Die Autoren erkennen dankbar die geschickte Unterstützung von Frau Trine W. Mikkelsen, Frau Lise M. Fitting, und das Personal an Påskehøjgaard. Das dänische Medical Research Council, die Lundbeck-Stiftung, und die Novo Nordisk Foundation finanziell unterstützten die Studie.

Tier Anästhesie und euthanesia wurden gemäß „Principles of Labortierhaltung“ (NIH Publikation Nr 86-23, überarbeitet 1985) und genehmigt vom dänischen Rat für Tierforschung Ethik durchgeführt. 1. Instrumente <ol><li> Sammeln Sie die Instrumente in dem Video dargestellt und in der Tabelle der Materialien aufgeführt. </li></ol> 2. Enthauptung HINWEIS: Die Anästhesie wurde durch eine intramuskuläre Injektion von 5 ml Midazolam (5 mg / ml) und 5 ml Ketamin (25 mg / ml) induziert. 5-10 min später, als das Tier sediert tief war, wurde eine kanülierte Ohrvene und eine letale Überdosis (100 mg / kg Körpergewicht) von Natriumpentobarbital (200 mg / ml) wurde intravenös verabreicht. Um sicherzustellen , dass das Tier vollständig eingeschläfert wurde, wurde der Kammschmerzreflex , wie durch Ettrup et al getestet. (2011) 20. Komplette Euthanasie wurde wie beschrieben gewährleistet inDie Ethik - Aussage über und durch eine transkardialer Perfusion mit 5 l isotonischer Kochsalzlösung folgte, wie von Ettrup et al. (2011) 20. Alle zeigten Verfahren sind Obduktion durchgeführt, Ausschließen der Notwendigkeit, dass die Wohlfahrtsmaßnahmen notwendig für die Langzeit Anästhesie und postprocedural Überleben. <ol><li> Köpft das Schwein durch einen hohen Kreis cervikale Inzision, ein chirurgisches Skalpell, direkt unterhalb dem Unterkieferwinkel (1A). </li><li> Immer noch mit dem chirurgischen Skalpell, weiterhin die Inzision ventral durch das weiche Gewebe des Halses einschließlich dem Larynx und die Speiseröhre, bis die knöcherne Wirbelsäule erreicht ist, etwa auf der Höhe des kraniozervikalen Übergangs. </li><li> Voraus, um den Schnitt mit einem Chirurgisches Skalpell aus der vorderen Seite des kraniozervikalen Übergangs über dem anterioren Bogen von Atlas, und durch das vordere atlanto Membran, um dadurch den Spinalkanal Belichtungs- und das Rückenmark (1B). Gleichzeitig einen Assistenten bitten, den Schweinekörper vom Schwein Kopf zu ziehen, den Zugang zwischen der Schädelbasis und dem ersten Halswirbel zu erleichtern. </li><li> Weiterhin den chirurgischen Schnitt durch den Duralsack und dem Rückenmark (1B). Besondere Vorsicht, um sicherzustellen, dass ein vollständiger Querschnitt des Rückenmarks erreicht wird. HINWEIS: Bei Nichtbeachtung des vorhergehenden Schritt durchzuführen, in unerwünschtem Zuge auf das Rückenmark und Gehirn während der folgenden Schritte des Prozesses Dekapitation führen. </li><li> Zwangsweise erweitert den cranocervical Übergang an der Abschnittsebene (1C). Zur gleichen Zeit, verwenden, um das chirurgische Skalpell Abschnitt die restlichen Bänder atlanto das Gelenk zwischen dem Condylus occipitalis zu lösen und dem oberen artikulierten Prozess des Atlas. Trennt den Schweinekopf aus dem Körper. </li></ol><img alt="Abbildung 1" src="/files/ftp_upload/55511/55511fig1.jpg" style="font-size: 14px;" /> Abbildung 1: Minipig Enthaupten. (A) Halsschnitt (Pfeil, Kieferwinkel). (B) durch die Incision atlanto Bänder und die Dura-Rückenmark umgibt (SC) an dem kraniozervikalen Übergang (C1, anteriore Bogen von Atlas; OC, occipitalen Kondylen). (C) Der hintere Teil des Gelenkes atlanto wird durch eine kraftvolle Erweiterung (Pfeile) auf der Abschnittsebene freigegeben. <a href="http://ecsource.jove.com/files/ftp_upload/55511/55511fig1large.jpg" style="font-size: 14px;" target="_blank">Bitte klicken Sie hier</a> , um <a href="http://ecsource.jove.com/files/ftp_upload/55511/55511fig1large.jpg" style="font-size: 14px;" target="_blank">eine größere Version dieser Figur zu sehen.