Gli autori riconoscono con gratitudine l&#39;aiuto sapiente della signora Trine W. Mikkelsen, la signora M. Lise montaggio, e il personale a Påskehøjgaard. The Danish Medical Research Council, la Fondazione Lundbeck, e la Novo Nordisk Foundation sostenuti finanziariamente lo studio.

anestesia degli animali e euthanesia è stata eseguita in conformità con i &quot;Principi di laboratorio cura degli animali&quot; (NIH pubblicazione n ° 86-23, rivisto 1985) e approvato dal Consiglio danese per la ricerca etica animale. 1. Strumenti <ol><li> Raccogliere gli strumenti presentati nel video ed elencati nella tabella dei materiali. </li></ol> 2. Decapitazione NOTA: L&#39;anestesia è stata indotta mediante iniezione intramuscolare di 5 mL di midazolam (5 mg / ml) e 5 ml di ketamina (25 mg / mL). 5-10 minuti più tardi, quando l&#39;animale era profondamente sedato, una vena dell&#39;orecchio è stata incannulata e overdose letale (100 mg / kg di peso corporeo) di pentobarbital sodio (200 mg / ml) è stato somministrato per via endovenosa. Per garantire che l&#39;animale è stato completamente eutanasia, il dolore riflesso interdigitale è stato testato come mostrato da Ettrup et al. (2011) 20. euthanization completo è stato assicurato come descritto inl&#39;istruzione etica sopra e seguita da una perfusione transcardial con 5 L di soluzione salina isotonica, come dimostrato da Ettrup et al. (2011) 20. Tutte le procedure vengono eseguite dimostrate postmortem, precludendo la necessità per le precauzioni necessarie per l&#39;anestesia benessere a lungo termine e la sopravvivenza postprocedurale. <ol><li> Decapitare il maiale da un alto un&#39;incisione circolare cervicale, utilizzando un bisturi chirurgico, appena sotto l&#39;angolo mandibolare (Figura 1A). </li><li> Ancora con il bisturi chirurgico, continuare l&#39;incisione anteriormente attraverso il tessuto molle del collo, compresa la laringe e dell&#39;esofago, finché la colonna vertebrale ossea viene raggiunto, approssimativamente al livello della giunzione cranio cervicale. </li><li> Avanzare il taglio con un bisturi chirurgico dal lato anteriore della giunzione craniocervicale, sopra l&#39;arco anteriore dell&#39;atlante, e attraverso la membrana atlanto anteriore, esponendo così il canale vertebrale e il midollo spinale (Figura 1B). Allo stesso tempo chiedere un assistente di tirare il corpo del maiale lontano dalla testa di maiale per facilitare l&#39;accesso tra la base del cranio e la prima vertebra cervicale. </li><li> Continuare l&#39;incisione chirurgica attraverso il sacco durale e il midollo spinale (Figura 1B). Prestare particolare attenzione per garantire che una sezione trasversale completa del midollo spinale è raggiunto. NOTA: La mancata esecuzione del passaggio precedente può portare a trazione indesiderati sul midollo spinale e del cervello durante le seguenti fasi del processo di decapitazione. </li><li> Forza estendere la giunzione cranocervical a livello di sezione (Figura 1C). Al tempo stesso, utilizzare il bisturi chirurgico alla sezione restanti legamenti atlanto per rilasciare l&#39;articolazione tra i condili occipitali e il processo articoli superiore dell&#39;atlante. Separare la testa di maiale dal corpo. </li></ol><img alt="Figura 1" src="/files/ftp_upload/55511/55511fig1.jpg" style="font-size: 14px;" /> Figura 1: minipig decapitazione. (A) del collo incisione (freccia, angolo mandibolare). (B) incisione attraverso i legamenti atlanto e il midollo spinale dura circondato (SC) alla giunzione craniocervicale (C1, arco anteriore dell&#39;atlante, OC, condilo occipitale). (C) La parte posteriore dell&#39;articolazione atlanto viene rilasciato da un&#39;estensione forte (frecce) a livello di sezione. <a href="http://ecsource.jove.com/files/ftp_upload/55511/55511fig1large.jpg" style="font-size: 14px;" target="_blank">Si prega di cliccare qui per vedere una versione più grande di questa figura.