PCH Mission Support Funds supported the research reported in this manuscript. The authors would like to thank Daniel Griffiths for taking the images used in the figures.

The Megabrain technique has been optimized using whole and hemisected brains from male adult C57Bl6 mice5, juvenile Sprague-Dawley rats, and adult Sprague-Dawley rats that were transcardially perfused with 1x phosphate buffered saline (PBS) followed by 4% paraformaldehyde6. Similar outcomes can be accomplished in other strains of mouse and rat, in both sexes and at different ages. The study to generate data for this manuscript used juvenile Sprague-Dawley rats and was approved by the University of Arizona Institutional Animal Care and Use Committee, and experimental animals were cared for according the Guide For the Care and Use of Laboratory Animals7.
1. Cryoprotection of Perfused Brains
<ol>
	<li>Following successful transcardial perfusion6, collect the full brain6,8 from animals and post-fix by placing into a 25 mL vial of 4% paraformaldehyde in PBS for 24 h. 
	CAUTION: Paraformaldehyde is a toxic tissue fixative; handle with care. Transfer paraformaldehyde into a specifically labeled waste container that identifies its concentration and volume inside a chemical fume hood and then store in the chemical waste cabinet until disposed by Chemical Safety or properly trained personnel.</li>
	<li>After 24 h has elapsed, in a fume hood, remove brains from 4% paraformaldehyde using a spatula. Transfer the brain into a vial of approximately 20 mL of 15% sucrose solution in 1x Tris-buffered saline (TBS) until brain sinks to the bottom of the vial (approximately 24 h).</li>
	<li>Following this, transfer the brain to a vial of approximately 20 mL of 30% sucrose solution in 1x TBS, until the brain sinks to the bottom of the vial (approximately 24&#8211;48 h).</li>
</ol>
2. Brain Freezing
<ol>
	<li>Place a 500 mL glass beaker on a bed of dry ice in an ice bucket. Pour 300&#8211;400 mL of isopentane (2 methyl-butane) into the glass beaker.</li>
	<li>Allow the temperature of the isopentane to reach between -45&#160;&#176;C and -50&#160;&#176;C. Closely monitor and maintain this temperature range with a thermometer, keeping it constant throughout the procedure. Ensure that temperature readings are taken from the middle of the solution, and not while touching the bottom or side of the beaker.</li>
	<li>On the benchtop, fill a disposable embedding mold (see materials list) approximately 1/2 full of Optimal Cutting Temperature (OCT; see Table of Materials) Compound. Ensure that there are no air bubbles in the OCT. If there are any air bubbles remove them with a spatula or needle.</li>
	<li>Remove the first brain from the vial of 30% sucrose with a spatula. At this point, either freeze brains whole or hemisect, depending on the study design.
	<ol>
		<li>For hemisected brains, use a new razor blade to make a clean cut through the tissue to separate the hemispheres. If not required for the study, remove the cerebellum and olfactory bulbs at this stage. If the cerebellum is needed for the study, it is suggested to cut a flat area at the posterior end of the brain to aid the brain in standing straight in the mold. If the study only requires tissue from a localized region, a rodent brain matrix can be used to block the brain based on known coordinates.</li>
	</ol>
	</li>
	<li>Give brains a numeric naming system. Draw a diagram as shown in Figure 1. Write the number of the first brain to be placed in the Megabrain mold in position 2, leaving position 1 as a blank space to aid in quick identification of the orientation in which the brains were frozen.</li>
	<li>Using blunt forceps or tweezers, pick up the brain to be placed in position 1. Orient the brain with the side to be cut first facing upwards. Lower the brain into the OCT in the appropriate location, using the tweezers to adjust its position until it stands independently.</li>
	<li>Repeat steps 2.3&#8211;2.5 for the remaining brains/hemispheres to be frozen in positions 3&#8211;10. Avoid positioning brains/hemispheres in a symmetrical layout.</li>
	<li>Review all 9 brains in the Megabrain mold. Once the brains are standing independently, upright, and correctly oriented in the OCT (as shown in Figure 2B), add more OCT to the mold (until the top of the brains are covered). Again, ensure that there are no visible air bubbles in the OCT.</li>
	<li>Use the forceps to hold the corner of the mold, ensuring the forceps do not become partially submerged in the OCT (Figure 3A). Being careful to keep the mold level, lower the mold into the isopentane (-45&#160;&#176;C to -50&#176;C) so that the bottom third of the mold is submerged. Hold it here to allow any bubbles in the OCT to rise to the surface and away from the tissue (Figure 3B).</li>