</a> 3. Schädelöffnung <ol><li> Positionieren Sie den Schweinekopf auf einem Tisch. </li><li> Führte einen dorsalen Längsinzision mit einem chirurgischen Skalpell durch die Haut und das darunterliegende Weichgewebe von der Rückseite des Mauls, über den Scheitel des Kopfes, und nach unten durch den hinteren Teildie Okzipitalregion. </li><li> Belichten des dorsalen und posterioren Teil des Schädels durch das Weichgewebe zu entfernen, um den anfänglichen Einschnitt mit einem chirurgischen Skalpell entfernt lateral. </li><li> Lassen Sie den Schläfenmuskel bilateral aus dem Schädel (2A) mit einem chirurgischen Skalpell. Stellen Sie sicher, dass der hintere Hinterhauptsbein ist aus weichem Gewebe gereinigt. </li><li> Verwenden Sie die posterior Eingang des Foramen Magnum okzipitalen Knochen mit einem Kerrison Knochenstanze und Knochen Rongeurs belichten und die Dura-bedeckten Cerebellum (2B) zu entfernen. </li><li> Kehrt zu der freiliegenden vorderen Seite des Schädels und wählen einen Eintrittspunkt in dem Stirnbein, direkt vor den Augen. An diesem Punkt wird eine Knochenmeißel mit einem Hammer den Schädel eindringt und die Stirnhöhle (2C) eingegeben werden . </li><li> Verwenden, um die Ausdehnung der Stirnhöhle das dorsoposterior Entfernen des äußeren Schädel Lamina mit einer Knochen-Rongeur oder Knochenstanze zu fördern und setzen die inneren, dünnenknöcherner Schädel Lamina das Cerebrum Belag (Figur 2D). </li><li> Öffnet sanft den inneren knöchernen Schädel lamina ventral mit einem Hammer und einem Meißel Knochen zur Dura bedeckte Cerebrum (Abbildung 2E) zu belichten. </li><li> Weiterhin die Knochenentfernung seitlich einen Knochen Meißel und einen Knochen - Rongeur durch die zeitliche und Scheitelbein , um das endgültige dorsoposterior Teil des Schädels freizugeben, der sich zwischen den bereits belichteten Teile der dura bedeckten Cerebrum und Cerebellum (Figur 2F). HINWEIS: Es ist oft möglich, im letzten Schritt dieses Verfahrens, den Meißel zu verwenden, öffnen Sie die restlichen hinteren Schädelknochen von einer Seite zu brechen, so wie man eine Tür öffnet. </li></ol><img alt="Figur 2" src="/files/ftp_upload/55511/55511fig2.jpg" style="opacity: 0.9; font-size: 14px;" /> Abbildung 2: Minipig Schädelöffnung. (A) Die Exposition der dorsoposterior Schädeloberfläche, einschließlich der Entfernung des okzipitalen und temporalen Muskeln. (B) Entfernen des Hinterhauptbeins (CB, dura bedeckten Cerebellum; OB, okzipitalen Knochen; OC, occipitalen Kondylen, und SC, Rückenmark). (C) ein Hammer und ein Meißel Knochen verwendet , um den Schädel ventral zu durchdringen und die Stirnhöhle auf der Höhe der Augen ein. (D) Das Ausmaß der Stirnhöhle (FS) werden verwendet , um die äußeren dicken Schädelknochen (1) zu entfernen, eine inneren dünnen Knochenplättchen (2) , die die Cerebrum aussetzt. (E) Entfernen der dünnen Knochen Lamina, Freilegung der Dura bedeckten Cerebrum (Pfeil). (F) Schließlich wird ein Hammer und ein Knochenmeißel verwendet , um die ventral seitlich anschließen und die hinteren Schädelöffnungen. <a href="http://ecsource.jove.com/files/ftp_upload/55511/55511fig2large.jpg" style="font-size: 14px;" target="_blank">Bitte klicken Sie hier</a> , um <a href="http://ecsource.jove.com/files/ftp_upload/55511/55511fig2large.jpg" style="font-size: 14px;" target="_blank">eine größere Version dieser Figur zu sehen.