Name: Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging (Video) | JoVE Journal
Uploaded: 2015-09-18T14:00:00.000+00:00
Duration: 6 min 5 s
Description: Learn to prepare colliculo-thalamocortical brain slices in mice using double angled cutting and flavoprotein imaging. This method preserves auditory pathway integrity for studying neural connectivity.

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5 minutes

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>Approximately 30 min before slicing, prepare high sucrose cutting solution and low calcium aCSF for incubation of the slice prior to recording or imaging.</li>
		<li>Lay out all necessary tools (large blunt end scissors, small spring scissors, anatomic forceps, large scissors or guillotine, iris scissors, jewelers forceps, 10 ml&#160;syringe with 27G&#160;x 1/2 inch needle, small piece of 1 cm x 2 cm filter paper, and a broken tipped Pasteur pipette for slice transfer) for perfusion and slicing, and prepare perfusion tray.</li>
		<li>Set up incubating chamber with oxygenated aCSF in a 32 oC water bath and a culture dish filled with cutting solution.</li>
	</ol>
	</li>
	<li>Prepare cutting stage for slicing.
	<ol>
		<li>Cut a small piece of 3% agar approximately 1 cm3 to use as a backstop for the brain and 1.5 cm x 1.5 mm x 3 mm for a bump to be used to prop up the IC.</li>
		<li>Glue the backstop onto the stage with cyanoacrylate adhesive as well as the bump on the right side of the backstop such that they form an 80o angle.</li>
	</ol>
	</li>
	<li>Remove the brain.
	<ol>
		<li>Deeply anesthetize a p12-p20 (ideally p14-18) mouse with ketamine 100 mg/kg and xylaxine 3 mg/kg (or comparable ethics committee approved procedure). 
		Note: Confirm proper anesthesia level via lack of response to toe pinch. As the animal is under anesthesia briefly, ophthalmic ointment is unnecessary.</li>
		<li>Using blunt end scissors, expose the ribcage from the xiphoid process to the neck.
		<ol>
			<li>Find the xiphoid process, and make a horizontal cut of approximately 1.5 cm.</li>
			<li>Make two vertical cuts from the ends of the previous horizontal cut to the shoulders, approximately 1.5 cm each.</li>
		</ol>
		</li>
		<li>Cut through the diaphragm and the costochondral junctions to expose the heart.</li>
		<li>With small spring scissors, make a small, 2-5 mm cut in the right atrium, from the ventral to dorsal side of the heart.</li>
		<li>Using a 10 ml&#160;syringe with a 27G&#160;x 1/2 inch needle, inject the left ventricle and quickly perfuse the animal with high sucrose cutting solution.</li>
		<li>Once the blood runs clear, use larger scissors to remove the head. 
		Note: We have not systematically assessed the utility of perfusion. However, in our experience, transcardiac perfusion in mice this young can be done with nearly 100% success, and does have the benefit of eliminating red blood cells, which can fluoresce and interfere with imaging.</li>
		<li>Cut the skin down the midline to expose the skull.</li>
		<li>Using bent iris scissors, cut the skull between the eyes, then starting from the cut between the eyes, carefully cut from anterior to posterior along the midline suture taking care not to damage the brain underneath. 
		Note: The dura usually comes off with the skull. If necessary, remove the dura before carefully removing the brain.</li>
		<li>Using jewelers forceps, pry open the skull and carefully remove the brain, then place the brain in cutting solution. Take care to avoid damaging the cortices.</li>
	</ol>
	</li>
</ol>
2. Preparing Brain for Slicing
<ol>
	<li>Preparing brain for mounting on vibratome stage.
	<ol>
		<li>Using a slide marked with two lines at 90o and a diagonal line at 17o from the top left to bottom right, intersecting where the two lines meet, place the brain dorsal side up, using a razor blade, remove 2-4 mm of the rostral end brain creating a flat surface.</li>
		<li>Place the brain caudal side up on the newly created flat surface, and align the dorsal surface of the brain with the horizontal and the midline of the brain with the vertical line.</li>
		<li>Align the razor blade with the 17o line, tilt the razor at a 30o angle and remove approximately 3 mm of the right cortex in a double diagonal cut (17o from horizontal plane and 60o from the coronal).</li>
	</ol>
	</li>
	<li>Mounting brain on vibratome stage.
	<ol>
		<li>Place a small piece of filter paper 1 cm x 2 cm on the ventral side of the brain so that the long dimension is perpendicular to the midline.</li>
		<li>Carefully apply a small amount of cyanoacrylate adhesive to the area in front of the backstop (and to the left of the bump).</li>
		<li>Place the double diagonally-cut brain face on to the glue so that the caudal part of the brain and hindbrain are propped upon the bump and the right side of the brain is against the backstop.</li>
