Name: modification of a colliculo-thalamocortical mouse brain slice, incorporating 3-d printing of chamber components and multi-scale optical imaging
Uploaded: 2015-09-18T14:00:00
Description: Watch this Scientific Journal Video about Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging at JoVE.com

This work was partially supported by National Institute of Deafness and Other Communications Disorders Awards R03-DC-012125 to D. A. Llano and F31-DC-013501 to B. J. Slater as well as the Carver Foundation.
The authors would like to thank Jason MacLean and Matthew Banks for technical advice with calcium imaging.

Alle procedurer blev godkendt af Institutional Animal Care og brug Udvalg på University of Illinois. Alle dyr blev opstaldet i dyr pasningsmuligheder er godkendt af American Association for Akkreditering af Laboratory Animal Care. Ethvert forsøg på at minimere antallet af dyr, der anvendes, og for at mindske lidelserne i alle faser af undersøgelsen. 1. Forberedelse til og Fjernelse af hjernen fra mus til udskæring <ol><li> Forbered perfusion og skive inkubation. <ol><li> Ca. 30 minutt...

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This protocol describes improvements upon a previously described colliculo-thalamocortical brain slice in p12-20 mouse to study information flow in the auditory system1. This method has a number of advantages over other, similar, brain slice preparations by retaining connections between more brain areas in a single slice, which gives investigators new tools to understand the interaction and interplay between auditory nuclei in the forebrain. There have been a few key modifications in this protocol, compared to...

