The authors would like to thank the donors and their families for their generous gift. Thank you to Mr. Xiao Xuan Li who recorded the video and helped with video editing; Ms. Hannah Lewis and Mr. Louis Szabo for providing technical support; and Professor Jan Provis for reviewing the video and providing input to the video content.

All methods described here have been approved by the Human Research Ethics Committee of the Australian National University. To create the ventricular model we used the Klingler fiber dissection technique12,14. The Klingler technique is a tactile dissection method that involves removing small portions of the gray matter of the cortex and peeling off bundles of nerve fibers, thus providing a step-by-step guide through the tissue layers from the surface to the deep structures of the brain.
NOTE: The brain specimen used to demonstrate this protocol in the accompanying video and images was carefully removed from a formalin-embalmed human cadaver obtained from the body donor program of the Medical School, Australian National University. The donor had no known history of neuropathological disease. After removal of the dura mater, the brain was stored in 10% ethanol solution at room temperature for three years.
1. Preparation
<ol>
	<li>Obtain a whole brain from an embalmed human cadaver and remove the dura mater and store the brain in 10% ethanol at room temperature prior to dissection. 
	Caution: Use personal protection equipment in a well-ventilated room in accordance to local guidelines when handling. Ensure all participants are familiar with the institutional procedures for the safe handling and disposal of a scalpel and sharp objects before commencing the dissection protocol.</li>
	<li>Prepare the following instruments: scissors, forceps, scalpel blades (No. 15 and No. 22), metal probe, and the blunt end of a metal scalpel handle (Figure 1). Use the blunt end of the scalpel handle to minimise damage to the delicate nerve fibers and conserve the major white matter fiber tracts (Figure 2)13.</li>
	<li>Position the brain so that its ventral surface is facing upwards.</li>
</ol>
2. Dissection Procedure
NOTE: The dissection takes approximately 2 to 3 h to complete
<ol>
	<li>Remove the arachnoid mater and associated vasculature from both cerebral hemispheres using a pair of atraumatic (blunt) forceps.</li>
	<li>Gently lift the cerebellum and locate the inferior colliculi. Place the scalpel blade (No. 15) attached to a long scalpel handle just caudal to the inferior colliculi and cut axially through the brainstem. Keep the blade as close to horizontal as possible to avoid damaging the cerebellum. Take care to preserve the tectum of the midbrain.</li>
	<li>Position the brain to view the left or right lateral fissure. Starting at the supramarginal gyrus, use the blunt end of the scalpel handle to gently remove the superficial cortical layers. Gently move forward first above, then below the lateral sulcus to reveal the horizontal association fiber bundles running in the parietal, frontal, and temporal lobes, respectively.</li>
	<li>Follow the direction of the fibers arching around the posterior border of the insula connecting the superior and inferior longitudinal fasciculi to reveal the arcuate fasciculus.</li>
	<li>Anteriorly, gently remove the remaining superficial cortical layers of the middle temporal and inferior frontal gyri to reveal the uncinate fascicular fibers that connect the temporal and frontal lobes</li>
	<li>Identify the short gyri of the insular cortex and then remove the insula. Next remove the extreme capsule and claustrum to reveal the underlying external capsule. Note the bulge formed by the lentiform nucleus deep to the capsule. Moving towards the dorsal surface of the cortex, reveal the fibers of the corona radiata (Figure 4).</li>
	<li>Remove the remaining cortex and underlying white matter on the dorsal surface of the brain to reach the cingulate gyrus. Continue to use the blunt-end of the scalpel handle to remove the cingulate cortex to reveal the cingulum, the white matter tracts connecting the anterior perforated substance with the parahippocampal gyrus.</li>
	<li>Use the same technique to remove the cingulum from posterior to anterior to reveal the corpus callosum, composed of commissural fibers connecting the two cerebral hemispheres. The dorsum of the body (trunk) of the corpus callosum will now be visible (Figure 6).</li>
	<li>Repeat steps 2.3 to 2.8 on the contralateral cerebral hemisphere.</li>
	<li>Palpate and identify the extent of the lateral ventricle on one of the hemispheres. Using a probe, puncture the lateral wall of the ventricle at the site of the collateral trigone. Using a size 24 blade (attached to a No. 4 scalpel handle) enter through the puncture-site and cut inferiorly to open up the entire length of the inferior horn of the lateral ventricle.</li>
	<li>Now return to the ventricular collateral trigone to extend the cut superiorly towards the splenium of the corpus callosum (dotted line in Figure 5).</li>
	<li>Repeat steps 2.10 and 2.11 on the other hemisphere.</li>
	<li>Open the body of the lateral ventricle by continuing the incision from the trigone rostrally using a cut approximately 3 cm parallel to the corpus callosum in both hemispheres (dotted lines in Figure 6).</li>
	<li>Join the two parallel incisions in each hemisphere rostrally at the level of the genu and caudally at the level of the splenium of the corpus callosum. Using forceps, held in the non-dominant hand, gently lift the corpus callosum at the splenium. With a small sharp pair of scissors, held in the dominant hand, separate the splenium from the underlying septum pellucidum. Once you have reached the rostral end of the body, cut the corpus callosum and remove it.</li>
	<li>Nestle the ventral surface of the brain lengthwise on the palm of your non-dominant hand to stabilize the occipital and temporal areas (posterior part). At the same time, use your dominant hand to firmly but gently hold the anterior end of the brain by placing your opposed fingers and thumb on the lentiform nuclei of both sides of the brain.</li>
	<li>Using gentle pulling and twisting motions, physically separate the anterior and posterior parts of the brain taking special care to keep the choroid plexus intact. It is recommended that a colleague be present to guide the separation and gently section any remaining connecting tissues during the process using a scalpel.</li>
</ol>