		<li>Delicately press down on the brain, ensuring that the entire surface is in contact with the slicing chamber.</li>
	</ol>
	</li>
</ol>
3. Obtaining the Colliculo-thalamocortical Slice
<ol>
	<li>Quickly take the cutting stage and place in the vibratome, fill the stage with cutting solution.</li>
	<li>Align the blade with the top (ventral side) of the brain.</li>
	<li>Remove 1-1.5 mm from the top of the brain, remove 300-500 &#181;m slices and assess depth after removal. 
	Note: When the IC, the MGB, and the lateral geniculate nucleus (LGN) are all visible, and the dentate gyrus forms a &#39;C&#39; shape take one to two 600 &#181;m slices. See Figure 3A.</li>
	<li>Place slices in heated (32 oC) holding chamber.</li>
</ol>
4. Imaging of the Slice
<ol>
	<li>Preparation for flavoprotein autofluorescence imaging.
	<ol>
		<li>Prepare aCSF for perfusion of slice in a standard electrophysiological recording chamber, bubble aCSF with 95% O2/5% CO2.</li>
		<li>Pull glass electrode for stimulation. Note that multiple different stimulating electrodes and configurations (glass, tungsten, carbon fiber, monopolar, bipolar) all work very well in conjunction with flavoprotein autofluorescence imaging.</li>
		<li>Turn on computer, camera, micromanipulator, light source, stimulation software, image capture software.</li>
		<li>Perfuse the recording chamber with oxygenated aCSF, place custom bridge (design available in Supplemental Materials) to elevate slice in chamber. 
		Note: Perfusion rate can vary with any individual set up; 5-15 ml/min is used here.</li>
		<li>Place slice in chamber, lower the liquid level in the chamber to prevent the slice from lifting off the bridge while placing specially constructed harp over slice, taking care to avoid placing harp strings over pathways of interest (Figure 1A). Increase liquid level to approximately 1-2 mm above slice to maintain aCSF flow above and below slice. 
		Note: If repositioning is necessary, use the broken end Pasteur pipette or allow the aCSF level to rise and use forceps with extreme care.</li>
		<li>Insert silver chloride electrode into glass electrode filled with aCSF, insert the glass electrode into micromanipulator and connect to stimulus isolator, and carefully place glass electrode in IC/white matter leading to thalamus(see Figure 1A).</li>
	</ol>
	</li>
	<li>Flavoprotein autofluorescence image acquisition.
	<ol>
		<li>Collect images at 4 Hz (using an infinity-corrected 2X macro objective (NA 0.13)) and a camera for 105 sec&#160;while electrically stimulating (each stimulation consists of 1 sec&#160;of 40 Hz 2 msec pulses tissue at 0.05 Hz five times) while illuminating the tissue with blue light 470-490 nm and capturing above 515 nm. Ensure that the image is neither too dark, nor blown out see Figure 3 left column for reference. 
		Note: Collection time, collection frequency, and stimulation frequency can be changed to suit the experiment, the custom program included in supplementary materials can be modified as such.</li>
		<li>Export images to Matlab, and using the custom written program available in supplementary materials to analyze spectral power of the images at 0.05 Hz. The resulting image will show connected pathways. 
		Note: The custom program uses a fast Fourier transform of the pixel values in the time series to produce the image.</li>
	</ol>
	</li>
	<li>Preparation for calcium imaging.
	<ol>
		<li>Prepare small incubation chamber using a 3 cm culture dish and raised culture membrane to allow aCSF to reach both the top and bottom of the slice, and place on a heating pad to maintain temperature at approximately 32 oC.</li>
		<li>Fill small incubation chamber with warm aCSF and slowly bubble with 95% O2/5% CO2.</li>
		<li>Mix 2 &#181;l&#160;of pluronic F-127 acid and 50 &#181;g of Fura-2AM dissolved in 48 &#181;l&#160;of DMSO.</li>
		<li>Place slice in small incubation chamber on raised membrane and carefully using a micropipette add staining mixture directly above the MGB (or other structure of interest).</li>
		<li>Cover slices to prevent bleaching prior to imaging, and allow slices to incubate for 45 min - 1 hr.</li>
		<li>Remove slices to heated holding chamber for 10-15 min&#160;to wash off excess material.</li>
		<li>Follow steps 4.1.1 to 4.1.6 for preparation to stimulate and image the colliculo-thalamocortical slice.</li>
	</ol>
	</li>
	<li>Calcium image acquisition.
	<ol>
		<li>Collect images at 10 Hz (using an 20X water immersion objective) for 25 sec&#160;while electrically stimulating (each stimulation consists of one 2 msec pulse) tissue at 0.2 Hz five times while illuminating the tissue with 365 nm light and capturing fluorescence above 510 nm.</li>
		<li>Export images to Matlab and using the custom written program available in supplementary materials to analyze spectral power of the images at 0.2 Hz. The resulting image will show active cells.</li>
	</ol>
	</li>
</ol>