	<li>After 30 s, lower the entire mold into the isopentane and release from the forceps, leaving the Megabrain submerged for at least 3 min (Figure 3C). Ensure that mold is kept level, so brains remain upright and equidistant (Figure 3D).</li>
	<li>After 3 min, use the forceps to remove the mold containing the frozen, OCT embedded brains (Figure 3E) from the isopentane.</li>
	<li>Immediately, wrap the mold and its contents in aluminum foil and label it with the animal numbers or Megabrain identification. Touch the frozen Megabrain as sparingly as possible to avoid thawing the tissue.</li>
	<li>This Megabrain can now be transferred to a -20&#160;&#176;C freezer and stored for a minimum of 24 h. 
	NOTE: Protocol can be paused here and the Megabrain can be stored frozen until cryosectioning takes place.</li>
</ol>
3. Preparing the Megabrain for Sectioning on the Cryostat
<ol>
	<li>Set the cabinet temperature of the cryostat to -19&#160;&#176;C. Make sure this temperature is reached before continuing. Throughout sectioning, ensure that the cabinet temperature remains between -18&#160;&#176;C and -20&#160;&#176;C.</li>
	<li>Take the Megabrain from the -20&#160;&#176;C freezer and place it into the cryostat. Also, place a chuck of the appropriate size dimensions, and two thin tipped paintbrushes into the cryostat. Leave both the Megabrain and the chuck in the cryostat for at least 15 min to come to the temperature of the cryostat.</li>
	<li>Appropriately label slides with the Megabrain identification number and the slide number. Lay these slides on a slide warmer (35&#8211;45 &#176;C).</li>
	<li>Unwrap the Megabrain from the aluminum foil. Use a razor blade to vertically cut the corners of the mold to allow easy removal of the block of OCT from the mold (Figure 4A).</li>
	<li>Dispense a thin layer (approximately 3 mm thick) of OCT onto the chuck.</li>
	<li>Quickly, with minimal contact between fingers and the OCT, place the Megabrain on the chuck, in the orientation desired. Large forceps can also be used for this step to minimize thawing. Before starting to section, leave the Megabrain mounted chuck in the cryostat for 15&#8211;20 min to allow the OCT to freeze completely.</li>
	<li>Once the base layer of OCT has frozen, apply a 2 mm thick layer OCT around the sides of the Megabrain and allow this to run down from the sides onto the chuck. This helps secure the Megabrain to the chuck. Again, before continuing, leave the Megabrain mounted chuck in the cryostat for 15&#8211;20 min to allow the OCT to freeze.</li>
	<li>Use a razor blade to remove any excess OCT from the sides or bottom of the chuck (Figure 4B).</li>
</ol>
4. Cryosectioning the Megabrain
<ol>
	<li>Before starting, ensure that a beaker containing at least 20 mL 1x PBS is accessible.</li>
	<li>Position the chuck, mounted with the Megabrain, into the chuck head on the cryostat (Figure 4B).</li>
	<li>Set the cryostat to cut at the desired thickness. The current methodology has been optimized for 10 &#181;m to 50 &#181;m sections.</li>
	<li>Trim the OCT and tissue as needed to reach the desired brain region for tissue collection (Figure 4C).</li>
	<li>When ready to collect tissue, lower the anti-roll plate until it rests on the stage and turn the cryostat handle to take a single section. Slowly lift off the anti-roll plate, being careful that the sectioned Megabrain is not attached to the anti-roll plate and does not fall off the stage Figure 4D).</li>
	<li>Gently use the paintbrushes to unroll the Megabrain section until it is lying flat on the cryostat stage (Figure 5A). (If needed, hold the OCT flat using the paintbrushes to prevent rolling).</li>
	<li>Remove slide from slide warmer and hover the slide, label-side down, over the Megabrain section on the stage allowing the section to stick to the slide.</li>
	<li>Quickly apply a drop of 1x PBS to each tissue section on the slide and do not allow these to dry out until they have been brushed flat (see step 4.9).</li>
	<li>Use a fine tipped paintbrush dipped in 1x PBS to brush out any bubbles in the tissue and unfold the tissue so it is lying flat on the slide (Figure 5B). Take care not to change the position of the brain sections and thus lose the relative position to the other tissue sections. Manipulate the tissue with care to avoid tears. Keep brain sections away from the edges of the slide, as peripheral space is required for a coverslip and PAP pen application in some staining protocols.</li>
	<li>Place the slide back onto the slide warmer and dry for 45 min. Cover as needed to prevent dust from settling on the tissue. Once dry, store the slides in at -80&#160;&#176;C until further use.</li>
</ol>