</a> 3. Teschio di apertura <ol><li> Posizionare la testa di maiale su un tavolo. </li><li> Effettuare un&#39;incisione longitudinale dorsale con un bisturi chirurgico attraverso la pelle e il tessuto molle sottostante dal retro del muso, sul vertice della testa, e giù attraverso la parte posterioreregione occipitale. </li><li> Esporre la parte dorsale e posteriore del cranio rimuovendo il tessuto molle situata lateralmente alla incisione iniziale con un bisturi chirurgico. </li><li> Rilasciare il muscolo temporale bilateralmente dal cranio (Figura 2A) con un bisturi chirurgico. Assicurarsi che l&#39;osso occipitale posteriore è pulita dei tessuti molli. </li><li> Utilizzare l&#39;entrata posteriore del foro occipitale per rimuovere l&#39;osso occipitale con Kerrison osso punzone e Bone Rongeurs ed esporre il cervelletto dura coperto (Figura 2B). </li><li> Ritorno al lato anteriore esposta del cranio e selezionare un punto di ingresso nel dell&#39;osso frontale, proprio di fronte agli occhi. A questo punto, utilizzare uno scalpello osso con un martello per penetrare il cranio e immettere il seno frontale (Figura 2C). </li><li> Utilizzare l&#39;entità del seno frontale per favorire la rimozione della lamina dorsoposterior cranio esterno con una rongeur ossea o punzone ed esporre l&#39;interno, sottilecranio lamina ossea che copre il cervello (Figura 2D). </li><li> Aprire delicatamente la lamina interna cranio osseo anteriormente con un martello e scalpello osso per esporre cervello dura coperto (Figura 2E). </li><li> Continuare la rimozione di osso lateralmente usando uno scalpello osseo e una rongeur osso attraverso l&#39;osso temporale e parietale al fine di rilasciare la parte dorsoposterior finale del cranio, situato tra le parti già esposte del cervello e nel cervelletto dura coperto (Figura 2F). NOTA: E &#39;spesso possibile, durante la fase finale di questa procedura, per usare lo scalpello per rompere il restante posteriore ossa del cranio da una parte all&#39;altra, proprio come uno apre una porta. </li></ol><img alt="figura 2" src="/files/ftp_upload/55511/55511fig2.jpg" style="opacity: 0.9; font-size: 14px;" /> Figura 2: apertura cranio cavia. (A) esposizione della dorsoposteriosuperficie del cranio R, tra cui la rimozione del occipitale e muscoli temporali. (B) La rimozione dell&#39;osso occipitale (CB, dura coperta di cervelletto, OB, osso occipitale, OC, condilo occipitale, e SC, midollo spinale). (C) un martello e uno scalpello osso sono usati per penetrare il cranio anteriormente e per entrare nel seno frontale a livello degli occhi. (D) La portata del seno frontale (FS) viene utilizzato per rimuovere il cranio esterna dell&#39;osso spessore (1), esponendo una sottile lamina ossea interna (2) che copre il cervello. (E) La rimozione del sottile lamina ossea, esponendo il cervello dura coperto (freccia). (F) Infine, un martello e uno scalpello osso vengono utilizzati per il collegamento laterale anteriore e le aperture posteriori cranio. <a href="http://ecsource.jove.com/files/ftp_upload/55511/55511fig2large.jpg" style="font-size: 14px;" target="_blank">Si prega di cliccare qui per vedere una versione più grande di questa figura.</a> 4. Cervello Removal <ol><li> Utilizzare pinze chirurgiche per sollevare la dura e creare un dolce un&#39;incisione vicino alla venoso seno longitudinale superiore utilizzando un bisturi chirurgico fine (Figura 3A). </li><li> Utilizzare micro-forbice o un coltello dura per aprire ulteriormente la dura madre che copre la superficie dorsale del cervello. NOTA: Particolare cura deve essere presa quando si rimuove la dura corrispondente al tentorio cerebellare (figura 3B), come conservazione di questo foglio durale impedirà successiva rimozione cervello. </li><li> Posizionare la testa di maiale verticalmente (Figura 3C). </li><li> Utilizzare lo scalpello osso o un dissettore per rilasciare cervello ventroanterior per via smussa del bulbo olfattivo dal pavimento dura coperto della cavità cranica (Figura 3D). </li><li> Utilizzare un bisturi chirurgico fino al punto chiasma ottico esposta (Figura 3E). Esporre e la sezione del peduncolo ipofisario ei nervi oculomotori. </li><li> Rilasciare il tronco cerebrale ventrale sezionando LOwer nervi cranici (Figura 3F) con una multa bisturi chirurgico. Garantire che tentorio cerebellare durale stato completamente inciso (figura 3B), in quanto questa foglia durale sarà altrimenti tagliare il tronco cerebrale durante il processo di rilascio. </li></ol><img alt="Figura 3" src="/files/ftp_upload/55511/55511fig3.jpg" style="font-size: 14px;" /> Figura 3: rimozione cervello cavia. Apertura (A) durale con pinze chirurgiche e un coltello dura. (B) Bisogna fare attenzione a incidere completamente la foglia durale (freccia), che si trova tra il cervello e il cervelletto. (C) La testa di maiale è in posizione verticale per una migliore visualizzazione delle strutture di base del cranio e al fine di gravità per aiutare lo spostamento previsto del cervello. (D) Un dissettore o uno scalpello osso viene usato per alleviare il bulbo olfattivo dalla sezione smussata dalla dura madre-coverebase del cranio d. (E) La dissezione viene continuata in direzione posteriore lungo la base del cranio per l&#39;esposizione e sezionamento del chiasma ottico (freccia), gambo infundibolare e nervi oculomotori. (F) Il rilascio cervello è completato con la sezione dei nervi cranici inferiori come si allontanano dalla superficie ventrale del tronco cerebrale (III, oculomotore, IV, nervo trocleare, V, nervo trigemino e VI, abducente nervi). <a href="http://ecsource.jove.com/files/ftp_upload/55511/55511fig3large.jpg" style="font-size: 14px;" target="_blank">Si prega di cliccare qui per vedere una versione più grande di questa figura.