This work was funded by support through the McNair Foundation, NARSAD, and NINDS grant R00NS064171-03.

1. In situ brain fixation
*Prepare a 10 ml syringe (28 gauge needle) filled with phosphate buffered saline (PBS). 
*Prepare a 10 ml syringe (28 gauge needle) filled with 4% paraformaldehyde (PFA) in PBS. Reserve an additional 5-10 ml of PFA/PBS for post fixation.

<ol>
<li>Inject experimental mouse intraperitoneally with a lethal dose of Avertin or Nembutal.</li>
<li>Once the mouse is sedated, wet the abdomen with ethanol and secure the mouse to the bottom of a tray layered with cork, wax, or silicone elastomer using dissection pins. With abdomen facing up, secure the four paws to the surface - spreading them as wide as possible.</li>
<li>Grab the skin with forceps at the level of the sternum, and cut crosswise to expose the liver. Cut laterally and then up through the ribs and diaphragm. Remove the rib tissue flap and continue cutting until the heart is accessible.</li>
<li>Pierce or snip the right atrium to drain circulated blood.</li>
<li>Flush residual blood from circulatory system by perfusion of PBS through left ventricle.</li>
<li> Accessing the same needle hole, fix whole mouse by subsequent perfusion of PBS/PFA through left ventricle.</li>
</ol>
2. Dissection and brain extraction 
<ol>
<li>Remove the skin from head, neck, and cranium by cutting around ear canal and eye orbits, followed by pulling skin anteriorly to expose the cranium. 
*See Figure 2 for an illustration of steps 2.2) to 2.5)</li>
<li>Using a bone scissors, cut crosswise from eye the socket through the nasal turbinate bones. Do this for each side. The crosscut should be anterior to the eyes and olfactory bulbs.</li>
<li>Cut lengthwise from each ear canal to eye socket.</li>
<li>Using a small dissection scissors, cut the occipital bone plate overlying the cerebellum from one ear canal to the other.</li>
<li>With the same dissection scissors, cut anteriorly along the midline to the level of the olfactory bulbs.</li>
<li>Using forceps, remove each bone plate from the brain, taking care to remove any connective tissue so that the brain remains intact when lifting off the bone.</li>
<li>Snip optic and cranial nerves, cut the top cervical vertebrae, and remove the brain into fixative.</li>
<li>Post-fix for 1-2 hrs at 4&deg; C. Wash 3 times 15 min each in PBS.</li>
</ol>
3. Agarose embedding
<ol>
<li>Melt 2% high-strength, low-gel point agarose type I-B (Sigma catalog number: A0576) in PBS saline with microwave oven.</li>
<li>Transfer molten agarose to a test tube. Place test tube in a warm incubator or water bath (40&deg;-41&deg; C) until temperature equilibration.</li>
<li>Glue fixed brain tissue onto the plunger of the specimen syringe with cyanoacrylate glue (Figure 3A). Brush a thin layer of saturated sucrose-PBS solution on the surface of the brain tissue to facilitate the later release of the agarose ring from the brain slices.</li>
<li>Draw down the plunger with brain tissue into the plunger housing (Figure 3B).</li>
<li>Pipette or pour warm agarose into the plunger housing, covering the brain tissue. Avoid trapping air bubbles into the agarose. Air bubbles in the agarose will compromise the slice quality.</li> 
<li>Clamp the specimen syringe with a cold (0&deg; to -25&deg; C) chilling block for 1 minute to set the agarose (Figure 3C).</li> 
</ol>
4. Sectioning brain slice with the Compresstome
<ol>
<li>Assemble the specimen syringe containing the brain tissue into the Compresstome (Figure 3D).</li>
<li>Align the edge of the razor blade closely with the outlet of the specimen syringe (Figure 3E).</li>
<li>Fill up the buffer tank with PBS solution.</li>
<li>Rotate the micrometer one slice thickness forward to extrude the agarose-brain tissue block out of the specimen syringe.</li>
<li>Press the &#x201C;start&#x201D; button on the control unit of the Compresstome to initiate the sectioning process.</li> 
<li>Repeat steps 4.4) to 4.5) until desired slices are obtained at determine thickness.</li> 
<li>Harvest brain slices with brush or pipette to vials or staining wells filled with PBS (Figure 3F).</li>
</ol>
5. Optical Clearing
<ol>
<li>Prepare a 75% vol:vol solution of glycerol:PBS.</li>
<li>Pour (or pipette) off half the volume of PBS wash from the collected brain slices.</li>
<li>Add back the volume removed with glycerol/PBS.</li>
<li>Allow to equilibrate with gentle mixing at 4&deg; C until the slices sink.</li>
<li>Repeat steps 6.2) to 6.4) until a final glycerol concentration reaches 75%.</li>
<li> Replace solution with 90% glycerol in PBS and allow to equilibrate. 
*Note: Take care not to introduce air bubbles while adding glycerol solutions. Pour solutions slowly and mix gently, since air bubbles will interfere with uniform equilibration and will be carried over to slide mounting.</li>
</ol>
6. Slice mounting for imaging
<ol>
<li>Cut double-sided seal adhesive (SA-S-1L, Grace Bio-Labs) into an open rectangle to fit the viewable area of a micro slide (Superfrost Plus, 25 X 75 X 1 mm, cat. # 48311-703, VWR) with sides 2-3 mm wide.</li>
<li>Place brain slices inside the center of the adhesive tape rectangle using a pipette or paintbrush. Remove excess glycerol with gentle aspiration.</li>
<li>Arrange brain slices on slide in desired order with a paintbrush.</li>
<li>Gently lower cover glass (24 X 60 mm, cat. # 48383-139, VWR) onto the adhesive tape, taking care not to introduce air bubbles. Gently apply pressure onto cover glass to ensure contact with adhesive tape.</li>
<li>Aspirate excess glycerol from edges between slide and coverglass.</li>
<li>Seal slide and coverslip using clear nail polish.</li>
<li>Allow to dry before imaging, or store long-term in slide boxes at 4&deg; C.</li>
</ol>
7. Representative Results: 
Processing, imaging, and analyzing fluorescently labeled brain tissue have become indispensible to the study of neurobiology. Many of these investigations require sophisticated genetic manipulations to obtain reporter expression in targeted neuronal subsets, followed by both low- and high-resolution image analyses. Often experimental and technical limitations make it impossible to obtain these types of data from the same animal, or in a rapid manner. Preparation of optically-cleared, semi-thick brain sections for fluorescent imaging aids in this challenge. An example of an intact thick brain slice labeled with a genetically modified rabies virus engineered to express EGFP, and imaged using epifluorescent and confocal microscopy is shown in Figure 4 and Figure 5 respectively. For epifluorescent imaging we used a Leica M205 FA, and for confocal image acquisition we used a Zeiss LSM 510. Due to the relative simplicity of the attached protocol, this method is capable of yielding useful image data from tissue expressing fluorescent reporters with a turnaround time of less than one day, and is compatible with both low- and high-resolution light microscopy. 
If the investigator chooses to incorporate additional postmortem labeling methods, immunohistochemical staining, or make thinner sections, the protocol lengthens accordingly. However, the method described above represents a simple and relatively high-throughput method for screening patterns of vital reporter expression in intact brain. 
<img src="/files/ftp_upload/2807/2807fig1.jpg" alt="Figure 1"/> Figure 1. Flow chart diagraming the fixation, dissection, slicing, clearing, and mounting procedure of semi-thick brain slices.
<img src="/files/ftp_upload/2807/2807fig2.jpg" alt="Figure 2"/> Figure 2. Diagram of sequential cutting steps to extract the intact mouse brain. 
<img src="/files/ftp_upload/2807/2807fig3.jpg" alt="Figure 3"/> Figure 3. Steps to mount and slice brain tissue using the Compresstome. A) Placement of brain tissue onto cutting plunger using superglue. B) Drawing down the mounted brain tissue into the plunger for agarose embedding. C) Solidifying an agarose brain plug using a chilled compression block. D) Insertion of agarose brain plug and plunger into Compresstome cutting chamber. E) Alignment of razorblade to the plunger device. F) Cutting and collection of brain slices into Compresstome buffer chamber.
<img src="/files/ftp_upload/2807/2807fig4.jpg" alt="Figure 4"/> Figure 4. Light microscopy images of a thick slice from glycerol-cleared brain tissue through the frontal cortex expressing enhanced green fluorescent protein (EGFP). A) Coronal slice through a mouse brain (200 um thick) labeled with a viral vector expressing EGFP and imaged at low resolution using an epifluorescent stereoscope. Scale bar, 2 mm. 
<img src="/files/ftp_upload/2807/2807fig5.jpg" alt="Figure 5"/> Figure 5. High magnification image of fluorescently labeled layer 5/6 cortical neurons in a cleared thick brain slice. A) High resolution confocal image of a maximum projection Z-stack (150 um thick) through the region highlighted in (Figure 4). Scale bar, 25 um.