I auditive system, selv om der er væsentlig bearbejdning af information mellem sensoriske periferi og ringere colliculus, der er betydelig yderligere behandling, før den når den auditive cortex. Vi ved meget lidt om, hvordan at behandlingen er gjort, og derfor lidt om, hvordan at transformation tillader hjernen at fortolke indkommende sensoriske informationer. Med undtagelse af lugtesansen, hver af sanserne har en meget lignende organisation med perifere signaler oprindeligt videreudsendelse med high fidelity, som falder, når signalet stiger til cortex. Cortex sender derefter fremspring til de nedre strukturer til yderligere at modulere den indkommende information. Dette komplekse system er blevet undersøgt i en række forskellige måder i vivo såvel som i en række in vitro-præparater. I førstnævnte, alle forbindelser er intakte, så forskeren at sonden et sæt af forbindelser, mens styring af sanseindtryk og målingproduktion i et givet område. Med denne fremgangsmåde, er der lidt at ingen kontrol over det store udvalg af andre indgange, herunder andre sensoriske inputs, ophidselse og opmærksomhed, hvilket giver anledning til en stærkt kompliceret output. In vitro har hjerneskiver blevet skåret til at fange enten et enkelt sæt af fremskrivninger eller to forbundne områder i hjernen, som giver forskerne til at stimulere og evaluere forskellige afferente eller hjerneområder. Disse er ofte enten thalamus eller tectothalamic skiver hvor enten input til thalamus eller thalamus og dens output til cortex er bevaret 2-5. Disse præparater giver mulighed for en bred vifte af farmakologiske, elektriske og optogenetic manipulationer. Men med kun to områder af hjernen, de primært vurdere overførsel af information og mangler evnen til at vurdere omdannelsen af ​​oplysninger, når den passerer gennem thalamus. Også netmave-thalamiske projektion, som kan spille en rolle i opmærksomhed graduering 6-9 er prESENT i dette udsnit. Her demonstrerer vi forbedringer i forhold til vores tidligere fremstilling 1, hvilket tillader investigator styring af forskellige input til thalamus at give et unikt perspektiv på, hvordan thalamus porte og filtre information. Vi par denne roman forberedelse skive med flavoprotein autofluorescens billeddannelse til vurdering skive tilslutningsmuligheder og storstilet aktiveringsanalyse, calcium billeddannelse i thalamus for neuronal befolkning analyse og enkelt celle optagelse for at måle effekten af ​​de forskellige indgange på en enkelt celle niveau. For at hjælpe med at opretholde disse omfattende forbindelser har vi også udviklet en række ændringer af den normale skive anker (aka &quot;harpe&quot;) til at holde hjernen skive på plads, og en bro til at ophøje den skive for øget perfusion. Harpen er designet i en modificeret hestesko form til at omgive skive og give mulighed for tilpasses fastgørelsespunkter for harpe strygere. Tre strenge erfastgjort således, at i) man ligger vandret langs den mediale kant af skiven, ii) en strækker sig fra kaudale kant af IC til den kaudale kant af AC og iii) en forløber diagonalt fra den mediale kant af udsnittet til et område rostrale til AC (se figur 1A). Små fordybninger i rammen til limning (med cyanoacrylat lim) på harpe strygere giver mulighed for en nedsat mængde af pres på skive for at hjælpe med at opretholde slice integritet (se figur 1B). Ved at bruge tredimensionelle udskrivning, er vi i stand til brugerdefinerede design harper til vores unikke specifikationer, såvel som broer, der tillader ideel strøm af over og under vævet kunstig cerebrospinalvæske (aCSF). Det fastholder også store områder til let at trænge ind i væv til patch clamp elektrofysiologi.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>High sucrose cutting solution</td>
			<td></td>
			<td></td>
			<td>in mM: 206 sucrose, 10.0 MgCl2, 11.0 glucose, 1.25 NaH2PO4, 26 NaHCO3, 0.5 CaCl2, 2.5 KCl, pH 7.4</td>
		</tr>
		<tr>
			<td>Low calcium aCSF</td>
			<td></td>
			<td></td>
			<td>in mM: 126 NaCl, 3.0 MgCl2, 10.0 glucose, 1.25 NaH2PO4, 26 NaHCO3, 1.0 CaCl2, 2.5 KCl, pH 7.4</td>
		</tr>
		<tr>
			<td>aCSF</td>
			<td></td>
			<td></td>
			<td>in mM: 126 NaCl, 2.0 MgCl2, 10.0 glucose, 1.25 NaH2PO4, 26 NaHCO3, 2.0 CaCl2, 2.5 KCl, pH 7.4</td>
		</tr>
		<tr>
			<td>Stimulus Isolator</td>
			<td>World Precision Instruments</td>
			<td>A360</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Life Technologies</td>
			<td>D12345</td>
			<td>Lot: 1572C502</td>
		</tr>
		<tr>
			<td>Fura-2AM</td>
			<td>Life Technologies</td>
			<td>F1201</td>
			<td>Lot: 144912</td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Life Technologies</td>
			<td>P3000MP</td>
			<td>Lot: 1499369</td>
		</tr>
		<tr>
			<td>Large culture dish</td>
			<td>Fisherbrand</td>
			<td>08-757-13</td>
			<td>100x15mm culture dish</td>
		</tr>
		<tr>
			<td>Small culture dish</td>
			<td>Falcon</td>
			<td align="right">353001</td>
			<td>35x10mm culture dish</td>
		</tr>
		<tr>
			<td>Raised culture membrane</td>
			<td>Millicell</td>
			<td>PICMORG50</td>
			<td>Used to maintain oxygenated fluid perfusion on both sides of slice.</td>
		</tr>
		<tr>
			<td>Flavoprotein imaging fluorescence cube</td>
			<td>Olympus</td>
			<td>UMNIB</td>
			<td>470&#8211;490 nm excitation, 505 nm dichroic, 515 nm emission long pass.&#160; We have found that virtually any green fluorescence protein filter cube will work here.</td>
		</tr>
		<tr>
			<td>Calcium imaging fluorescence cube</td>
			<td>Omega Optical</td>
			<td>BX-18</td>
			<td>XF1005 365nm exitation, XF2001 400nm dichroic, XF3080 510nm emission</td>
		</tr>
		<tr>
			<td>Agar for blocking brain</td>
			<td></td>
			<td></td>
			<td>3% by weight in water</td>
		</tr>
		<tr>
			<td>Viper si Stereo Lithography Apparatus</td>
			<td>3D Systems</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

modification of a colliculo-thalamocortical mouse brain slice, incorporating 3-d printing of chamber components and multi-scale optical imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Et eksempel på colliculo thalamus musehjerne skive opnået i P15 mus er vist i figur 2. Den ideelle skive vil indeholde de fire store midthjernen og forhjernen auditive strukturer IC, MGB, TRN og AC, der alle aktiveres, når IC stimuleres (Figur 2A). Brug af Fourier-analyse, er den spektrale effekt målt ved den elektriske stimulation frekvens, med tilsluttet hjerneområder, der viser aktivitet, der er periodisk og medføres på stimulationsfrekvensen 10. Denne protokol muli...

Watch this Scientific Journal Video about Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging at JoVE.com

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

Brug af flere vinkler til at skære musen hvalp hjernen, forbedrer vi på en tidligere beskrevne akut hjerne skive som indfanger forbindelserne mellem de fleste af de store auditive midthjernen og forhjernen strukturer.

Ændring af et Colliculo thalamus Mouse Brain Slice, Omfattende 3D-print af Afdeling Komponenter og Multi-skala Optisk Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

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