<ol>
	<li>Lyck, L., Dalmau, I., Chemnitz, J., Finsen, B., Schroder, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+markers+for+quantitative+studies+of+neurons+and+glia+in+human+neocortex.">Immunohistochemical markers for quantitative studies of neurons and glia in human neocortex.</a> Journal of Histochemistry and Cytochemistry. 56 (3), 201-221 (2008).</li><li>Fritz, P., Wu, X., Tuczek, H., Multhaupt, H., Schwarzmann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+in+immunohistochemistry.+A+research+method+or+a+diagnostic+tool+in+surgical+pathology?.">Quantitation in immunohistochemistry. A research method or a diagnostic tool in surgical pathology?.</a> Pathologica. 87 (3), 300-309 (1995).</li><li>Walker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+immunohistochemistry--issues+concerning+methods,+utility+and+semiquantitative+assessment+I.">Quantification of immunohistochemistry--issues concerning methods, utility and semiquantitative assessment I.</a> Histopathology. 49 (4), 406-410 (2006).</li><li>Evilsizor, M. N., Ray-Jones, H. F., Lifshitz, J., Ziebell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer+for+immunohistochemistry+on+cryosectioned+rat+brain+tissue:+example+staining+for+microglia+and+neurons.">Primer for immunohistochemistry on cryosectioned rat brain tissue: example staining for microglia and neurons.</a> Journal of Visualized Experiments. (99), (2015).</li><li>Harrison, J. 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=Resolvins+AT-D1+and+E1+differentially+impact+functional+outcome,+post-traumatic+sleep,+and+microglial+activation+following+diffuse+brain+injury+in+the+mouse.">Resolvins AT-D1 and E1 differentially impact functional outcome, post-traumatic sleep, and microglial activation following diffuse brain injury in the mouse.</a> Brain, Behavior, and Immunity. 47, 131-140 (2015).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of Visualized Experiments. (65), (2012).</li><li>Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M., Altman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+bioscience+research+reporting:+the+ARRIVE+guidelines+for+reporting+animal+research.">Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research.</a> PLoS Biology. 8 (6), e1000412 (2010).</li><li>Kennedy, H. S., Puth, F., Van Hoy, M., Le Pichon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+removing+the+brain+and+spinal+cord+as+one+unit+from+adult+mice+and+rats.">A method for removing the brain and spinal cord as one unit from adult mice and rats.</a> Lab Animal (NY). 40 (2), 53-57 (2011).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematoxylin+and+eosin+staining+of+tissue+and+cell+sections.">Hematoxylin and eosin staining of tissue and cell sections.</a> CSH Protocols. 2008, (2008).</li><li>Ji, X., 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+Impact+of+Repeated+Freeze-Thaw+Cycles+on+the+Quality+of+Biomolecules+in+Four+Different+Tissues.">The Impact of Repeated Freeze-Thaw Cycles on the Quality of Biomolecules in Four Different Tissues.</a> Biopreservation and Biobanking. 15 (5), 475-483 (2017).</li><li>Pegg, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+history+and+principles+of+cryopreservation.">The history and principles of cryopreservation.</a> Seminars in Reproductive Medicine. 20 (1), 5-13 (2002).</li></ol>

The authors have no disclosures.

