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.

All procedures were approved by the Institutional Animal Care and Use Committee at the University of Illinois. All animals were housed in animal care facilities approved by the American Association for Accreditation of Laboratory Animal Care. Every attempt was made to minimize the number of animals used and to reduce suffering at all stages of the study.
1. Preparation for and Removal of Brain from Mouse for Slicing
<ol>
	<li>Prepare for perfusion and slice incubation.
	<ol>
		<li>Approximat...

<ol>
	<li>Llano, D. A., Slater, B. J., Lesicko, A. M., Stebbings, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+auditory+colliculothalamocortical+brain+slice+preparation+in+mouse.">An auditory colliculothalamocortical brain slice preparation in mouse.</a> J. Neurosci. 111, 197-207 (2014).</li><li>Agmon, A., Connors, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thalamocortical+responses+of+mouse+somatosensory+(barrel)+cortex+in+vitro.">Thalamocortical responses of mouse somatosensory (barrel) cortex in vitro.</a> Neuro. 41, 365-379 (1991).</li><li>Cruikshank, S. J., Rose, H. J., Metherate, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+Thalamocortical+Synaptic+Transmission+In+Vitro.">Auditory Thalamocortical Synaptic Transmission In Vitro.</a> J Neurophysiol. 87, 361-384 (2002).</li><li>Lee, C. C., Sherman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topography+and+physiology+of+ascending+streams+in+the+auditory+tectothalamic+pathway.">Topography and physiology of ascending streams in the auditory tectothalamic pathway.</a> PNAS. 107, 372-377 (2010).</li><li>MacLean, J. N., Fenstermaker, V., Watson, B. O., Yuste, 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+visual+thalamocortical+slice.">A visual thalamocortical slice.</a> Nat meth. 3, 129-134 (2006).</li><li>Crabtree, J. W., Isaac, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+intrathalamic+pathways+allowing+modality-related+and+cross-modality+switching+in+the+dorsal+thalamus.">New intrathalamic pathways allowing modality-related and cross-modality switching in the dorsal thalamus.</a> J. Neurosci. 22, 8754-8761 (2002).</li><li>McAlonan, K., Cavanaugh, J., Wurtz, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attentional+Modulation+of+Thalamic+Reticular+Neurons.">Attentional Modulation of Thalamic Reticular Neurons.</a> J. Neurosci. 26, 4444-4450 (2006).</li><li>Weese, G. D., Phillips, J. M., Brown, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attentional+Orienting+Is+Impaired+by+Unilateral+Lesions+of+the+Thalamic+Reticular+Nucleus+in+the+Rat.">Attentional Orienting Is Impaired by Unilateral Lesions of the Thalamic Reticular Nucleus in the Rat.</a> J. Neurosci. 19, 10135-10139 (1999).</li><li>Zikopoulos, B., Barbas, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prefrontal+Projections+to+the+Thalamic+Reticular+Nucleus+form+a+Unique+Circuit+for+Attentional+Mechanisms.">Prefrontal Projections to the Thalamic Reticular Nucleus form a Unique Circuit for Attentional Mechanisms.</a> J. Neurosci. 26, 7348-7361 (2006).</li><li>Kalatsky, V. A., Stryker, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Paradigm+for+Optical+Imaging:+Temporally+Encoded+Maps+of+Intrinsic+Signal.">New Paradigm for Optical Imaging: Temporally Encoded Maps of Intrinsic Signal.</a> Neuron. 38, 529-545 (2003).</li><li>Llano, D. A., Turner, J., Caspary, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diminished+cortical+inhibition+in+an+aging+mouse+model+of+chronic+tinnitus.">Diminished cortical inhibition in an aging mouse model of chronic tinnitus.</a> J. Neurosci. 32, 16141-16148 (2012).</li><li>Ehret, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+studies+on+auditory+development+in+mammals+in+relation+to+higher+nervous+system+functioning.">Behavioural studies on auditory development in mammals in relation to higher nervous system functioning.</a> Acta otolaryngol. 99, 31-40 (1985).</li><li>Mikaelian, D., Ruben, R. <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+hearing+in+the+normal+CBA-J+mouse:+correlation+of+physiological+observations+with+behavioral+responses+and+with+cochlear+anatomy.">Development of hearing in the normal CBA-J mouse: correlation of physiological observations with behavioral responses and with cochlear anatomy.</a> Acta. 59, 451-461 (1965).</li></ol>

The authors have nothing to disclose.