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M., 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+ventricular+system+of+the+brain:+a+comprehensive+review+of+its+history,+anatomy,+histology,+embryology,+and+surgical+considerations.">The ventricular system of the brain: a comprehensive review of its history, anatomy, histology, embryology, and surgical considerations.</a> Childs Nerv Syst. 30 (1), 19-35 (2014).</li><li>Drapkin, Z. A., Lindgren, K. A., Lopez, M. J., Stabio, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+assessment+of+a+new+3D+neuroanatomy+teaching+tool+for+MRI+training.">Development and assessment of a new 3D neuroanatomy teaching tool for MRI training.</a> Anat Sci Educ. 8 (6), 502-509 (2015).</li><li>Ruisoto Palomera, P., JuanesM&#233;ndez, J. 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The authors declare that they have no conflict of interest.

The purpose of this paper was to devise a dissection guide for dissemination to teachers and students that could be used to enhance teaching and learning of the deep ventricular and periventricular structures of the human brain. We have devised a step-by-step guide with accompanying images, together with a video resource, that can be used to aid understanding of the morphology of the ventricles and their associated structures. The dissection technique itself is not new. Fiber dissection has been previously used for the s...

Many students struggle to comprehend the negative spaces of the ventricular system, located deep within the human brain1,2. Commonly used resources available for students to study the ventricles provide relatively crude representations of the intricate 3D relationships of these deep cerebral structures. Understanding the 3D anatomy of the ventricular system and related structures is particularly important in neurosurgery because access to the ventricular system is one of the most utilised techniques to measure intracranial pressure, decompress the ventricular system, and administer medications3. In addition, rapid advancements in medical imaging have necessitated the development of skills in the interpretation of 3D anatomy.
Two-dimensional (2D) sections of the brain in different planes are typically used to visualise the deep brain structures that form the boundaries of the negative ventricular spaces4. However, 2D slices of the brain alone are insufficient to enable students to understand the full extent of the 3D architecture of the ventricles and the fine details of the region such as fiber bundles connecting the cortex and subcortical structures5. Consequently, educators have to rely on the students&#39; own ability to compute a comprehensible 3D conception of the ventricles4. Students who struggle with spatial awareness find it extremely difficult to create this 3D image. Whilst plastic models and ventricular casts provide a 3D representation of the ventricular system, they fail to demonstrate the comprehensive relationships that form the boundaries of the ventricles. Students often mindlessly remove parts of the plastic model to access the ventricular system and understand its interconnections. In this process, they frequently overlook the detailed relative positions of each structure and lose understanding of their relationships (e.g.&#160;formation of the roof of the lateral ventricles by the corpus callosum).