<ol><li>Llano, D. A., Slater, B. J., Lesicko, A. M., Stebbings, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+auditory+colliculothalamocortical+brain+slice+preparation+in+mouse%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Thalamocortical+responses+of+mouse+somatosensory+%28barrel%29+cortex+in+vitro%5BTitle%5D)+AND+%22Neuro%22%5BJournal%5D)">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/pubmed?term=((Auditory+Thalamocortical+Synaptic+Transmission+In+Vitro%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">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/pubmed?term=((Topography+and+physiology+of+ascending+streams+in+the+auditory+tectothalamic+pathway%5BTitle%5D)+AND+%22PNAS%22%5BJournal%5D)">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/pubmed?term=((A+visual+thalamocortical+slice%5BTitle%5D)+AND+%22Nat+meth%22%5BJournal%5D)">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/pubmed?term=((New+intrathalamic+pathways+allowing+modality-related+and+cross-modality+switching+in+the+dorsal+thalamus%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Attentional+Modulation+of+Thalamic+Reticular+Neurons%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Attentional+Orienting+Is+Impaired+by+Unilateral+Lesions+of+the+Thalamic+Reticular+Nucleus+in+the+Rat%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Prefrontal+Projections+to+the+Thalamic+Reticular+Nucleus+form+a+Unique+Circuit+for+Attentional+Mechanisms%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((New+Paradigm+for+Optical+Imaging%3A+Temporally+Encoded+Maps+of+Intrinsic+Signal%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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/pubmed?term=((Diminished+cortical+inhibition+in+an+aging+mouse+model+of+chronic+tinnitus%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Behavioural+studies+on+auditory+development+in+mammals+in+relation+to+higher+nervous+system+functioning%5BTitle%5D)+AND+%22Acta+otolaryngol%22%5BJournal%5D)">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/pubmed?term=((Development+of+hearing+in+the+normal+CBA-J+mouse%3A+correlation+of+physiological+observations+with+behavioral+responses+and+with+cochlear+anatomy%5BTitle%5D)+AND+%22Acta%22%5BJournal%5D)">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><tbody><tr><td>High sucrose cutting solution</td><td></td><td></td><td>in mM: 206 sucrose, 10.0 MgCl&lt;sub&gt;2&lt;/sub&gt;, 11.0 glucose, 1.25 NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 26 NaHCO&lt;sub&gt;3&lt;/sub&gt;, 0.5 CaCl&lt;sub&gt;2&lt;/sub&gt;, 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 MgCl&lt;sub&gt;2&lt;/sub&gt;, 10.0 glucose, 1.25 NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 26 NaHCO&lt;sub&gt;3&lt;/sub&gt;, 1.0 CaCl&lt;sub&gt;2&lt;/sub&gt;, 2.5 KCl, pH 7.4</td></tr><tr><td>aCSF</td><td></td><td></td><td>in mM: 126 NaCl, 2.0 MgCl&lt;sub&gt;2&lt;/sub&gt;, 10.0 glucose, 1.25 NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 26 NaHCO&lt;sub&gt;3&lt;/sub&gt;, 2.0 CaCl&lt;sub&gt;2&lt;/sub&gt;, 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>100 x 15 mm culture dish</td></tr><tr><td>Small culture dish</td><td>Falcon</td><td>353001</td><td>35 x10 mm 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&amp;ndash;490 nm excitation, 505 nm dichroic, 515 nm emission long pass.&amp;nbsp; 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 365 nm exitation, XF2001 400 nm dichroic, XF3080 510 nm 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

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...

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 ...