It should be considered during this procedure that the temperature of the Megabrain and its surroundings must be constantly monitored to prevent thawing and re-freezing of the tissue. The brain can only be removed from the -20 &#176;C freezer up to 3 times as every time it is touched and left in the cryostat, with a warmer fluctuating temperature, the tissue thaws and refreezes, causing a jelly like texture and abnormal tissue integrity10. Therefore, it is optimal to cut the Megabrain all at once....

Here, we present the protocol for a novel method, which we call Megabrain, developed to cryosection multiple rodent brains simultaneously for downstream immunohistochemical procedures. A Megabrain allows for the production of single slides containing tissue from multiple animals. This technique has been optimized to cut coronal sections from 9 adult rat hemispheres, or 5 adult full brains, simultaneously. Therefore, the technique is most applicable in large immunohistochemical studies or other analyses done on slide-mounted brain tissue from a large cohort of animals.
Immunohistochemistry involves the use of specific antibodies directed against proteins of interest to understand and characterize their expression and cellular changes in specific tissue1,2,3. The use of immunohistochemistry is prevalent in neuroscience research, among other scientific disciplines, aiding in the cellular and molecular understanding of the brain4. Large-scale studies involving many animals and brain sections can be both resource and time intensive. As such, there is a multifaceted rationale behind the development of the Megabrain: to reduce time spent cryosectioning, mounting, and staining tissue, while consequently using less reagents. Moreover, the ability to streamline the process and stain multiple brains in the same round helps to alleviate some of the variability between staining batches, a limitation of immunohistochemistry3. In addition, sectioning a Megabrain is a time saving alternative to sectioning individual frozen rodent brains and allows for rapid comparison of tissue between animals or even treatment groups by microscopy techniques.

<table>
	<tbody>
		<tr>
			<td>Andwin scientific tissue-tek CRYO-OCT compound (case of 12)</td>
			<td>Fisher Scientific</td>
			<td>14-373-65</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Shandon Peel-A-Way Disposable Embedding Molds</td>
			<td>Fisher Scientific</td>
			<td>18-41</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand High Precision Straight Broad Strong Point Tweezers/Forceps</td>
			<td>Fisher Scientific</td>
			<td>12-000-128</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand 20mL HDPE Scintillation Vials with Polypropylene Cap</td>
			<td>Fisher Scientific</td>
			<td>03-337-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose, poly bottle 2.5 kg</td>
			<td>Fisher Scientific</td>
			<td>S2-212</td>
			<td>Both 15% and 30% sucrose concentrations need to be made up.</td>
		</tr>
		<tr>
			<td>2-Methylbutane (Certified), Fisher Chemical</td>
			<td>Fisher Scientific</td>
			<td>O3551-4</td>
			<td></td>
		</tr>
		<tr>
			<td>PYREX Tall-Form Beakers</td>
			<td>Fisher Scientific</td>
			<td>02-546E</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand General Purpose Liquid-in-Glass Partial Immersion Thermometers (-50&#176; to +50&#176;C)</td>
			<td>Fisher Scientific</td>
			<td>13-201-642</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Scoopula Spatula</td>
			<td>Fisher Scientific</td>
			<td>14-357Q</td>
			<td></td>
		</tr>
		<tr>
			<td>STANLEY Razor Blade</td>
			<td>Grainger</td>
			<td>4A807</td>
			<td></td>
		</tr>
		<tr>
			<td>Edge-Rite Microtome blades</td>
			<td>Fisher Scientific</td>
			<td>14-070-60</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides (1&#34; frost) - white</td>
			<td>Fisher Scientific</td>
			<td>22-034-979</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco&#160;PBS (Phosphate Buffered Saline) 10X, pH 7.2</td>
			<td>Fisher Scientific</td>
			<td>70-013-032</td>
			<td>Dilute to 1X&#160; before use</td>
		</tr>
		<tr>
			<td>15 piece fine paint brushes</td>
			<td>Amazon</td>
			<td>B079J12ZRV</td>
			<td></td>
		</tr>
		<tr>
			<td>PAP pen</td>
			<td>abcam</td>
			<td>ab2601</td>
			<td></td>
		</tr>
		<tr>
			<td>Chuck Shown in Figure 4 was custom made by a lab technician, however similar sizes are available to order from other companies commercially.</td>
			<td>Electron Microscopy Sciences</td>
			<td>EMS065</td>
			<td></td>
		</tr>
	</tbody>
</table>