The development of new computerised teaching tools has addressed some of these limitations. However, many of these models are limited to static text and images and do not take advantage of the interactivity offered by these new technologies7,8. Whilst interactive technologies enable the user to rotate 3D computer models to study multiple viewpoints, this can confuse some users especially novices who find it challenging to orientate structures6. Furthermore, interactive computer resources have been shown to be less effective in teaching more complex anatomical structures6. Thus, one of the challenges in neuroanatomy education is to provide students with resources that enable them to adequately visualize the ventricles and appreciate their 3D structure and anatomical relationships including the delicate associative, projection, and commissural fiber bundles that form complex relationships with the periventricular structures2.
Dissection has been shown to be an excellent educational method for learning anatomy7,8. A recent study provides evidence of the benefits of student dissection in learning neuroanatomy. In 2016, Rae et al. found improved short-term and long-term retention of neuroanatomy knowledge in students participating in dissections9. Whilst advances in technology continue to improve the accuracy and interactivity of 3D computer models, the knowledge acquired through hands-on dissection cannot be replicated digitally at the present time10.
In this study, we aimed to produce a reproducible dissection of a human brain. We chose a fiber dissection method because that allows preservation of the delicate fiber bundles and periventricular gray matter structures to better define the negative space of the ventricles.
Here we present a comprehensive step-by-step guide for creating a prosection model of the ventricles and periventricular structures together with an accompanying training video for use in neuroanatomy teaching and learning. These resources can be used for teaching and learning the neuroanatomy of the brain by both educators and students.

<table border="0" cellpadding="0" cellspacing="0" width="615">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Scalpel Blade No 15</td>
			<td style="width:113px;">Swann-Morton</td>
			<td style="width:119px;">0205</td>
			<td style="width:167px;">Scalpel blade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scalpel Blade No 11</td>
			<td style="width:113px;">Swann-Morton</td>
			<td style="width:119px;">0203</td>
			<td>Scalpel blade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scalpel Blade No 24</td>
			<td style="width:113px;">Swann-Morton</td>
			<td style="width:119px;">0211</td>
			<td>Scalpel blade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Long Scalpel handle No3L</td>
			<td style="width:113px;">Swann-Morton</td>
			<td style="width:119px;">0913</td>
			<td>Scalpel handle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Short Scalpel handle No4G</td>
			<td style="width:113px;">Swann-Morton</td>
			<td style="width:119px;">0934</td>
			<td>Scalpel handle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scissors</td>
			<td style="width:113px;"></td>
			<td style="width:119px;"></td>
			<td>Scissors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Atraumatic Forceps</td>
			<td style="width:113px;"></td>
			<td style="width:119px;"></td>
			<td>Atraumatic forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Toothed Forceps</td>
			<td style="width:113px;"></td>
			<td style="width:119px;"></td>
			<td>Toothed forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Genelyn Arterial Enhanced</td>
			<td style="width:113px;">GMS Inovations</td>
			<td style="width:119px;">AE-475</td>
			<td>Arterial embalming media</td>
		</tr>
	</tbody>
</table>

exploring deep space - uncovering the anatomy of periventricular structures to reveal the lateral ventricles of the human brain

