Name: rabies necropsy techniques in large and small animals
Uploaded: 2019-07-30T13:00:00
Description: Watch this Scientific Journal Video about Rabies Necropsy Techniques in Large and Small Animals at JoVE.com

We are grateful to&#160;the New York State Department of Health Wadsworth Center for supporting this project. We also would like to acknowledge the support from&#160;Amy Willsey and&#160;Frank Blaisdell&#160;of Department of Health Wadsworth Center, and&#160;LL Ranch, Altamont, NY.&#160;

All methods described were approved by the Wadsworth Center Institutional Animal Care and Use Committee (IACUC).
1. Preparation
<ol>
	<li>Don PPE, at minimum eye protection (glasses or face shield), surgical or N-95 mask, and non-latex gloves.</li>
	<li>Prepare work area, ideally a bio-safety cabinet (BSC), with a disposable work surface covering (e.g., kraft paper or absorbent pads) and clean necropsy instruments (Figure 1).</li>
	<li>Place the specimen on the ...

<ol>
	<li>Centers for Disease Control and Prevention (CDC). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=West+Nile+virus+activity+-+United+States,+2009.">West Nile virus activity - United States, 2009.</a> MMWR Morbidity and Mortality Weekly Report. 59 (25), 769-772 (2010).</li><li>McDaniel, C. J., Cardwell, D. M., Moeller, R. B., Gray, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humans+and+cattle:+A+review+of+bovine+zoonoses.">Humans and cattle: A review of bovine zoonoses.</a> Vector Borne and Zoonotic Diseases. 14 (1), 1-19 (2014).</li><li>Zoonotic diseases. Merck Veterinary Manual Available from: <a target="_blank" href="https://www.merckvetmanual.com/public-health/zoonoses/zoonotic-diseases">https://www.merckvetmanual.com/public-health/zoonoses/zoonotic-diseases</a> (2019)</li><li>Wenner, L., Pauli, U., Summermatter, K., Gantenbein, H., Vidondo, B., Posthaus, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerosol+generation+during+bone-sawing+procedures+in+veterinary+autopsies.">Aerosol generation during bone-sawing procedures in veterinary autopsies.</a> Veterinary Pathology. 54 (3), 425-436 (2017).</li><li>Green, F. H. Y., Yoshida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+aerosols+generated+during+autopsy+procedures+and+their+potential+role+as+carriers+of+infectious+agents.">Characteristics of aerosols generated during autopsy procedures and their potential role as carriers of infectious agents.</a> Applied Occupational and Environmental Hygiene. 5 (12), 853-858 (1990).</li><li>Barrat, J., Meslin, F. X., Kaplan, M. M., Koprowski, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+technique+for+the+collection+and+shipment+of+brain+specimens+for+rabies+diagnosis.">Simple technique for the collection and shipment of brain specimens for rabies diagnosis.</a> Laboratory techniques in Rabies 4th Edition. , 425-427 (1996).</li><li>Ness, S. L., Bain, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+perform+an+equine+field+necropsy.">How to perform an equine field necropsy.</a> American Association of Equine Practitioners. 55, 313-316 (2009).</li><li>. Sample requirements for TSE testing and confirmation &#8211; EURL guidance Available from: <a target="_blank" href="https://science.vla.gov.uk/tse-lab-net/documents/tse-oie-rl-samp.pdf">https://science.vla.gov.uk/tse-lab-net/documents/tse-oie-rl-samp.pdf</a> (2019)</li><li>. Rabies reports Available from: <a target="_blank" href="https://www.wadsworth.org/programs/id/rabies/reports">https://www.wadsworth.org/programs/id/rabies/reports</a> (2019)</li><li>. Protocol for postmortem diagnosis of rabies in animals by direct fluorescent antibody testing: A minimum standard for rabies diagnosis in the United States Available from: <a target="_blank" href="https://www.cdc.gov/rabies/pdf/rabiesdfaspv2.pdf">https://www.cdc.gov/rabies/pdf/rabiesdfaspv2.pdf</a> (2019)</li><li>Miller, L. D., Davis, A. J., Jenny, A. L., Fekadu, M., Whitfield, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surveillance+for+lesions+of+bovine+spongiform+encephalopathy+in+U.S.+cattle.">Surveillance for lesions of bovine spongiform encephalopathy in U.S. cattle.</a> Developments in Biological Standardizations. 80, 119-121 (1993).</li><li>Andrews, C., Gerdin, J., Patterson, J., Buckles, E. L., Fitzgerald, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eastern+equine+encephalitis+in+puppies+in+Michigan+and+New+York+states.">Eastern equine encephalitis in puppies in Michigan and New York states.</a> Journal of Veterinary Diagnostic Investigation. 30 (4), 633-636 (2018).</li><li>Appler, K., Brunt, S., Jarvis, J. A., Davis, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clarifying+indeterminate+results+on+the+rabies+direct+fluorescent+antibody+test+using+real-time+reverse+transcriptase+polymerase+chain+reaction.">Clarifying indeterminate results on the rabies direct fluorescent antibody test using real-time reverse transcriptase polymerase chain reaction.</a> Public Health Reports. 134 (1), 57-62 (2019).</li><li>Rupprecht, C. E., Fooks, A. R., Abela-Ridder, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+7.+Brain+removal.">Chapter 7. Brain removal.</a> Laboratory techniques in Rabies 5th Edition. , 67-72 (2018).</li></ol>