The overall goal of this procedure is to prepare the Calot Thal cortical slice with four major auditory structures, the inferior colus, the auditory thalamus, the auditory portion of the thalamic reticular nucleus, and the auditory cortex. This is accomplished by first removing the brain from the mouse. The second step is to block the brain in preparation for slicing.Next, cut the brain to obtain slices containing the Calot thal cortical connection. The final step is to use flavoprotein imaging to assess connectivity. Ultimately, the brain slice containing the carotal cortical connection can be used for a variety of experiments to better understand information processing in the auditory regions of the mid and four brain Visual demonstration of this method is critical as the steps to block the brain before slicing are difficult to learn because it requires a complex double angled cut, which is key to obtaining intact projections.Demonstrating the technique will be BJ Slater, a graduate student in my laboratory, To prepare the cutting stage for slicing, cut a small piece of 3%auger of approximately one cubic centimeter to be used as a backstop for the brain, and another piece of 1.5 centimeter by 1.5 millimeter by three millimeters to be used as a bump to support the inferior colliculus. Next, glue the backstop onto the stage with cyanoacrylate adhesive, and then glue the bump on the right side of the backstop such that they form an 80 degree angle on a slide marked with two lines at 90 degrees and a diagonal line at 17 degrees from the top left to the bottom right place the brain dorsal side up at the intersecting point of the two lines. Using a razor blade, remove two to four millimeters of the rostral end of the brain to create a flat surface.Next place the brain coddle side up on the newly created flat surface. Align the dorsal surface of the brain with a horizontal line and the midline of the brain with a vertical line marked on the slide. Then align the razor blade with a 17 degree line.Tilt the razor at a 30 degree angle and remove approximately three millimeters of the right cortex in a double diagonal cut to mount the brain on the vibrato stage. Place a small piece of filter paper on the ventral side of the brain so that the long dimension is perpendicular to the midline. Carefully apply a small amount of cyanoacrylate adhesive to the area in front of the backstop and to the left of the bump.Subsequently, place the double diagonally cut brain side onto the glue so that the coddle part of the brain and the hind brain are propped up onto the bump, and the right side of the brain is against the backstop. In this procedure, quickly place the cutting stage in the vibrato, build a stage with cutting solution. Next, align the blade with the top of the brain.Remove one to 1.5 millimeters from the top of the brain. Then slice and remove more slices of 300 to 500 micrometers while moving deeper. When the inferior colliculus, the medial ate body and the lateral ICT nucleus are all visible, the dentate gyrus should appear as a C shape and the structure should be in alignment.Obtain about two slices of 600 micrometers. These are the carotal cortical slices. After that, transfer them to the 32 degree Celsius holding chamber.To acquire the Flavoprotein Autofluorescence image, collect the images at four hertz for 105 seconds. Illuminate the tissue with blue light 470 to 490 nanometers and capture above 515 nanometers ensuring that the image is neither too dark nor blown out. Then use electrical stimulation to activate the tissue to acquire a calcium image.Collect images at 10 hertz for 25 seconds. Illuminate the tissue with 365 nanometers light and capture fluorescence above 510 nanometers. Again, ensuring the image is neither too dark nor blown out.Then use electrical stimulation to activate the cells. This image shows the confirmation of carotal cortical connection in the brain slice by Flavoprotein Autofluorescence. Electrical stimulation given at 0.05 hertz to the inferior colus was imaged at four hertz and four a processed to show the power at that stimulation.Frequency, the auditory thalamus, thalamic, reticular, nucleus, and auditory cortex are synaptically activated. Here is an unconnected slice. Electrical stimulation of the inferior colus with activation was seen only in the auditory thalamus.The entire pathway was not captured likely due to one of the cuts being slightly out of plane. With Alam cortical projections. This image shows atypical activation of the auditory cortex.Electrical stimulation of the inferior colus activated the auditory cortex without visible signal. In the auditory thalamus, it is likely that the thalamus was still activated, but it was not on the image surface. After watching this video, you should have a good understanding of how to obtain AAL thalamic cortical slice, and to assess its connectivity.

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Neuroscience

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

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.

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

The video demonstrates the preparation of colliculo-thalamocortical brain slices from mouse pup brains. An isolated brain is aligned on a marked slide and cut at an angle to align the auditory midbrain and forebrain in the same plane. The brain is then sliced using a vibratome, and the slices containing the major auditory midbrain and forebrain structures are transferred to a holding chamber for preservation.

Preparation of Colliculo-Thalamocortical Slices from a Mouse Pup Brain

This video demonstrates the brain's neural activity recording through an optical window in transgenic mice. The mice express a fluorescent protein that enables the detection of neural activities. A small portion of the skull is carefully removed to install an optical window, and the skull is then covered with black cement to block light before imaging.

Installation of Optical Windows in the Brain of Transgenic Mice to Image Neural Activities

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Using MRI scans (human), 3D imaging software, and immunohistological analysis, we document changes to the brain’s lateral ventricles. Longitudinal 3D mapping of lateral ventricle volume changes and characterization of periventricular cellular changes that occur in the human brain due to aging or disease are then modeled in mice.

3D Modeling of the Lateral Ventricles and Histological Characterization of Periventricular Tissue in Humans and Mouse

Here, we present a procedure for performing large-scale Ca2+ imaging with cellular-resolution across multiple cortical layers in freely moving mice. Hundreds of active cells can be observed simultaneously using a miniature, head-mounted microscope coupled with an implanted prism probe.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

Calcium Imaging in Mouse Superior Colliculus

Learn to prepare colliculo-thalamocortical brain slices in mice using double angled cutting and flavoprotein imaging. This method preserves auditory pathway integrity for studying neural connectivity.

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging (Video) | JoVE Journal

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

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