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this challenging. Because the ventricles are negative spaces located deep within the brain, the only way to understand their anatomy is by appreciating their boundaries formed by related structures. Looking at a 2D representation of these spaces, in any of the cardinal planes, will not enable visualisation of all of the structures that form the boundaries of the ventricles. Thus, using 2D sections alone requires students to compute their own mental image of the 3D ventricular spaces. The aim of this study was to develop a reproducible method for dissecting the human brain to create an educational resource to enhance student understanding of the intricate relationships between the ventricles and periventricular structures. To achieve this, we created a video resource that features a step-by-step guide using a fiber dissection method to reveal the lateral and third ventricles together with the closely related limbic system and basal ganglia structures. One of the advantages of this method is that it enables delineation of the white matter tracts that are difficult to distinguish using other dissection techniques. This video is accompanied by a written protocol that provides a systematic description of the process to aid in the reproduction of the brain dissection. This package offers a valuable anatomy teaching resource for educators and students alike. By following these instructions educators can create teaching resources and students can be guided to produce their own brain dissection as a hands-on practical activity. We recommend that this video guide be incorporated into neuroanatomy teaching to enhance student understanding of the morphology and clinical relevance of the ventricles.

This method of dissection exposes the ventricular system by separating the brain into an anterior and a posterior part (Figure 7 and Figure 8). The posterior part offers an internal view to the collateral trigone from which the posterior and inferior horns can be seen extending to the occipital and temporal lobes, respectively (Figure 8). In the inferior/temporal horn the hippocampus...

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Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

This paper demonstrates the effective use of a fiber dissection method to reveal the superficial white matter tracts and periventricular structures of the human brain, in three-dimensional space, to aid student comprehension of ventricular morphology.

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this ...

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Research

JoVE Journal

Neuroscience

16.5K Views.  Australian National University. This paper demonstrates the effective use of a fiber dissection method to reveal the superficial white matter tracts and periventricular structures of the human brain, in three-dimensional space, to aid student comprehension of ventricular morphology.

Video: Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

Mouse Models of Periventricular Leukomalacia

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature ...

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

Manual Drainage of the Zebrafish Embryonic Brain Ventricles

Cerebrospinal fluid (CSF) is a protein rich fluid contained within the brain ventricles. It is present during early vertebrate embryonic development and persists throughout life. Adult CSF is thought to cushion the brain, remove waste, and carry secreted molecules1,2. In the adult and older embryo, the majority of CSF is made by the choroid plexus, a series of highly vascularized secretory regions located adjacent to the brain ventricles3-5. In zebrafish, the choroid plexus is fully formed at 144 hours post fertilization (hpf)6. Prior to this, in both zebrafish and other vertebrate embryos including mouse, a significant amount of embryonic CSF (eCSF) is present . These data and studies in chick suggest that the neuroepithelium is secretory early in development and may be the major source of eCSF prior to choroid plexus development7.
eCSF contains about three times more protein than adult CSF, suggesting that it may have an important role during development8,9. Studies in chick and mouse demonstrate that secreted factors in the eCSF, fluid pressure, or a combination of these, are important for neurogenesis, gene expression, cell proliferation, and cell survival in the neuroepithelium10-20. Proteomic analyses of human, rat, mouse, and chick eCSF have identified many proteins that may be necessary for CSF function. These include extracellular matrix components, apolipoproteins, osmotic pressure regulating proteins, and proteins involved in cell death and proliferation21-24. However, the complex functions of the eCSF are largely unknown.
We have developed a method for removing eCSF from zebrafish brain ventricles, thus allowing for identification of eCSF components and for analysis of the eCSF requirement during development. Although more eCSF can be collected from other vertebrate systems with larger embryos, eCSF can be collected from the earliest stages of zebrafish development, and under genetic or environmental conditions that lead to abnormal brain ventricle volume or morphology. Removal and collection of eCSF allows for mass spectrometric analysis, investigation of eCSF function, and reintroduction of select factors into the ventricles to assay their function. Thus the accessibility of the early zebrafish embryo allows for detailed analysis of eCSF function during development.

We present a method to collect cerebrospinal fluid (CSF) and to create a system which lacks CSF within the embryonic zebrafish brain ventricular system. This allows for further examination of CSF composition and its requirement during embryonic brain development.