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...

In the auditory system, although there is substantial processing of information between the sensory periphery and the inferior colliculus, there is considerable additional processing before it reaches the auditory cortex. We know very little about how that processing is done and therefore little about how that transformation allows the brain to interpret incoming sensory information. With the exception of olfaction, each of the senses has a very similar organization with peripheral signals initially being relayed with high fidelity which declines as the signal ascends to the cortex. The cortex then sends projections to the lower structures to further modulate the incoming information. This complex system has been studied in a variety of ways in vivo as well as in a number of in vitro preparations. In the former, all connections are intact, enabling the researcher to probe any set of connections, while controlling the sensory input and measuring output in any given area. With this approach, there is little to no control of the large variety of other inputs, including other sensory inputs, arousal, and attention, giving rise to an intensely complex output. In vitro, brain slices have been cut to capture either a single set of projections, or two connected brain areas, which allow researchers to stimulate and evaluate various afferents or brain areas. These are often either thalamocortical or tectothalamic slices where either the input to the thalamus or the thalamus and its output to the cortex are preserved2-5. These preparations allow for a wide variety of pharmacological, electrical, and optogenetic manipulations. However with only two brain regions, they primarily evaluate the transfer of information and lack the ability to evaluate the transformation of information as it passes through the thalamus. Also the reticulo-thalamic projection, which may play a role in attention modulation6-9 is present in this slice. Here we demonstrate improvements upon our previous preparation1, which allows the investigator control of various inputs to the thalamus to give a unique perspective of how the thalamus gates and filters information. We couple this novel slice preparation with flavoprotein autofluorescence imaging for assessing slice connectivity and large-scale activation analysis, calcium imaging in the thalamus for neuronal population analysis, and single cell recording to measure the impact of the various inputs on a single cell level.
To assist in maintaining these widespread connections we have also developed a number of modifications of the normal slice anchor (a.k.a. &#8220;harp&#8221;) for holding the brain slice in place and a bridge to elevate the slice for enhanced perfusion. The harp is designed in a modified horseshoe shape to surround the slice and allow for customizable attachment points for the harp strings. Three strings are attached such that i) one lies horizontally along the medial edge of the slice, ii) one extends from the caudal edge of the IC to the caudal edge of the AC and iii) one extends diagonally from the medial edge of the slice to an area rostral to the AC (see Figure 1A). Small indentations in the frame for gluing (with cyanoacrylate glue) of the harp strings allow for a decreased amount of pressure on the slice to help maintain slice integrity (see Figure 1B). By using three dimensional printing, we are able to custom design harps to our unique specifications, as well as bridges which allow for ideal flow of artificial cerebrospinal fluid (aCSF) above and below the tissue. This also maintains large areas for light to penetrate the tissue for patch clamp electrophysiology.

<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.

An example of colliculo-thalamocortical mouse brain slice obtained in P15 mouse is shown in figure 2. The ideal slice will contain the four major midbrain and forebrain auditory structures IC, MGB, TRN, and AC, which are all activated when the IC is stimulated (Figure 2A). Using Fourier analysis, the spectral power is measured at the electrical stimulation frequency, with connected brain regions showing activity that is periodic and entrained at the stimulation frequency10. Th...

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

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

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 ...

modification-colliculo-thalamocortical-mouse-brain-slice

Research

JoVE Journal

Neuroscience

8.2K Views.  University of Illinois Urbana-Champaign. Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.