</a> 4. Gehirn Removal <ol><li> Verwenden chirurgische Zange der Dura heben und einen sanften Einschnitt nahe dem venösen Sinus sagittalis superior mit einem feinen Chirurgisches Skalpell (3A) zu erstellen. </li><li> Verwenden Mikro Schere oder ein Dura-Messer weiter die Dura öffnen Abdecken der dorsale Oberfläche des Gehirns. HINWEIS: Besondere Vorsicht ist geboten, wenn die Dura an das Klein tentorium (3B) entsprechende Entfernung, wie Erhaltung dieses dural Blattes wird nachfolgende Gehirnentfernung zu verhindern. </li><li> Platzieren Sie den Schweinekopf vertikal (3C). </li><li> Verwenden , um die Knochenmeißel oder Dissektor die ventroanterior Cerebrum zur Freigabe durch stumpfe Dissektion des Riechkolbens aus dem Dura-bedeckten Boden der Schädelhöhle (Abbildung 3D). </li><li> Verwenden , um ein feines Chirurgisches Skalpell Abschnitt des belichteten Chiasma (Figur 3E). Freizulegen, und den Abschnitt Hypophysenstiels und die oculomotor Nerven. </li><li> Lassen Sie die ventrale brainstem durch die lo sectioningWer Hirnnerven (Abbildung 3F) mit einem feinen chirurgischen Skalpell. Sicherstellen , dass die duralen cerebellar tentorium vollständig eingeschnitten wurde (3B), da diese sonst durch durale Blatthirnstamm während des Freisetzungsprozesses geschnitten wird. </li></ol><img alt="Abbildung 3" src="/files/ftp_upload/55511/55511fig3.jpg" style="font-size: 14px;" /> Abbildung 3: Minipig Hirnentfernung. (A) Dural Öffnung mit chirurgischer Pinzette und ein dura Messer. (B) Es muss vollständig genommen werden , um den Dural Blatt (Pfeil) einzuschneiden, zwischen Gehirn- und Cerebellum. (C) Der Schweinekopf positioniert ist , vertikal zur besseren Visualisierung der Schädelbasisstrukturen und damit die Schwerkraft in der beabsichtigten Verschiebung des Gehirns zu unterstützen. (D) Ein Dissektor oder ein Knochenmeißel wird verwendet , um den Riechkolben durch stumpfe Sektion von den Dura-covere zu entlastend Schädelbasis. (E) Die Präparation wird in einer posterioren Richtung entlang der Schädelbasis für die Belichtung und Sektionierung des Chiasma (Pfeil), infundibular Stiel und oculomotor Nerven fortgesetzt. (F) Das Gehirn Freisetzung wird mit dem Abschnitt der unteren Hirnnerven abgeschlossen , wie sie von der ventralen Oberfläche der brainstem abgehen (III, Oculomotorius; IV, Trochlearis; V, Trigeminus und VI, Abducens). <a href="http://ecsource.jove.com/files/ftp_upload/55511/55511fig3large.jpg" style="font-size: 14px;" target="_blank">Bitte klicken Sie hier</a> , um <a href="http://ecsource.jove.com/files/ftp_upload/55511/55511fig3large.jpg" style="font-size: 14px;" target="_blank">eine größere Version dieser Figur zu sehen.</a> 5. Hypophysen Removal <ol><li> Identifizieren Sie den Hypophysenstiel und seine Umgebung dural Blatt (die diapragma sellae) im Schädelboden (4A). </li><li> Einzuschneiden die durale Blatt lateral den Hypophysenstiels ein feines chirurgisches Skalpell (4B) verwendet wird . </li><li> Verwenden Sie einen Dissektor die Grube zu lösenuitary und hebt ihn aus der Hypophyse fossa (4C). </li></ol><img alt="Abbildung 4" src="/files/ftp_upload/55511/55511fig4.jpg" style="opacity: 0.9; font-size: 14px;" /> Abbildung 4: Minipig Hypophysen Entfernung. (A) Die Hypophyse fossa (*) in dem Schädelboden identifiziert (1, Bulbus olfactorius, 2, Chiasma; und PF, hintere Schädelgrube). (B) Die durale Abdeckung (sellar diagphragm, (Pfeil)) seitlich eingeschnitten. (C) Die Hypophyse (Pfeil) mit einem Dissektor freigegeben und aus der Hypophyse Fossa angehoben. Maßstabsbalken (AC) = 10 mm. <a href="http://ecsource.jove.com/files/ftp_upload/55511/55511fig4large.jpg" style="font-size: 14px;" target="_blank">Bitte klicken Sie hier</a> , um <a href="http://ecsource.jove.com/files/ftp_upload/55511/55511fig4large.jpg" style="font-size: 14px;" target="_blank">eine größere Version dieser Figur zu sehen.</a>