Cerebrospinal fluid  (CSF) is a protein rich fluid contained within the brain ventricles. It is  present during early vertebrate embryonic development and ...

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

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional units of the ventricles are ciliated epithelial cells termed ependymal cells, which line the ventricles and through ciliary action are capable of generating laminar flow of CSF at the ventricle surface. This monolayer of ependymal cells also provides barrier and filtration functions that promote exchange between brain interstitial fluids (ISF) and circulating CSF. Biochemical changes in the brain are thereby reflected in the composition of the CSF and destruction of the ependyma can disrupt the delicate balance of CSF and ISF exchange. In humans there is a strong correlation between lateral ventricle expansion and aging. Age-associated ventriculomegaly can occur even in the absence of dementia or obstruction of CSF flow. The exact cause and progression of ventriculomegaly is often unknown; however, enlarged ventricles can show regional and, often, extensive loss of ependymal cell coverage with ventricle surface astrogliosis and associated periventricular edema replacing the functional ependymal cell monolayer. Using MRI scans together with postmortem human brain tissue, we describe how to prepare, image and compile 3D renderings of lateral ventricle volumes, calculate lateral ventricle volumes, and characterize periventricular tissue through immunohistochemical analysis of en face lateral ventricle wall tissue preparations. Corresponding analyses of mouse brain tissue are also presented supporting the use of mouse models as a means to evaluate changes to the lateral ventricles and periventricular tissue found in human aging and disease. Together, these protocols allow investigations into the cause and effect of ventriculomegaly and highlight techniques to study ventricular system health and its important barrier and filtration functions within the brain.

Using MRI scans (human), 3D imaging software, and immunohistological analysis, we document changes to the brain&#8217;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.

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Investigating the Function of Deep Cortical and Subcortical Structures Using Stereotactic Electroencephalography: Lessons from the Anterior Cingulate Cortex

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure involves the chronic placement of multiple depth electrodes into regions of the brain typically inaccessible via subdural grid electrode placement. SEEG thus provides a unique opportunity to investigate brain function. In this paper we demonstrate how SEEG can be used to investigate the role of the dorsal anterior cingulate cortex (dACC) in cognitive control. We include a description of the SEEG procedure, demonstrating the surgical placement of the electrodes. We describe the components and process required to record local field potential (LFP) data from consenting subjects while they are engaged in a behavioral task. In the example provided, subjects play a cognitive interference task, and we demonstrate how signals are recorded and analyzed from electrodes in the dorsal anterior cingulate cortex, an area intimately involved in decision-making. We conclude with further suggestions of ways in which this method can be used for investigating human cognitive processes.

Stereotactic Electroencephalography (SEEG) is an operative technique used in epilepsy surgery to help localize seizure foci. It also affords a unique opportunity to investigate brain function. Here we describe how SEEG can be used to investigate cognitive processes in human subjects.

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) experiments. Determining the appropriate protocol to follow throughout the sample preparation process can be difficult as each step must be optimized to comply with the unique characteristics of the analytes of interest. This process involves not only finding a compatible matrix that can desorb and ionize the molecules of interest efficiently, but also selecting the appropriate matrix deposition technique. For example, a wet matrix deposition technique, which entails dissolving a matrix in solvent, is superior for desorption of most proteins and peptides, whereas dry matrix deposition techniques are particularly effective for ionization of lipids. Sublimation has been reported as a highly efficient method of dry matrix deposition for the detection of lipids in tissue by MALDI IMS due to the homogeneity of matrix crystal deposition and minimal analyte delocalization as compared to many wet deposition methods 1,2. Broadly, it involves placing a sample and powdered matrix in a vacuum-sealed chamber with the samples pressed against a cold surface. The apparatus is then lowered into a heated bath (sand or oil), resulting in sublimation of the powdered matrix onto the cooled tissue sample surface. Here we describe a sublimation protocol using 1,5-diaminonaphthalene (DAN) matrix for the detection and visualization of gangliosides in the rat brain using MALDI IMS.

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...