<ol>
	<li>Pfister, H., Lichtman, J., Reid, 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> The Connectome Project. , (2009).</li><li>Briggman, K. L., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+neural+circuit+reconstruction+with+volume+electron+microscopy+techniques.">Towards neural circuit reconstruction with volume electron microscopy techniques.</a> Curr Opin Neurobiol. 16, 562-570 (2006).</li><li>Micheva, K. D., Smith, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Array+tomography:+a+new+tool+for+imaging+the+molecular+architecture+and+ultrastructure+of+neural+circuits.">Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits.</a> Neuron. 55, 25-36 (2007).</li><li>Arenkiel, B. R., Ehlers, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+genetic+and+imaging+technologies+for+circuit+based+neuroanatomy.">Molecular genetic and imaging technologies for circuit based neuroanatomy.</a> Nature. 461, 900-907 (2009).</li><li>Capecchi, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altering+the+genome+by+homologous+recombination.">Altering the genome by homologous recombination.</a> Science. 244, 1288-1292 (1989).</li><li>Luo, L., Callaway, E. M., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+neural+circuits.">Genetic dissection of neural circuits.</a> Neuron. 57, 634-660 (2008).</li><li>Shimomura, O., Johnson, F., Saiga, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction,+purification,+and+properties+of+aequorin,+a+bioluminescent+protein+from+the+luminous+hydromedusan,+Aequorea.">Extraction, purification, and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea.</a> J. Cell Comp Physiol. 59, 223-239 (1962).</li><li>Chalfie, M., Tu, Y., Euskirchen, G., Ward, W., Prasher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+fluorescent+protein+as+a+marker+for+gene+expression.">Gene fluorescent protein as a marker for gene expression.</a> Science. 263, 802-805 (1994).</li><li>Giepmans, B. N., Adams, S. R., Ellisman, M. H., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fluorescent+toolbox+for+assessing+protein+location+and+function.">The fluorescent toolbox for assessing protein location and function.</a> Science. 312, 217-224 (2006).</li><li>Micheva, K. D., Smith, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Array+tomography:+a+new+tool+for+imaging+the+molecular+architecture+and+ultrastructure+of+neural+circuits.">Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits.</a> Neuron. 55, 25-36 (2007).</li><li>Svoboda, K., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+two-photon+excitation+microscopy+and+its+applications+to+neuroscience.">Principles of two-photon excitation microscopy and its applications to neuroscience.</a> Neuron. 50, 823-839 (2006).</li></ol>

