The authors acknowledge with gratitude the skillful assistance of Mrs. Trine W. Mikkelsen, Mrs. Lise M. Fitting, and the staff at P&#229;skeh&#248;jgaard. The Danish Medical Research Council, the Lundbeck Foundation, and the Novo Nordisk Foundation financially supported the study.

Animal anesthesia and euthanesia was performed in accordance with &#34;Principles of laboratory animal care&#34; (NIH publication No. 86-23, revised 1985) and approved by the Danish Council for Animal Research Ethics.
1. Instruments
<ol>
	<li>Collect the instruments presented in the video and listed in the Table of Materials.</li>
</ol>
2. Decapitation
NOTE: Anesthesia was induced by an intramuscular injection of 5 mL of midazolam (5 mg/mL) and 5 mL of ketamine (25 mg/mL). 5-10 min later, when the animal was deeply sedated, an ear vein was cannulated and a lethal overdose (100 mg/kg of bodyweight) of sodium pentobarbital (200 mg/mL) was given intravenously. To ensure that the animal was completely euthanized, the interdigital pain reflex was tested as shown by Ettrup et al. (2011)20. Complete euthanization was ensured as described in the ethics statement above and followed by a transcardial perfusion with 5 L of isotonic saline, as demonstrated by Ettrup et al. (2011)20. All the demonstrated procedures are performed postmortem, precluding the need for the welfare precautions necessary for long-term anesthesia and postprocedural survival.
<ol>
	<li>Decapitate the pig by a high circular cervical incision, using a surgical scalpel, just below the mandibular angle (Figure 1A).</li>
	<li>Still with the surgical scalpel, continue the incision anteriorly through the soft tissue of the neck, including the larynx and the esophagus, until the bony spinal column is reached, approximately at the level of the craniocervical junction.</li>
	<li>Advance the cut with a surgical scalpel from the anterior side of the craniocervical junction, above the anterior arc of atlas, and through the anterior atlantooccipital membrane, thereby exposing the spinal canal and the spinal cord (Figure 1B). Simultaneously ask an assistant to pull the pig body away from the pig head to ease the access between the skull base and the first cervical vertebra.</li>
	<li>Continue the surgical incision through the dural sac and the spinal cord (Figure 1B). Take special care to ensure that a complete transverse section of the spinal cord is achieved. 
	NOTE: Failure to perform the previous step may result in unwanted traction on the spinal cord and brain during the following steps of the decapitation process.</li>
	<li>Forcefully extend the cranocervical junction at the section level (Figure 1C). At the same time, use the surgical scalpel to section the remaining atlantooccipital ligaments to release the articulation between the occipital condyles and the upper articulate process of atlas. Separate the pig head from the body.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/55511/55511fig1.jpg" style="font-size: 14px;" /> 
Figure 1: Minipig decapitation.&#160;(A) Neck incision (arrow, mandibular angle). (B) Incision through the atlantooccipital ligaments and the dura-surrounded spinal cord (SC) at the craniocervical junction (C1, anterior arc of atlas; OC, occipital condyle). (C) The posterior part of the atlantooccipital articulation is released by a forceful extension (arrows) at the section level.&#160;<a href="http://cloudfront.jove.com/files/ftp_upload/55511/55511fig1large.jpg" style="font-size: 14px;" target="_blank">Please click here to view a larger version of this figure.</a>
3. Skull Opening
<ol>
	<li>Position the pig head on a table.</li>
	<li>Make a dorsal longitudinal incision with a surgical scalpel through the skin and the underlying soft tissue from the back of the snout, over the vertex of the head, and down through the posterior part of the occipital region.</li>
	<li>Expose the dorsal and posterior part of the skull by removing the soft tissue located lateral to the initial incision with a surgical scalpel.</li>
	<li>Release the temporal muscle bilaterally from the skull (Figure 2A) with a surgical scalpel. Make sure that the posterior occipital bone is cleaned of soft tissue.</li>
	<li>Use the posterior entrance of the foramen magnum to remove the occipital bone with a Kerrison bone punch and bone rongeurs and expose the dura-covered cerebellum (Figure 2B).</li>
	<li>Return to the exposed anterior side of the skull and select an entry point in the frontal bone, just in front of the eyes. At this point, use a bone chisel with a hammer to penetrate the skull and enter the frontal sinus (Figure 2C).</li>
	<li>Use the extent of the frontal sinus to further the dorsoposterior removal of the outer skull lamina with a bone rongeur or bone punch and expose the inner, thin bony skull lamina covering the cerebrum (Figure 2D).</li>
	<li>Gently open the inner bony skull lamina anteriorly with a hammer and a bone chisel to expose the dura-covered cerebrum (Figure 2E).</li>
	<li>Continue the bone removal laterally using a bone chisel and a bone rongeur through the temporal and parietal bone in order to release the final dorsoposterior part of the skull, located between the already exposed parts of the dura-covered cerebrum and cerebellum (Figure 2F). 
	NOTE: It is often possible, during the final step of this procedure, to use the chisel to break open the remaining posterior skull bone from one side, just like one opens a door.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/55511/55511fig2.jpg" style="opacity: 0.9; font-size: 14px;" /> 
Figure 2: Minipig skull opening.&#160;(A) Exposure of the dorsoposterior skull surface, including the removal of the occipital and temporal muscles. (B) Removal of the occipital bone (CB, dura-covered cerebellum; OB, occipital bone; OC, occipital condyle; and SC, spinal cord). (C) A hammer and a bone chisel are used to penetrate the skull anteriorly and to enter the frontal sinus at the level of the eyes. (D) The extent of the frontal sinus (FS) is used to remove the outer thick skull bone (1), exposing an inner thin bone lamina (2) covering the cerebrum. (E) Removal of the thin bone lamina, exposing the dura-covered cerebrum (arrow). (F) Finally, a hammer and a bone chisel are used to laterally connect the anterior and the posterior skull openings.&#160;<a href="http://cloudfront.jove.com/files/ftp_upload/55511/55511fig2large.jpg" style="font-size: 14px;" target="_blank">Please click here to view a larger version of this figure.</a>
4. Brain Removal
<ol>
	<li>Use surgical forceps to lift the dura and create a gentle incision close to the venous superior sagittal sinus using a fine surgical scalpel (Figure 3A).</li>
	<li>Use micro-scissor or a dura-knife to further open the dura covering the dorsal surface of the brain. 
	NOTE: Special care must be taken when removing the dura corresponding to the cerebellar tentorium (Figure 3B), as preservation of this dural leaf will prevent subsequent brain removal.</li>
	<li>Position the pig head vertically (Figure 3C).</li>
	<li>Use the bone chisel or a dissector to release the ventroanterior cerebrum by blunt dissection of the olfactory bulb from the dura-covered floor of the cranial cavity (Figure 3D).</li>
	<li>Use a fine surgical scalpel to section the exposed optic chiasm (Figure 3E). Expose and section the pituitary stalk and the oculomotor nerves.</li>
	<li>Release the ventral brainstem by sectioning the lower cranial nerves (Figure 3F) with a fine surgical scalpel. Ensure that the dural cerebellar tentorium was completely incised (Figure 3B), as this dural leaf will otherwise cut through the brainstem during the release process.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/55511/55511fig3.jpg" style="font-size: 14px;" /> 
Figure 3: Minipig brain removal.&#160;(A) Dural opening with surgical forceps and a dura knife. (B) Care must be taken to completely incise the dural leaf (arrow), located between the cerebrum and the cerebellum. (C) The pig head is positioned vertically for better visualization of skull base structures and in order for gravity to assist in the intended displacement of the brain. (D) A dissector or a bone chisel is used to relieve the olfactory bulb by blunt section from the dura-covered skull base. (E) The dissection is continued in a posterior direction along the skull base for exposure and sectioning of the optic chiasm (arrow), infundibular stalk, and oculomotor nerves. (F) The brain release is completed with the section of the lower cranial nerves as they depart from the ventral surface of the brainstem (III, oculomotor nerve; IV, trochlear nerve; V, trigeminal nerve; and VI, abducens nerve).&#160;<a href="http://cloudfront.jove.com/files/ftp_upload/55511/55511fig3large.jpg" style="font-size: 14px;" target="_blank">Please click here to view a larger version of this figure.</a>
5. Pituitary Removal
<ol>
	<li>Identify the pituitary stalk and its surrounding dural leaf (the diapragma sellae) in the skull floor (Figure 4A).</li>
	<li>Incise the dural leaf lateral to the pituitary stalk using a fine surgical scalpel (Figure 4B).</li>
	<li>Use a dissector to release the pituitary and lift it out of the pituitary fossa (Figure 4C).</li>
</ol>
<img alt="Figure 4" src="/files/ftp_upload/55511/55511fig4.jpg" style="opacity: 0.9; font-size: 14px;" /> 
Figure 4:&#160;Minipig pituitary removal. (A) The pituitary fossa (*) is identified in the skull floor (1, olfactory bulb; 2, optic chiasm; and PF, posterior cranial fossa). (B) The dural covering (sellar diagphragm, (arrow)) is incised laterally. (C) The pituitary (arrow) is released with a dissector and lifted out of the pituitary fossa. Scale bar (A-C) = 10 mm.&#160;<a href="http://cloudfront.jove.com/files/ftp_upload/55511/55511fig4large.jpg" style="font-size: 14px;" target="_blank">Please click here to view a larger version of this figure.</a>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathological+investigation+of+different+MCAO+modalities+and+impact+of+autologous+bone+marrow+mononuclear+cell+administration+in+an+ovine+stroke+model.">Histopathological investigation of different MCAO modalities and impact of autologous bone marrow mononuclear cell administration in an ovine stroke model.</a> Transl Stroke Res. 2, 279-293 (2011).</li><li>Jortner, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+return+of+the+dark+neuron.+A+histological+artifact+complicating+contemporary+neurotoxicologic+evaluation.">The return of the dark neuron. A histological artifact complicating contemporary neurotoxicologic evaluation.</a> Neurotoxicology. 27, 628-634 (2006).</li></ol>

The authors have nothing to disclose.

Most experimental neuroscience studies are performed in small animal species, such as mice and rats, where access to the CNS is facilitated by a thin skull- and dural-thickness. However, in larger experimental animals like pigs1,4,8, sheep32, and non-human primates, the considerable thickness of these structures necessitates the use of robust instruments (table of materials) and proper entry points for sk...

Translational neuroscience studies in pigs have become increasingly popular during the last two decades. The large size of the pig brain enables the use of conventional clinical brain imagers and the direct use and testing of neurosurgical procedures and equipment from the human clinic1,2,3,4,5,6,7,8. In the last 20 years, pigs, especially minipigs (e.g., G&#246;ttingen minipig), have been used to examine neuromodulatory treatment modalities, such as stem cell transplantation; viral vector transfection; and deep brain stimulation directed towards Parkinson disease, obesity, depression, and Alzheimer disease2,6,9,10,11,12,13,14,15,16,17. This has been followed by the development of stereotaxic and surgical approaches to manipulate the minipig CNS3,18,19,20,21. The instituted CNS changes have been evaluated in live animals using brain imaging (PET10,13,22,24 and MR23), cystometry11,12,25, gait analysis17, neurological evaluation9,17, and postmortem examination based on histology and stereological analysis14,15,17,26,27,31. However, postmortem analysis requires the exposure and removal of the pig brain, which is not an easy task, as a thick, bony skull and a fibrous dural covering surround the pig brain.
The goal of this paper and instructional video is to describe how the postmortem pig brain and pituitary may be exposed and removed in an intact state in 15-20 min using non-motorized surgical tools. The instructional video and photographic illustrations show male minipigs (age: 6 months, bodyweight: 20-25 kg) used for an anatomical study on the minipig pituitary gland.

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			<td height="21" style="height:21px;width:216px;">Heavy Scalpel Handle #4</td>
			<td style="width:191px;">FST (Fine Science Tools)</td>
			<td style="width:149px;">10008-13</td>
			<td style="width:481px;">Good for skin incision and soft tissue removal</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Non-Sterile Scalpel Blades #23</td>
			<td style="width:191px;">FST&#160;</td>
			<td>10023-00</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Scalpel Handle #7</td>
			<td style="width:191px;">FST&#160;</td>
			<td>10007-12</td>
			<td>Optimal for dural incision and precision work</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Non-Sterile Scalpel Blades #11</td>
			<td style="width:191px;">FST&#160;</td>
			<td>10011-00</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Surgical Forceps</td>
			<td style="width:191px;">FST&#160;</td>
			<td>11024-18</td>
			<td>The tip of the surgical forceps ensure a firm grip&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kerrison Bone Punch</td>
			<td style="width:191px;">Aesculap Neurosurgery</td>
			<td style="width:149px;">FF713R</td>
			<td>Must be robust, bite size 3-5 mm</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Bone Rongeur</td>
			<td>Aesculap Neurosurgery</td>
			<td style="width:149px;">MD615</td>
			<td>Must be robust, bite size 15 x 5 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bone Rongeur</td>
			<td>Aesculap Neurosurgery</td>
			<td style="width:149px;">FO551R</td>
			<td>Must be robust, bite size 25 x 15 mm&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bone Chisel</td>
			<td>Lawton</td>
			<td>67-0335</td>
			<td>The size of the chisel head should not exceed 20 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mallet (Hammer)</td>
			<td>Millarco</td>
			<td>5624108</td>
			<td>Weigth 300 g, length 30 cm, head hit area size 2 x 2 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-Scissor</td>
			<td>FST&#160;</td>
			<td>14002-14&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissector</td>
			<td>Aesculap Neurosurgery</td>
			<td style="width:149px;">OL165R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">&#160;G&#246;ttingen minipigs</td>
			<td style="width:191px;">&#160;Ellegaard G&#246;ttingen Minipigs A/S, Denmark</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Euthanimal</td>
			<td style="width:191px;"></td>
			<td style="width:149px;"></td>
			<td>pentobarbital</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Ketamine</td>
			<td style="width:191px;">Pfizer</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Midazolam&#160;</td>
			<td style="width:191px;">Hameln Pharmaceuticals</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
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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.

To prevent the tissue material from drying out, it is recommended to store the removed brain and pituitary in a jar filled with fixative or isotonic saline immediately after macroscopic analysis has been performed. The tissue material may be stored in the fixative for years, whereas storage in isotonic saline, even in a refrigerator, will lead to tissue decay with time.
The removed pituitary may also be directly frozen by immers...

Watch this Scientific Journal Video about Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal at JoVE.com

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

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 ...

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15.3K Views.  Aalborg University Hospital. 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.

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

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

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Co-analysis of Brain Structure and Function using fMRI and Diffusion-weighted Imaging

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when studying the brain, as the functional role that a brain area fulfills may be largely defined by its connections to other brain areas. In this report, we describe a novel, non-invasive approach for relating brain structure and function using magnetic resonance imaging (MRI). This approach, a combination of structural imaging of long-range fiber connections and functional imaging data, is illustrated in two distinct cognitive domains, visual attention and face perception. Structural imaging is performed with diffusion-weighted imaging (DWI) and fiber tractography, which track the diffusion of water molecules along white-matter fiber tracts in the brain (Figure 1). By visualizing these fiber tracts, we are able to investigate the long-range connective architecture of the brain. The results compare favorably with one of the most widely-used techniques in DWI, diffusion tensor imaging (DTI). DTI is unable to resolve complex configurations of fiber tracts, limiting its utility for constructing detailed, anatomically-informed models of brain function. In contrast, our analyses reproduce known neuroanatomy with precision and accuracy. This advantage is partly due to data acquisition procedures: while many DTI protocols measure diffusion in a small number of directions (e.g., 6 or 12), we employ a diffusion spectrum imaging (DSI)1, 2 protocol which assesses diffusion in 257 directions and at a range of magnetic gradient strengths. Moreover, DSI data allow us to use more sophisticated methods for reconstructing acquired data. In two experiments (visual attention and face perception), tractography reveals that co-active areas of the human brain are anatomically connected, supporting extant hypotheses that they form functional networks. DWI allows us to create a "circuit diagram" and reproduce it on an individual-subject basis, for the purpose of monitoring task-relevant brain activity in networks of interest.

We describe a novel approach for simultaneous analysis of brain function and structure using magnetic resonance imaging (MRI). We assess brain structure with high-resolution diffusion-weighted imaging and white-matter fiber tractography. Unlike standard structural MRI, these techniques allow us to directly relate anatomical connectivity to functional properties of brain networks.

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when ...

A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, hippocampus, and the associated parahippocampal regions (perirhinal, entorhinal,&#160;and parahippocampal proper). Unlike most other tracing protocols available, typically focusing on certain MTL areas (e.g., amygdala and/or hippocampus), the integrative perspective adopted by the present tracing guidelines allows for clear localization of all MTL subregions. By integrating information from a variety of sources, including extant tracing protocols separately targeting various MTL structures, histological reports, and brain atlases, and with the complement of illustrative visual materials, the present protocol provides an accurate, intuitive, and convenient guide for understanding the MTL anatomy. The need for such tracing guidelines is also emphasized by illustrating possible differences between automatic and manual segmentation protocols. This knowledge can be applied toward research involving not only structural MRI investigations but also structural-functional colocalization and fMRI signal extraction from anatomically defined ROIs, in healthy and clinical groups alike.

The present work provides a comprehensive set of guidelines for manually tracing the medial temporal lobe (MTL) structures. This protocol can be applied to research involving structural and/or combined structural-functional magnetic resonance imaging (MRI) investigations of the MTL, in both healthy and clinical groups.

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

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 ...

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

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 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 ...

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.
Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological&#160;approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...