (SOM +) e.

Siamo qui condividiamo la nostra strategia per ottenere registrazioni singola unità da interneuroni inibitori geneticamente classificati nel topo corticale anestetizzato, utilizzando un metodo optogenetic sviluppato da Lima et al. Tabella 1 i dettagli cocktail anestetico suggerito, Ketamina-Medetomidina-acepromazina (1. &quot; KMA &quot;). La Figura 1 illustra un microelettrodo di tungsteno, preparato per la registrazione. La figura 2 contiene uno s...

Watch this Scientific Journal Video about A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons at JoVE.com

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

Una guida per<em&gt; In vivo</em&gt; Singola unità di registrazione da Optogenetically Identificato corticale inibitorio interneuroni

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

a-guide-to-vivo-single-unit-recording-from-optogenetically-identified

Research

JoVE Journal

Neuroscience

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

19.0K Views.  University of Oregon. Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching. 

Video: A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

Singola unità In vivo Registrazioni dal chiasma ottico di Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

Ricerca

Neuroscienze

Recording Behavioral Responses to Reflection in Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

Registrazione di risposte comportamentali alla riflessione in gamberi di fiume

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

Impianto chirurgico di elettrodi neurali cronici per la registrazione di attività singola unità e segnali Electrocorticographic

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

La registrazione simultanea dei segnali di calcio da parte dei neuroni identificati e comportamento alimentare di Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

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.

Retrograda Etichettatura fluorescente Consente mirata extracellulare singola unità di registrazione da parte dei neuroni identificati<em&gt; In vivo</em

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

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

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

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

La registrazione dell&#39;attività elettrica dai neuroni identificati nel cervello intatto di pesci transgenici

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

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

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

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

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

Intracellulare di registrazione, sensoriale Campo Mapping, e la coltura neuroni identificata nel Leech, Hirudo medicinalis</i

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

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Sottotipo-selettivo elettroporazione della corticale interneuroni

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

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

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

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

Whole-cell patch-clamp registrazioni da Morphologically- e neurochimico-identificati interneuroni dell&#39;ippocampo

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

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Registrazione simultanea elettrofisiologici e micro-iniezioni di agenti inibitori nel cervello del roditore

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...