simultaneous cryosectioning of multiple rodent brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

A positive end result to this procedure is tissue that lies flat on the slide, with no bubbles or tears, in the orientation in which they were frozen. Tissue sections are evenly spaced apart and easily identifiable due to good placement of the brain in the OCT and good notation as demonstrated in Figure 1. Assuming that the brain tissue was collected from animals of a similar age, and that the tissue was properly aligned in the OCT, sections collected on a sl...

Watch this Scientific Journal Video about Simultaneous Cryosectioning of Multiple Rodent Brains at JoVE.com

Simultaneous Cryosectioning of Multiple Rodent Brains

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...

simultaneous-cryosectioning-of-multiple-rodent-brains

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Neuroscience

23.3K Views.  University of Arizona College of Medicine. Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Video: Simultaneous Cryosectioning of Multiple Rodent Brains

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.

Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of scientific and clinical development. The use of flexible polymer electrode arrays can support long-lasting recording, but the same mechanical properties that allow for longevity of recording make multiple insertions and integration into a chronic implant a challenge. Here is a methodology by which multiple polymer electrode arrays can be targeted to a relatively spatially unconstrained set of brain areas.
The method utilizes thin-film polymer devices, selected for their biocompatibility and capability to achieve long-term and stable&#160;electrophysiologic recording interfaces. The resultant implant allows accurate and flexible targeting of anatomically distant regions, physical stability for months, and robustness to electrical noise. The methodology supports up to sixteen serially inserted devices across eight different anatomic targets. As previously demonstrated, the methodology is capable of recording from 1024 channels. Of these, the 512 channels in this demonstration used for single neuron recording yielded 375 single units distributed across six recording sites. Importantly, this method also can record single units for at least 160 days.
This implantation strategy, including temporarily bracing each device with a retractable silicon insertion shuttle, involves tethering of devices at their target depths to a skull-adhered plastic base piece that is custom-designed for each set of recording targets, and stabilization/protection of the devices within a silicone-filled, custom-designed plastic case. Also covered is the preparation of devices for implantation, and design principles that should guide adaptation to different combinations of brain areas or array designs.

Described below is a method for implantation of multiple polymer electrode arrays across anatomically distant brain regions for chronic electrophysiological recording in freely moving rats. Preparation and surgical implantation are described in detail, with emphasis on design principles to guide adaptation of these methods for use in other species.

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of ...

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

Free-floating Immunostaining of Mouse Brains

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the regulation of energy metabolism and other neurobiological processes. However, the quality, reliability, and reproducibility of brain histology results may vary among laboratories. For each staining experiment, it is necessary to optimize the key procedures based on differences in species, tissues, targeted proteins, and the working conditions of the reagents. This paper demonstrates a reliable workflow in detail, including intra-aortic perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging, which can be followed easily by researchers in this field.
Also discussed are how to modify these procedures to satisfy the individual needs of researchers. To illustrate the reliability and efficiency of this protocol, perineuronal nets were stained with biotin-labeled Wisteria florbunda agglutinin (WFA) and arginine vasopressin (AVP) with an anti-AVP antibody in the mouse brain. Finally, the critical details for the entire procedure have been addressed, and the advantages of this protocol compared to those of other protocols. Taken together, this paper presents an optimized protocol for free-floating immunostaining of mouse brain tissue. Following this protocol makes this process easier for both junior and senior scientists to improve the quality, reliability, and reproducibility of immunostaining studies.

This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging.