The authors would like to acknowledge Jim Knoblauch and Martin-Booth Hodges of the College of Agricultural, Consumer and Environmental Sciences Information Technology and Communication Services for their expertise in shooting, recording, and editing audio and video.

Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Illinois at Urbana-Champaign
NOTE: Prior to euthanasia, the pig was anesthetized via intramuscular injection with a combination of telazol:ketamine:xylazine (50.0 mg of tiletamine HCl plus 50.0 mg of zolazepam HCl reconstituted with 2.50 mL of ketamine HCl (100 g/L) and 2.50 mL of xylazine (100 g/L) and administered at 0.06 mg/kg BW). Once anesthetized, the pig was euthanized via intracardiac administration of sodium pentobarbital (390 mg/mL administered at 1 mL/5 kg BW). For brain dissection, it is recommended that the method of euthanasia be chosen based on the desired analytical procedure of the tissue. The method of euthanasia should cause as little damage to the brain as possible.
1. Extraction of the pig brain
<ol>
	<li>Following humane euthanasia, decapitate the pig by cutting above the nape of the neck, between the first and second vertebra (atlas and axis, respectively).</li>
	<li>Secure the head in an immobilized bench vise modified to include spikes. Ensure that the head is completely immobilized before proceeding.
	<ol>
		<li>Using a scalpel, make a sagittal cut along the midline of the skull that continues to the posterior of the head.</li>
		<li>Make a second (transverse) cut on the posterior of the head.</li>
		<li>Make a third (transverse) cut on the posterior end of the snout and in-line with the eyes. Cut the skin as far away from the skull as possible to ensure easy access to the skull.</li>
	</ol>
	</li>
	<li>Using a bone saw, make two anterior-to-posterior cuts lateral to the midline, extending from the eyes to the apex of the skull curvature. Bevel the saw towards the midline and cut just deep enough to penetrate the skull.
	<ol>
		<li>Repeat the above process on the perpendicular sides, creating a rectangular &#34;window&#34; in the skull.</li>
	</ol>
	</li>
	<li>Using a meat hook, pry the rectangular section of the skull off. Begin by placing the hook into one of the corners of the section and apply upward pressure to loosen the skull piece. Take care to place the meat hook only at the level of the skull to prevent inadvertently penetrating the brain tissue.</li>
	<li>If any meningeal layers remain on the brain, use forceps and blunt scissors (or alternatively, a scalpel) to gently remove the layers without cutting into the brain.</li>
	<li>Use a scalpel to cut away muscle and fat at the posterior portion of the head, exposing the posterior portion of the skull.
	<ol>
		<li>Make two transverse cuts along the posterior of the skull. Be sure not to cut into brain tissue.</li>
		<li>Secure the head by placing a firm hand on the snout and apply a backwards pressure to pull the posterior portion of the skull off, exposing the cerebellum.</li>
	</ol>
	</li>
	<li>Remove the head from the bench vice and place on a surgical mat.</li>
	<li>Use a long slender tool, such as the blunt end of a scalpel, to remove the brain. Invert the head and use a gentle scooping motion to coax the brain out of the skull cavity without damaging the surface of the brain. 
	&#8203;NOTE: It will be necessary to sever cranial nerves to remove the brain. Do so gently and do not attempt to forcefully pull the brain out. The brain will fall out on its own when it is properly and completely detached from the skull and spine.</li>
</ol>
2. Dissection of the pig brain
NOTE: It may be helpful to use a brain atlas or fiber dissection guide14 as a visual representation during dissection procedures. Make sure dissected tissue samples are stored properly according to project-specific needs upon removal of each sample (described in more detail below). Additionally, please note that for the purposes of this video, all brain regions shown were dissected from the right hemisphere, but this may differ per laboratory based on experimental objectives.
<ol>
	<li>To remove the brainstem (predominantly the medulla), make a coronal cut caudal to the cerebellum.</li>
	<li>To remove the cerebellum, make a coronal cut posterior to the cortex. Isolate desired regions (e.g., vermis, flocculus, etc.) of the cerebellum from this sample. Be sure not to include any portions of the brain stem in this sample.</li>
	<li>Separate the two hemispheres of the brain by making a mid-sagittal cut along the longitudinal fissure. Make this cut as a continuous motion to prevent causing damage to the cortex.</li>
	<li>To remove the midbrain, dissect a desired amount of tissue just ventral to the superior and inferior colliculi.</li>
	<li>To remove the hippocampus, place the blunt end of a scalpel in the posterior portion of the corpus callosum and gently roll the hippocampus out, using a &#34;J&#34; motion starting from the posterior portion of the corpus callosum; an entire hippocampal horn is &#39;bean-shaped&#39;.</li>
	<li>The striatum (caudate nucleus is primarily shown) is a grey and white matter-striped region just below the corpus callosum and anterior to the hippocampus. Bevel the scalpel and remove this region, revealing striated tissue upon removal.</li>
	<li>To remove the medial prefrontal cortex, dissect tissue from the frontal gyrus to the corpus callosum, removing the most medial portion of that section. The right cortex should remain after removal of the medial prefrontal cortex.</li>
	<li>To remove the thalamus, remove the bulb-like structure at the center of the midsagittal surface of the brain and rostral to the midbrain. The thalamus has a spherical shape and will look slightly darker than the surrounding tissue.</li>
</ol>
3. Post-dissection
<ol>
	<li>Upon completion of the dissection, preserve tissue samples properly for subsequent analyses. Omission of this step will result in significant autolysis and degradation within as little as 20 minutes.</li>
	<li>Leave each brain region intact at the time of dissection if structural analyses will be performed, or mince the tissues to create a homogenous tissue sample prior to preservation. Common sample preservation methods for brain tissues include cryopreservation and chemical fixation using crosslinking agents15.</li>
	<li>For standard lab assays (e.g., gene or protein expression), immerse in liquid nitrogen or solutions that stabilize genetic material prior to long-term storage at -80 &#176;C to provide convenient and cost-effective preservation methods.</li>
	<li>If maintaining tissue structure is a priority, preserve brain tissues through traditional fixation methods (e.g., using aldehyde-based fixatives to crosslink proteins), either without or with perfusion of the animal or organ of interest prior to tissue dissection.</li>
</ol>

<ol>
	<li>Hughes, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swine+in+cardiovascular+research.">Swine in cardiovascular research.</a> Laboratory Animal Science. 36 (4), 348-350 (1986).</li><li>Yen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+Digestive+System+and+Nutritional+Physiology.">Anatomy of the Digestive System and Nutritional Physiology.</a> Biology of the Domestic Pig. , 31-63 (2001).</li><li>Pond, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+Pigs+and+People.">Of Pigs and People.</a> Swine Nutrition. , 3-24 (2001).</li><li>Odle, J., Lin, X., Jacobi, S. K., Kim, S. W., Stahl, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Suckling+Piglet+as+an+Agrimedical+Model+for+the+Study+of+Pediatric+Nutrition+and+Metabolism.">The Suckling Piglet as an Agrimedical Model for the Study of Pediatric Nutrition and Metabolism.</a> Annual Review of Animal Biosciences. 2 (1), 419-444 (2014).</li><li>Larsen, M. O., Rolin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+G&#246;ttingen+minipig+as+a+model+of+diabetes,+with+special+focus+on+type+1+diabetes+research.">Use of the G&#246;ttingen minipig as a model of diabetes, with special focus on type 1 diabetes research.</a> ILAR Journal. 45 (3), 303-313 (2004).</li><li>Lehmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+minipig+in+general+toxicology.">The minipig in general toxicology.</a> Scandinavian Journal of Laboratory Animal Science. 25, 59-62 (1998).</li><li>Richer, J., 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=Sacrococcygeal+and+transsacral+epidural+anesthesia+in+the+laboratory+pig.">Sacrococcygeal and transsacral epidural anesthesia in the laboratory pig.</a> Surgical Radiologic Anatomy. 20, 431-435 (1998).</li><li>Lind, N. 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+use+of+pigs+in+neuroscience:+Modeling+brain+disorders.">The use of pigs in neuroscience: Modeling brain disorders.</a> Neuroscience and Biobehavioral Reviews. 31 (5), 728-751 (2007).</li><li>Sauleau, P., Lapouble, E., Val-Laillet, D., Malbert, C. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pig+model+in+brain+imaging+and+neurosurgery.">The pig model in brain imaging and neurosurgery.</a> Animal. 3 (8), 1138-1151 (2009).</li><li>Dobbing, J., Sands, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+aspects+of+the+brain+growth+spurt.">Comparative aspects of the brain growth spurt.</a> Early Human Development. 311, 79-83 (1979).</li><li>Aurich, L. A., 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=Microsurgical+training+model+with+nonliving+swine+head.+Alternative+for+neurosurgical+education.">Microsurgical training model with nonliving swine head. Alternative for neurosurgical education.</a> Acta Cirurgica Brasileira. 29 (6), 405-409 (2014).</li><li>Bassi, T., Rohrs, E., Fernandez, K., Ornowska, M., Reynolds, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+brain+excision:+An+easier+method+to+harvest+the+pig's+brain.">Direct brain excision: An easier method to harvest the pig's brain.</a> Interdisciplinary Neurosurgery. 14, 37-38 (2018).</li><li>Bjarkam, C. R., 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=Exposure+of+the+pig+CNS+for+histological+analysis:+A+manual+for+decapitation,+skull+opening,+and+brain+removal.">Exposure of the pig CNS for histological analysis: A manual for decapitation, skull opening, and brain removal.</a> Journal of Visualized Experiments. 122, e55511 (2017).</li><li>Pascalau, R., Szabo, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibre+dissection+and+sectional+study+of+the+major+porcine+cerebral+white+matter+tracts.">Fibre dissection and sectional study of the major porcine cerebral white matter tracts.</a> Anatomia, Histologia, Embryologia. 46, 378-390 (2017).</li><li>McFadden, W. C., 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=Perfusion+fixation+in+brain+banking:+a+systematic+review.">Perfusion fixation in brain banking: a systematic review.</a> Acta Neuropathologica Communications. 7, 146 (2019).</li><li>F&#233;lix, B., 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=Stereotaxic+atlas+of+the+pig+brain.">Stereotaxic atlas of the pig brain.</a> Brain Research Bulletin. 49, 1 (1999).</li></ol>

The authors declare that they have no competing financial interests.

The techniques described herein were designed for pigs approximately 4 weeks of age. It is critical to perform these steps immediately after the pig has been humanely euthanized to ensure the integrity of brain tissue structure is maintained, especially when considering subsequent biochemical assays. It is helpful to use an atlas or fiber16 dissection guides when first learning the techniques. It is recommended that the experimenter practice several brain extractions and dissections prior to obtai...

The pig has been well documented and accepted as a translatable animal model for research in cardiovascular systems1, gastrointestinal systems2, nutrition3,4, diabetes5, toxicology6, and surgical techniques7. Use of the pig in neuroscience is beginning to increase, as PubMed searches for the keywords &#34;swine brain animal model&#34; result in four-times more results from 1996-2005 than the preceding 10 year period8, and even more results at present. A primary reason that the popularity of the pig model is expanding is due to its similarities in growth, structure, and function of the brain when compared with humans. In comparison to the human brain, the pig brain exhibits similar gyral patterning, vascularization and distribution of gray and white matter9. Moreover, the pig brain has been used in neuroimaging procedures, evoked potential recording, and in establishing neurosurgery techniques8,9. Unlike other animal models, however, the pig and human experience perinatal brain growth spurts, as opposed to pre- or post-natal growth spurts. At birth, the human and pig brain weigh approximately 27 and 25 percent of their adult brain weight, respectively, compared to the rat brain that weighs 12 percent of its adult brain weight and the rhesus monkey brain at 76 percent of adult weight10.
One reason the pig has been only slowly adopted as an animal model for neuroscience is because many researchers are unfamiliar with the animal in this context. Researchers may not be aware of its potential uses in the field or may not know the proper techniques required to use such a model. As use of the pig as a biomedical and preclinical model gains attention and use in neuroscience, it is necessary to establish standardized procedures of tissue removal to ensure accurate comparison of data across studies. Although dissection and surgical techniques involving the pig brain have been published elsewhere11,12,13, there is a need for simple and standardized protocols to collect pig brain tissue, especially for use in biochemical assays. As such, the aim of this video is to provide the knowledge necessary to allow researchers to perform a standardized brain extraction and dissection. This video illustrates one proper technique to remove the pig brain while keeping the cortex and brainstem intact, and subsequently review methods to dissect several key brain regions.

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extraction and dissection of the domesticated pig brain

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular systems, gastrointestinal systems, and nutrition, and the pig is increasingly being used as a large animal model in neuroscience. Furthermore, the pig is an accepted model to study neurodevelopment as it displays brain growth and development patterns similar to what occurs in humans. As a less common animal model in neuroscience, surgical and dissection procedures on pigs may not be as familiar or well-practiced among researchers. Therefore, a standardized visual protocol detailing consistent extraction and dissection methods may prove valuable for researchers working with the pig. The following video showcases a technique to remove the pig brain while keeping the cortex and brainstem intact and reviews methods to dissect several commonly investigated brain regions including the brainstem, cerebellum, midbrain, hippocampus, striatum, thalamus, and medial prefrontal cortex. The purpose of this video is to provide researchers with the tools and knowledge necessary to consistently perform a brain extraction and dissection on the four-week-old pig.

This section describes examples of results obtained after correct extraction and dissection of a 4-week-old pig brain. Figure 1 outlines the shape of each brain region for use as a guide during dissection. Part of the brainstem may remain in the skull after removal of the cerebellum (Figure 1B). This can be removed while isolating the desired region of the cerebellum. Table 1 displays the average weight (mean &#177; standard error of the mean) f...

Watch this Scientific Journal Video about Extraction and Dissection of the Domesticated Pig Brain at JoVE.com

Extraction and Dissection of the Domesticated Pig Brain

This protocol details the technique for removal of the pig brain in its entirety and dissection of several brain regions commonly studied in neuroscience.

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular ...

extraction-and-dissection-of-the-domesticated-pig-brain

Research

JoVE Journal

Neuroscience

19.8K Views.  University of Illinois at Urbana-Champaign. This protocol details the technique for removal of the pig brain in its entirety and dissection of several brain regions commonly studied in neuroscience. 

Video: Extraction and Dissection of the Domesticated Pig Brain

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain proper, that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis. The tissue harvested is suitable for biochemical and physiological analysis, and the MAV has been shown to be sensitive to damage produced by amphetamine and hyperthermia 1,2. As well, the major and minor cerebral vasculatures harvested in MAV are of potentially high interest when investigating concussive types of head trauma. The MAV dissected in this presentation consists of the pial and some of the arachnoid membrane (less dura) of the meninges and the major and minor cerebral surface vasculature. The choroid plexus dissected is the structure that resides in the lateral ventricles as described by Oldfield and McKinley3,4,5,6. The methods used for harvesting these two tissues also facilitate the harvesting of regional cortical tissue devoid of meninges and larger cerebral surface vasculature, and is compatible with harvesting other brain tissues such as striatum, hypothalamus, hippocampus, etc. The dissection of the two tissues takes from 5 to 10 min total. The gene expression levels for the dissected MAV and choroid plexus, as shown and described in this presentation can be found at GSE23093 (MAV) and GSE29733 (choroid plexus) at the NCBI GEO repository. This data has been, and is being, used to help further understand the functioning of the MAV and choroid plexus and how neurotoxic events such as severe hyperthermia and AMPH adversely affect their function.

This video presentation shows a method of harvesting the two most important highly vascular structures that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis.

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain ...

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

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 ...

Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal

Pigs have become increasingly popular in large-animal translational neuroscience research as an economically and ethically feasible substitute to non-human primates. The large brain size of the pig allows the use of conventional clinical brain imagers and the direct use and testing of neurosurgical procedures and equipment from the human clinic. Further macroscopic and histological analysis, however, requires postmortem exposure of the pig central nervous system (CNS) and subsequent brain removal. This is not an easy task, as the pig CNS is encapsulated by a thick, bony skull and spinal column. The goal of this paper and instructional video is to describe how to expose and remove the postmortem pig brain and the pituitary gland in an intact state, suitable for subsequent macroscopic and histological analysis.

The goal of this paper and instructional video is to describe how to expose and remove the postmortem pig brain and pituitary gland in an intact state, suitable for subsequent macroscopic and histological analysis.

Pigs have become increasingly popular in large-animal translational neuroscience research as an economically and ethically feasible substitute to ...

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 ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...