</a> 5. Rimozione pituitaria <ol><li> Identificare il peduncolo ipofisario ei suoi dintorni foglia durale (la sellae diapragma) nel pavimento cranio (Figura 4A). </li><li> Incidere la foglia laterali durale al peduncolo ipofisario utilizzando una multa bisturi chirurgico (Figura 4B). </li><li> Utilizzare un dissettore per rilasciare la fossauitary ed estrarlo dell&#39;ipofisi fossa (Figura 4C). </li></ol><img alt="Figura 4" src="/files/ftp_upload/55511/55511fig4.jpg" style="opacity: 0.9; font-size: 14px;" /> Figura 4: rimozione pituitaria cavia. (A) La fossa pituitaria (*) è identificato nel piano cranio (1, bulbo olfattivo, 2, chiasma ottico e PF, fossa cranica posteriore). (B) La copertura durale (diagphragm Sellar, (freccia)) viene incisa lateralmente. (C) L&#39;ipofisi (freccia) viene rilasciato con un dissettore e tirato fuori dalla fossa pituitaria. barra di scala (AC) = 10 mm. <a href="http://ecsource.jove.com/files/ftp_upload/55511/55511fig4large.jpg" style="font-size: 14px;" target="_blank">Si prega di cliccare qui per vedere una versione più grande di questa figura.</a>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+substantia+nigra+pars+compacta+of+the+minipig:+An+anatomical+and+stereological+study.">The substantia nigra pars compacta of the minipig: An anatomical and stereological study.</a> Brain Struct Funct. (4-5), 481-488 (2009).</li><li>S&#248;rensen, J. C., Bjarkam, C. R., Simonsen, C. Z., Danielsen, E., Geneser, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oriented+sectioning+of+irregular+tissue+blocks+in+relation+to+computerized+scanning+modalities.+Results+from+the+domestic+pig+brain.">Oriented sectioning of irregular tissue blocks in relation to computerized scanning modalities. Results from the domestic pig brain.</a> J Neurosci Methods. 104, 93-98 (2000).</li><li>Bjarkam, C. R., Pedersen, M., S&#248;rensen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+strategies+for+embedding,+orientation+and+sectioning+of+small+brain+specimens+enable+direct+correlation+to+MR-images,+brain+atlases,+or+use+of+unbiased+stereology.">New strategies for embedding, orientation and sectioning of small brain specimens enable direct correlation to MR-images, brain atlases, or use of unbiased stereology.</a> J Neurosci Methods. 108, 153-159 (2001).</li><li>Bjarkam, C. R., S&#248;rensen, J. C., Geneser, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+and+morphology+of+serotonin-immunoreactive+axons+in+the+hippocampal+region+of+the+New+Zealand+white+rabbit.+I.+Area+dentata+and+hippocampus+proper.">Distribution and morphology of serotonin-immunoreactive axons in the hippocampal region of the New Zealand white rabbit. I. Area dentata and hippocampus proper.</a> Hippocampus. 13 (1), 21-37 (2003).</li><li>Bjarkam, C. R., Glud, A. N., Orlowski, D., S&#248;rensen, J. C., Palomero-Gallagher, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+telencephalon+of+the+minipig,+cytoarchitecture+and+cortical+surface+anatomy.">The telencephalon of the minipig, cytoarchitecture and cortical surface anatomy.</a> Brain Struct Funct. , (2016).</li><li>Boltze, J., Nitzsche, B., Geiger, K. D., Schoon, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathological+investigation+of+different+MCAO+modalities+and+impact+of+autologous+bone+marrow+mononuclear+cell+administration+in+an+ovine+stroke+model.">Histopathological investigation of different MCAO modalities and impact of autologous bone marrow mononuclear cell administration in an ovine stroke model.</a> Transl Stroke Res. 2, 279-293 (2011).</li><li>Jortner, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+return+of+the+dark+neuron.+A+histological+artifact+complicating+contemporary+neurotoxicologic+evaluation.">The return of the dark neuron. A histological artifact complicating contemporary neurotoxicologic evaluation.</a> Neurotoxicology. 27, 628-634 (2006).</li></ol>