Jian-Qiang Kong is an employee of Precisionary Instruments, Inc., that manufactures a product used in this article.

Given the widespread application of using fluorescent proteins to target neuronal subsets for investigation via light microscopy, the need to rapidly screen, image, and analyze neural networks within intact brain tissue has become invaluable. 
Technical advances in the development of user-friendly viral vectors, in vivo electroporation techniques, and genetically modified mouse strains now provides a seemingly unlimited source of labeled cell types to investigate. However, image anal...

<table border="1">
	<tbody>
 <tr>
 	<th>Name of the item</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 	<td>bone scissors</td>
 <td>F.S.T.</td>
 <td>16044-10</td>
 <td>-or equivalent</td>
 </tr>
 <tr>
 	<td>dissection scissors</td>
 <td>F.S.T.</td>
 <td>14084-08</td>
 <td>-or equivalent</td>
 </tr>

 <tr>
 	<td>type I-B agarose</td>
 <td>Sigma</td>
 <td>A0576</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 	<td>Compresstome</td>
 <td>Precisionary Instruments</td>
 <td>VF-200</td>
 <td>-other vibratomes are compatible </td>
 </tr>

 <tr>
 	<td>double sided adhesive</td>
 <td>Grace Bio-Labs</td>
 <td>SA-S-1L</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 	<td>Superfrost Plus slides</td>
 <td>VWR</td>
 <td>48311-703</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 	<td>Cover glass</td>
 <td>VWR</td>
 <td>48383-139</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 	<td>glycerol</td>
 <td>EMD Chemicals Inc.</td>
 <td>GX0185-6</td>
 <td>-or equivalent</td>
 </tr>
	 </tbody>
</table>

a rapid approach to high-resolution fluorescence imaging in semi-thick brain slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Watch this Scientific Journal Video about A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices at JoVE.com

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

a-rapid-approach-to-high-resolution-fluorescence-imaging-semi-thick

Research

JoVE Journal

Neuroscience

16.2K Views.  Baylor College of Medicine (BCM). כאן אנו מתארים שיטה מהירה ופשוטה תאים תמונה שכותרתו fluorescently למחצה עבה פרוסות המוח. על ידי תיקון, חיתוך, ואת אופטית ניקוי רקמת המוח אנו מתארים כיצד הדמיה epifluorescent או confocal רגיל יכול לשמש כדי לחזות תאים בודדים ורשתות נוירונים בתוך רקמת העצבים ללא פגע.

Video: A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique cells regarding their extended ramified morphologies and consequently raise several methodological challenges for volume measurement. In the particular case of in vitro neuronal growth, the chosen methodology should include sub-micrometric axial resolution combined with full-field observation on time scales from minutes to hours or days. Unlike other methods like cell shape reconstruction using confocal imaging, electrically-based measurements or Atomic Force Microscopy, the recently developed Fluorescence eXclusion method (FXm) has the potential to fulfill these challenges. However, although being simple in its principle, implementation of a high-resolution FXm for neurons requires multiple adjustments and a dedicated methodology. We present here a method based on the combination of fluorescence exclusion, low-roughness multi-compartments microfluidic devices, and finally micropatterning to achieve in vitro measurements of local neuronal volume. The high resolution provided by the device allowed us to measure the local volume of neuronal processes (neurites) and the volume of some specific structures involved in neuronal growth, such as growth cones (GCs).

Volume is an important parameter regarding physiological and pathological characteristics of cells. We describe a fluorescent exclusion method allowing full-field measurement of in vitro neuronal volume with sub-micrometric axial resolution required for the analysis of neurites and dynamic structures implied in neuronal growth.

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...