<ol>
	<li>Lind, M. N., Moustgaard, A., Jelsing, J., Vajta, G., Cumming, P., Hansen, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+pigs+in+neuroscience:+Modeling+brain+disorders.">The use of pigs in neuroscience: Modeling brain disorders.</a> Neurosci Biobehav Rev. 31, 728-751 (2007).</li><li>Bjarkam, C. R., 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=Neuromodulation+in+a+minipig+model+of+Parkinson+disease.">Neuromodulation in a minipig model of Parkinson disease.</a> British J Neurosurg. 22 (Suppl. 1), S9-S12 (2008).</li><li>Bjarkam, C. R., Cancian, G., Glud, A. N., Ettrup, K. S., &#216;stergaard, L., 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=MRI-guided+stereotaxic+targeting+in+pigs+based+on+a+stereotaxic+localizer+box+fitted+with+an+isocentric+frame+and+use+of+SurgiPlan+computer-planning+software.">MRI-guided stereotaxic targeting in pigs based on a stereotaxic localizer box fitted with an isocentric frame and use of SurgiPlan computer-planning software.</a> J Neurosci Methods. 183 (2), 119-126 (2009).</li><li>Sauleau, P., Lapouble, E., Val-Laillet, D., Malbert, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pig+model+in+brain+imaging+and+neurosurgery.">The pig model in brain imaging and neurosurgery.</a> Animal. 3 (8), 1138-1151 (2009).</li><li>Bjarkam, C. R., 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=Safety+and+function+of+a+new+clinical+intracerebral+microinjection+instrument+for+stem+cells+and+therapeutics+examined+in+the+Göttingen+minipig.">Safety and function of a new clinical intracerebral microinjection instrument for stem cells and therapeutics examined in the Göttingen minipig.</a> Stereotact Funct Neurosurg. 88 (1), 56-63 (2010).</li><li>Fjord-Larsen, 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=Long-term+delivery+of+nerve+growth+factor+by+encapsulated+cell+biodelivery+in+the+minipig+basal+forebrain.">Long-term delivery of nerve growth factor by encapsulated cell biodelivery in the minipig basal forebrain.</a> Mol Therapy. 18 (12), 2164-2172 (2010).</li><li>S&#248;rensen, J. C., 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=Development+of+neuromodulation+treatments+in+a+large+animal+model+-+Do+neurosurgeons+dream+of+electric+pigs?.">Development of neuromodulation treatments in a large animal model - Do neurosurgeons dream of electric pigs?.</a> Prog Brain Res. 194, 97-103 (2011).</li><li>Dolezalova, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pig+models+of+neurodegenerative+disorders:+utilization+in+cell+replacement-based+preclinical+safety+and+efficacy+studies.">Pig models of neurodegenerative disorders: utilization in cell replacement-based preclinical safety and efficacy studies.</a> J Comp Neurol. 522 (12), 2784-2801 (2014).</li><li>Mikkelsen, M., Moller, A., Jensen, L. H., Pedersen, A., Harajehi, J. B., Pakkenberg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MPTP-induced+Parkinsonism+in+minipigs:+A+behavioral,+biochemical,+and+histological+study.">MPTP-induced Parkinsonism in minipigs: A behavioral, biochemical, and histological study.</a> Neurotoxicol Teratol. 21, 169-175 (1999).</li><li>Danielsen, E. 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=The+DaNEX+study+of+embryonic+mesencephalic,+dopaminergic+tissue+grafted+to+a+minipig+model+of+Parkinson's+disease:+Preliminary+findings+of+effect+of+MPTP+poisoning+on+striatal+dopaminergic+markers.">The DaNEX study of embryonic mesencephalic, dopaminergic tissue grafted to a minipig model of Parkinson's disease: Preliminary findings of effect of MPTP poisoning on striatal dopaminergic markers.</a> Cell Transplant. 9 (2), 247-259 (2000).</li><li>Dalmose, A., Bjarkam, C. R., S&#248;rensen, J. C., J&#248;rgensen, T. M., Djurhuus, 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=Effects+of+high+frequency+deep+brain+stimulation+on+urine+storage+and+voiding+function+in+conscious+minipigs.">Effects of high frequency deep brain stimulation on urine storage and voiding function in conscious minipigs.</a> Neurourol Urodyn. 23 (3), 265-272 (2004).</li><li>Dalmose, A., Bjarkam, C. R., Djurhuus, 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=Stereotactic+electrical+stimulation+of+the+pontine+micturition+center+in+the+pig.">Stereotactic electrical stimulation of the pontine micturition center in the pig.</a> Br J Urol. 95, 886-889 (2005).</li><li>Andersen, F., Watanabe, H., Bjarkam, C. R., Danielsen, E. H., Cumming, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pig+brain+stereotaxic+standard+space:+Mapping+of+cerebral+blood+flow+normative+values+and+effect+of+MPTP-lesioning.">Pig brain stereotaxic standard space: Mapping of cerebral blood flow normative values and effect of MPTP-lesioning.</a> Brain Res Bull. 66 (1), 17-29 (2005).