Gli autori non hanno nulla da rivelare.

La maggior parte degli studi di neuroscienze sperimentali vengono eseguiti in piccole specie animali, come topi e ratti, in cui l&#39;accesso al sistema nervoso centrale è facilitato da un sottile skull- e durale spessore. Tuttavia, in animali da esperimento più grandi come maiali 1, 4, 8, pecore 32, e primati non umani, l&#39;elevato spessore di queste strutture richiede l&#39;uso di strumenti robusti...

<html> studi neuroscienze traslazionale nei suini sono diventati sempre più popolari nel corso degli ultimi due decenni. Le grandi dimensioni del cervello di maiale consente l&#39;utilizzo di convenzionali imager cerebrali clinica e l&#39;uso diretto e sperimentazione di procedure neurochirurgiche ed attrezzature dalla clinica umana 1, 2, 3, 4, 5, 6, 7, 8. Negli ultimi 20 anni, maiali, specialmente maialino (ad esempio, Göttingen cavia), sono stati utilizzati per esaminare modalità di trattamento neuromodulatori, come il trapianto di cellule staminali; Vettore virale trasfezione; e la stimolazione cerebrale profonda diretta verso il morbo di Parkinson, l&#39;obesità, la depressione, e la malattia di Alzheimer 2, 6,= &quot;xref&quot;&gt; 9, 10, 11, 12, 13, 14, 15, 16, 17. Questo è stato seguito dallo sviluppo di approcci stereotassica e chirurgici per manipolare cavia CNS 3, 18, 19, 20, 21. I cambiamenti del SNC istituiti sono state valutate in animali vivi mediante imaging cerebrale (PET 10, 13, 22, 24 e MR 23), cistometria 11, 12, 25, analisi del passo17, la valutazione neurologica 9, 17, e l&#39;esame autoptico basato su istologia e analisi stereologica 14, 15, 17, 26, 27, 31. Tuttavia, l&#39;analisi post-mortem richiede l&#39;esposizione e la rimozione del cervello di maiale, che non è un compito facile, come una spessa cranio osseo e durale fibroso che copre circondano il cervello di maiale. L&#39;obiettivo di questo documento e video didattico è quello di descrivere come il cervello post-mortem di suini e dell&#39;ipofisi possono essere esposti e rimossi in uno stato integro in 15-20 minuti utilizzando strumenti chirurgici non motorizzati. Il video didattico e illustrazioni fotografiche mostrano cavie di sesso maschile (età: 6 mesi, a corpo libero: 20-25 kg) utilizzato per uno studio anatomico sulla ghiandola pituitaria cavia.

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			<td height="21" style="height:21px;width:216px;">Heavy Scalpel Handle #4</td>
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			<td style="width:481px;">Good for skin incision and soft tissue removal</td>
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			<td>Optimal for dural incision and precision work</td>
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			<td height="21" style="height:21px;">Non-Sterile Scalpel Blades #11</td>
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			<td style="width:191px;">Aesculap Neurosurgery</td>
			<td style="width:149px;">FF713R</td>
			<td>Must be robust, bite size 3-5 mm</td>
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			<td height="21" style="height:21px;">Bone Rongeur</td>
			<td>Aesculap Neurosurgery</td>
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			<td>Must be robust, bite size 15 x 5 mm</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bone Rongeur</td>
			<td>Aesculap Neurosurgery</td>
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			<td>Must be robust, bite size 25 x 15 mm&#160;</td>
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			<td height="21" style="height:21px;">Bone Chisel</td>
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			<td height="21" style="height:21px;">Mallet (Hammer)</td>
			<td>Millarco</td>
			<td>5624108</td>
			<td>Weigth 300 g, length 30 cm, head hit area size 2 x 2 cm</td>
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			<td height="21" style="height:21px;">Micro-Scissor</td>
			<td>FST&#160;</td>
			<td>14002-14&#160;&#160;</td>
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			<td height="21" style="height:21px;">Dissector</td>
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		<tr height="42">
			<td height="42" style="height:42px;">&#160;G&#246;ttingen minipigs</td>
			<td style="width:191px;">&#160;Ellegaard G&#246;ttingen Minipigs A/S, Denmark</td>
			<td style="width:149px;"></td>
			<td></td>
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			<td height="21" style="height:21px;">Euthanimal</td>
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			<td>pentobarbital</td>
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			<td height="21" style="height:21px;">Ketamine</td>
			<td style="width:191px;">Pfizer</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Midazolam&#160;</td>
			<td style="width:191px;">Hameln Pharmaceuticals</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
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</table>

exposure of the pig cns for histological analysis: a manual for decapitation, skull opening, and brain removal

Suini sono diventate sempre più popolari in larga animale ricerca traslazionale neuroscienze come sostituto economicamente ed eticamente fattibile primati non umani. La dimensione del cervello di grandi dimensioni del maiale permette l&#39;utilizzo di convenzionali imager cervello clinica e l&#39;uso diretto e la sperimentazione di procedure e attrezzature neurochirurgiche dalla clinica umana. Ulteriori analisi macroscopica e istologica, tuttavia, richiede l&#39;esposizione post mortem del maiale sistema nervoso centrale (SNC) e successiva rimozione del cervello. Questo non è un compito facile, come il maiale CNS è incapsulato da una fitta, il cranio ossuto e della colonna vertebrale. L&#39;obiettivo di questo documento e video didattico è quello di descrivere come esporre e rimuovere il cervello post-mortem di suini e la ghiandola pituitaria in uno stato integro, adatto per la successiva analisi macroscopica e istologica.

Per evitare che il materiale del tessuto si secchi, si consiglia di conservare il cervello rimosso e pituitaria in un vaso riempito di fissativo o soluzione salina isotonica immediatamente dopo l&#39;analisi macroscopica è stata eseguita. Il materiale tessuto può essere immagazzinato nel fissativo per anni, mentre stoccaggio in soluzione salina isotonica, anche in frigorifero, porterà a decadimento dei tessuti con il tempo. ...

Watch this Scientific Journal Video about Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal at JoVE.com

Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal

L&#39;obiettivo di questo documento e video didattico è quello di descrivere come esporre e rimuovere il post-mortem del cervello di maiale e ghiandola pituitaria in uno stato integro, adatto per la successiva analisi macroscopica e istologica.

L&#39;esposizione del Maiale CNS per l&#39;analisi istologica: Un manuale per la decapitazione, Teschio di apertura, e Cervello di rimozione

Pigs have become increasingly popular in large-animal translational neuroscience research as an economically and ethically feasible substitute to ...

exposure-pig-cns-for-histological-analysis-manual-for-decapitation

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Unit 1

The Axial Skeleton

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15.3K Views.  Aalborg University Hospital.  L'obiettivo di questo documento e video didattico è quello di descrivere come esporre e rimuovere il post-mortem del cervello di maiale e ghiandola pituitaria in uno stato integro, adatto per la successiva analisi macroscopica e istologica. 

Video: Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

Un lucido e rinforzato Assottigliato cranio-Window a lungo termine Imaging del cervello di topo

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Ricerca

Neuroscienze

Co-analysis of Brain Structure and Function using fMRI and Diffusion-weighted Imaging