</li><li>Glud, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+gene+transfer+in+the+minipig+CNS+using+stereotaxic+lentiviral+microinjections.">Direct gene transfer in the minipig CNS using stereotaxic lentiviral microinjections.</a> Acta Neurobiol Exp. 70 (3), 1-8 (2010).</li><li>Glud, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+MRI-guided+stereotaxic+viral+mediated+gene+transfer+of+alpha-synuclein+in+the+minipig+CNS.">Direct MRI-guided stereotaxic viral mediated gene transfer of alpha-synuclein in the minipig CNS.</a> Acta Neurobiol Exp. 71 (4), 508-518 (2011).</li><li>Ettrup, K. S., S&#248;rensen, J. C., Rodell, A., Alstrup, A. K. O., Bjarkam, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic+deep+brain+stimulation+influences+autonomic+and+limbic+circuitry+involved+in+the+regulation+of+aggression+and+cardiocerebrovascular+control+in+the+minipig.">Hypothalamic deep brain stimulation influences autonomic and limbic circuitry involved in the regulation of aggression and cardiocerebrovascular control in the minipig.</a> Stereotact Funct Neurosurg. 90 (5), 281-291 (2012).</li><li>Nielsen, M. 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=Continuous+MPTP+intoxication+in+the+minipig+results+in+chronic+parkinsonian+deficits.">Continuous MPTP intoxication in the minipig results in chronic parkinsonian deficits.</a> Acta Neurobiol Exp. 76, 198-210 (2016).</li><li>Bjarkam, C. R., 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+MRI-compatible+stereotaxic+localizer+box+enables+high-precision+stereotaxic+procedures+in+pigs.">A MRI-compatible stereotaxic localizer box enables high-precision stereotaxic procedures in pigs.</a> J Neurosci Methods. 139 (2), 293-298 (2004).</li><li>Bjarkam, C. R., Jorgensen, R. L., Jensen, K. N., Sunde, N. A. A., S&#248;rensen, J. C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+brain+stimulation+electrode+anchoring+using+BioGlue&#174;,+a+protective+electrode+covering,+and+a+titanium+microplate.">Deep brain stimulation electrode anchoring using BioGlue&#174;, a protective electrode covering, and a titanium microplate.</a> J Neurosci Methods. 168, 151-155 (2008).</li><li>Ettrup, K. 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=Basic+Surgical+Techniques+in+the+Minipig:+Intubation,+Transurethral+Bladder+Catheterization,+Femoral+Vessel+Catheterization,+and+Transcardial+Perfusion.">Basic Surgical Techniques in the Minipig: Intubation, Transurethral Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion.</a> J Vis Exp. (52), e2652 (2011).</li><li>Ettrup, K. S., Torn&#248;e, J., S&#248;rensen, J. C., Bjarkam, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+surgical+device+for+minimally+invasive+implantation+of+experimental+deep+brain+stimulation+leads+in+large+research+animals.">A surgical device for minimally invasive implantation of experimental deep brain stimulation leads in large research animals.</a> J Neurosci Methods. 200 (1), 41-46 (2011).</li><li>Danielsen, E. 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=Positron+emission+tomography+of+living+brain+in+minipigs+and+domestic+pigs.">Positron emission tomography of living brain in minipigs and domestic pigs.</a> Scand J Lab Anim Sci Suppl. 25 (1), 127-135 (1998).</li><li>R&#248;hl, 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=Time+evolution+of+cerebral+perfusion+and+ADC+measured+by+MRI+in+a+porcine+stroke+model.">Time evolution of cerebral perfusion and ADC measured by MRI in a porcine stroke model.</a> J Magn Reson Imaging. 15 (2), 123-129 (2002).</li><li>Cumming, P., Gillings, N. M., Jensen, S. B., Bjarkam, C. R., Gjedde, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+the+uptake+and+distribution+of+the+dopamine+D2/3+agonist+(R)-N-[1-11C]n-propylnorapomorphine+in+brain+of+healthy+and+MPTP-poisoned+Gottingen+miniature+pigs.">Kinetics of the uptake and distribution of the dopamine D2/3 agonist (R)-N-[1-11C]n-propylnorapomorphine in brain of healthy and MPTP-poisoned Gottingen miniature pigs.</a> Nucl Med Biol. 30 (5), 547-553 (2003).</li><li>Jensen, K. N., Deding, D., S&#248;rensen, J. C., Bjarkam, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+implantation+of+deep+brain+stimulation+electrodes+in+the+pontine+micturition+centre+of+the+minipig.">Long-term implantation of deep brain stimulation electrodes in the pontine micturition centre of the minipig.</a> Acta Neurochir. 151 (7), 785-794 (2009).</li><li>Rosendal, F., 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=Does+chronic+low+dose+treatment+with+ciclosporin+influence+the+brain?+A+histopathological+study+in+pigs.">Does chronic low dose treatment with ciclosporin influence the brain? A histopathological study in pigs.</a> Transplantation Proc. 37 (8), 3305-3308 (2005).</li><li>Nielsen, M. S., S&#248;rensen, J. C., Bjarkam, C. 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>

Die Autoren haben nichts zu offenbaren.

Die meisten experimentellen Neurowissenschaften Studien werden in kleinen Tierarten, wie Mäuse und Ratten durchgeführt, in denen den Zugang zum ZNS durch einen dünnen Schädel- und Dural-Dicke erleichtert wird. In größeren Versuchstieren , wie Schweine 1, 4, 8, Schafe 32, aber, und nicht-menschliche Primaten, erfordern die beträchtlichen Dicke dieser Strukturen , die die Verwendung von robusten Ins...

<html> Translationale Neurowissenschaften Studien bei Schweinen immer beliebter in den letzten zwei Jahrzehnten worden. Die große Größe des Schweinehirns ermöglicht die Verwendung von herkömmlich klinischen brain Imager und der unmittelbaren Nutzung und Prüfung von neurochirurgischen Verfahren und Geräten aus der menschlichen Klinik 1, 2, 3, 4, 5, 6, 7, 8. In den letzten 20 Jahren, Schweine, insbesondere Minipigs (zB minipig Göttingen), wurden verwendet , neuromodulatorischen Behandlungsmodalitäten, wie beispielsweise Stammzelltransplantation zu untersuchen; viraler Vektor Transfektion; und tiefe Hirnstimulation gerichtet gegen Parkinson - Krankheit, Übergewicht, Depression und Alzheimer Krankheit 2, 6,= &quot;Xref&quot;&gt; 9, 10, 11, 12, 13, 14, 15, 16, 17. Dies wurde durch die Entwicklung von Stereotaxierahmen und chirurgische Ansätze folgte dem minipig CNS 3, 18, 19, 20, 21 zu manipulieren. Die eingeleiteten CNS Veränderungen in lebenden Tieren unter Verwendung von brain imaging (PET 10, 13, 22, 24 und MR 23), Zystometrie 11, 12, 25, Ganganalyse evaluiert17, neurologische Bewertung 9, 17 und Obduktion basierend auf der Histologie und stereologischen Analyse 14, 15, 17, 26, 27, 31. Jedoch erfordert postmortale Analyse die Belichtung und die Entfernung des Schweinehirns, das keine leichte Aufgabe ist, als ein dicker, knöcherner Schädel und ein faseriger dural das Schweinehirn abdeckt umgeben. Das Ziel dieses Papiers und Lehr-Video ist zu beschreiben, wie die Obduktion Schweinehirn und Hypophyse können unter Verwendung von nicht-motorisierte chirurgische Instrumente in 15-20 min in einem intakten Zustand belichtet und entfernt werden. Das Lehr-Video und fotografische Abbildungen zeigen männlich minipigs (Alter: 6 Monate, Körpergewicht: 20-25 kg) für eine anatomische Studie über die minipig Hypophyse verwendet.