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when studying the brain, as the functional role that a brain area fulfills may be largely defined by its connections to other brain areas. In this report, we describe a novel, non-invasive approach for relating brain structure and function using magnetic resonance imaging (MRI). This approach, a combination of structural imaging of long-range fiber connections and functional imaging data, is illustrated in two distinct cognitive domains, visual attention and face perception. Structural imaging is performed with diffusion-weighted imaging (DWI) and fiber tractography, which track the diffusion of water molecules along white-matter fiber tracts in the brain (Figure 1). By visualizing these fiber tracts, we are able to investigate the long-range connective architecture of the brain. The results compare favorably with one of the most widely-used techniques in DWI, diffusion tensor imaging (DTI). DTI is unable to resolve complex configurations of fiber tracts, limiting its utility for constructing detailed, anatomically-informed models of brain function. In contrast, our analyses reproduce known neuroanatomy with precision and accuracy. This advantage is partly due to data acquisition procedures: while many DTI protocols measure diffusion in a small number of directions (e.g., 6 or 12), we employ a diffusion spectrum imaging (DSI)1, 2 protocol which assesses diffusion in 257 directions and at a range of magnetic gradient strengths. Moreover, DSI data allow us to use more sophisticated methods for reconstructing acquired data. In two experiments (visual attention and face perception), tractography reveals that co-active areas of the human brain are anatomically connected, supporting extant hypotheses that they form functional networks. DWI allows us to create a "circuit diagram" and reproduce it on an individual-subject basis, for the purpose of monitoring task-relevant brain activity in networks of interest.

We describe a novel approach for simultaneous analysis of brain function and structure using magnetic resonance imaging (MRI). We assess brain structure with high-resolution diffusion-weighted imaging and white-matter fiber tractography. Unlike standard structural MRI, these techniques allow us to directly relate anatomical connectivity to functional properties of brain networks.

Co-analisi della struttura e delle funzioni cerebrali con fMRI e Diffusion-weighted imaging

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when ...

A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, hippocampus, and the associated parahippocampal regions (perirhinal, entorhinal,&#160;and parahippocampal proper). Unlike most other tracing protocols available, typically focusing on certain MTL areas (e.g., amygdala and/or hippocampus), the integrative perspective adopted by the present tracing guidelines allows for clear localization of all MTL subregions. By integrating information from a variety of sources, including extant tracing protocols separately targeting various MTL structures, histological reports, and brain atlases, and with the complement of illustrative visual materials, the present protocol provides an accurate, intuitive, and convenient guide for understanding the MTL anatomy. The need for such tracing guidelines is also emphasized by illustrating possible differences between automatic and manual segmentation protocols. This knowledge can be applied toward research involving not only structural MRI investigations but also structural-functional colocalization and fMRI signal extraction from anatomically defined ROIs, in healthy and clinical groups alike.

The present work provides a comprehensive set of guidelines for manually tracing the medial temporal lobe (MTL) structures. This protocol can be applied to research involving structural and/or combined structural-functional magnetic resonance imaging (MRI) investigations of the MTL, in both healthy and clinical groups.

Un protocollo completo per Segmentazione manuale delle Strutture mediale del lobo temporale

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

Analizzando Synaptic Modulazione di<em&gt; Drosophila melanogaster</em&gt; Fotorecettori dopo l&#39;esposizione alla luce prolungata

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

High-throughput analisi del comportamento motorio nell'analisi di Isola della drosofila

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

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

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

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

Registrazione di plasticità sinaptica in fettine ippocampali Acute mantenuti in un piccolo volume riciclaggio - aspersione- e sistema di immersione-tipo camera

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

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

Romanzo passivo metodi di compensazione per la produzione rapida di trasparenza ottica nell'intero tessuto dello SNC

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Impianto cronico di tutto-corticale Electrocorticographic Array nel Marmoset comune

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Extraction and Dissection of the Domesticated Pig Brain

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular systems, gastrointestinal systems, and nutrition, and the pig is increasingly being used as a large animal model in neuroscience. Furthermore, the pig is an accepted model to study neurodevelopment as it displays brain growth and development patterns similar to what occurs in humans. As a less common animal model in neuroscience, surgical and dissection procedures on pigs may not be as familiar or well-practiced among researchers. Therefore, a standardized visual protocol detailing consistent extraction and dissection methods may prove valuable for researchers working with the pig. The following video showcases a technique to remove the pig brain while keeping the cortex and brainstem intact and reviews methods to dissect several commonly investigated brain regions including the brainstem, cerebellum, midbrain, hippocampus, striatum, thalamus, and medial prefrontal cortex. The purpose of this video is to provide researchers with the tools and knowledge necessary to consistently perform a brain extraction and dissection on the four-week-old pig.

This protocol details the technique for removal of the pig brain in its entirety and dissection of several brain regions commonly studied in neuroscience.

Estrazione e dissezione del cervello di maiale addomesticato

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular ...

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.
Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological&#160;approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

Imaging in vivo del tessuto cerebrale completamente attivo in larve di zebrafish svegli e giovani mediante rimozione del cranio e della pelle

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...