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			<td height="21" style="height:21px;width:216px;">Heavy Scalpel Handle #4</td>
			<td style="width:191px;">FST (Fine Science Tools)</td>
			<td style="width:149px;">10008-13</td>
			<td style="width:481px;">Good for skin incision and soft tissue removal</td>
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			<td height="21" style="height:21px;">Non-Sterile Scalpel Blades #23</td>
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			<td>10023-00</td>
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			<td height="21" style="height:21px;">Scalpel Handle #7</td>
			<td style="width:191px;">FST&#160;</td>
			<td>10007-12</td>
			<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>10011-00</td>
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			<td height="21" style="height:21px;width:216px;">Surgical Forceps</td>
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			<td>11024-18</td>
			<td>The tip of the surgical forceps ensure a firm grip&#160;</td>
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			<td height="21" style="height:21px;">Kerrison Bone Punch</td>
			<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>
			<td style="width:149px;">MD615</td>
			<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>
			<td style="width:149px;">FO551R</td>
			<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>
			<td>Lawton</td>
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			<td>The size of the chisel head should not exceed 20 mm</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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		<tr height="21">
			<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>
			<td>Aesculap Neurosurgery</td>
			<td style="width:149px;">OL165R</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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Euthanimal</td>
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			<td style="width:149px;"></td>
			<td>pentobarbital</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Ketamine</td>
			<td style="width:191px;">Pfizer</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<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

Schweine haben in Großtier translationalen neurowissenschaftlichen Forschung als wirtschaftlich und ethisch möglich Ersatz für nichtmenschliche Primaten immer beliebter geworden. Die große Größe des Gehirn des Schweins ermöglicht die Verwendung von herkömmlich klinischem Gehirn Imager und der direkten Nutzung und Prüfung von neurochirurgischen Verfahren und Geräten aus der menschlichen Klinik. Weitere makroskopische und histologische Analyse erfordert jedoch postmortem Exposition des Schweins Zentralnervensystem (ZNS) und die anschließende Entfernung des Gehirns. Dies ist keine einfache Aufgabe, da das Schwein CNS durch einen dicken, knöchernen Schädels und der Wirbelsäule eingekapselt ist. Das Ziel dieses Papier und Lehr-Video ist zu beschreiben, wie die Obduktion Schweinehirn und die Hypophyse in einem intakten Zustand, geeignet für die anschließende makroskopische und histologische Analyse belichten und zu entfernen.

Um das Gewebematerial vor dem Austrocknen zu verhindern, wird empfohlen, das Gehirn und Hypophyse entfernt in einem Glas mit Fixativ oder isotonischer Kochsalzlösung gefüllt zu speichern, unmittelbar nach makroskopischer Analyse durchgeführt wurde. Das Gewebematerial kann in der Fixiermittel für Jahre gespeichert werden, während der Lagerung in isotonischer Kochsalzlösung, auch in einem Kühlschrank wird auf Gewebezerfall mit der Zeit führen. <p class="jove_content" fo:keep-to...

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

Das Ziel dieses Papier und Lehr-Video ist zu beschreiben, wie die Obduktion Schweinehirn und Hypophyse in einem intakten Zustand, geeignet für die anschließende makroskopische und histologische Analyse belichten und zu entfernen.

Belichtung des Schweins CNS für die histologische Analyse: Ein Handbuch für Enthauptung, Schädel öffnen und Gehirn Removal

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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Anatomy and Physiology

Neuroscience

Unit 1

The Axial Skeleton

SCIENTISTS IN ACTION

15.3K Aufrufe.  Aalborg University Hospital.  Das Ziel dieses Papier und Lehr-Video ist zu beschreiben, wie die Obduktion Schweinehirn und Hypophyse in einem intakten Zustand, geeignet für die anschließende makroskopische und histologische Analyse belichten und zu entfernen. 

Serie: 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.

Eine polierte und verstärkte ausgedünnten Schädel Fenster für Langzeit-Imaging im Gehirn der Maus

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

Forschung

Neurowissenschaften

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-Analyse von Hirnstruktur und-funktion mittels fMRT und Diffusionsgewichtete 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.

Eine umfassende Protokoll für die manuelle Segmentierung der medialen Temporallappen Structures

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.

Analyse Synaptische Modulation von<em&gt; Drosophila melanogaster</em&gt; Photorezeptoren nach der Einwirkung von Längerer Licht

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.

Hochdurchsatz-Analyse des Bewegungsapparates Verhaltens in der Drosophila -Insel-Assay

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.

Aufnahme synaptische Plastizität in akuten Hippocampal Scheiben gepflegt in einem kleinvolumigen Recycling - Perfusions- und untertauchen-Typ-Kammer-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 ...

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.

Neuartige passiv Clearing Methoden für die schnelle Produktion von optischen Transparenz im gesamten ZNS-Gewebe

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.

Chronische Implantation des gesamten kortikalen Electrocorticographic Arrays in der Weißbüschelaffe

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.

Extraktion und Zerlegung des domestizierenden Schweinehirns

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.

In vivo Bildgebung von vollaktivem Hirngewebe bei wachen Zebrafischlarven und Jungtieren durch Schädel- und Hautentfernung

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