Specimens submitted for rabies necropsy often have a history of clinical signs compatible with a neurological illness. The presence of clinical illness may be associated with a variety of disorders, including zoonotic diseases, increasing the risk to staff of a laboratory acquired infection. To reduce these risks, techniques have been implemented that decrease the handling and manipulation of specimens.
The methods demonstrated represent a necropsy event to remove desired tissues from a single...

Working on the necropsy floor of a rabies laboratory is inherently dangerous. At times, specimens arrive with embedded porcupine quills, foreign objects including arrows/bullets/pellets or exposed bone shards that may penetrate the protective shipping wrap. Improper packaging can result in leakage, endangering individuals who unpack specimens. In addition to physical injury, necropsy technicians risk exposure to unknown zoonotic infectious agents from the CNS and body fluids of the specimens. Additionally, ectoparasites carried by the specimen may transmit other zoonotic diseases, as fleas and ticks are commonly seen on submitted animals. Depending on geographic location and species involved the diseases exposed vary. Arboviruses such as Eastern equine encephalitis virus (EEEV) or West Nile virus (WNV), tick-borne diseases including Lyme disease or tularemia, bacteria causing Q fever or tuberculosis, and infectious prions name a small number of the possible dangers1,2,3.
The purpose of these methods is to demonstrate safe and efficient necropsy techniques using instruments that minimize the potential for aerosolization unlike power tools or saws4,5. Commonly, the necropsy of small animals in the rabies laboratory requires cutting away the cranial muscles and using a hammer and chisel to open the caudal dorsal portion of the calvarium6. Removing this area of calvarium exposes the hind brain, including the entire cerebellum and cranial brain stem. Modified necropsy techniques may be performed on the ventral part of the skull, avoiding the large cranial muscles and thicker regions of the skull. However, these modified necropsy techniques are only possible when the specimen is without cervical vertebrae.
Similarly, brain tissue in large animals can be removed by separating the cranial muscles and opening the caudal dorsal portion of the skull7. Considerable effort is required to expose the cerebellum and brain stem as the skulls of larger animals are generally thicker. To avoid penetrating the skull, the head of a large animal is positioned so the ventro-caudal portion of the skull is facing the technician. Using modified instruments, the cerebellum and brain stem are removed through the foramen magnum. This is similar to the sample acquisition method recommended by the TSE European Union Reference Laboratory for Transmissible Spongiform Encephalopathy (TSE) investigations8. Cranial vertebrae should be removed beforehand to provide access to the foramen magnum.
Application of these techniques are beneficial to suitably trained technicians in rabies laboratories. As the rabies laboratory receives samples of various sizes, from juvenile bats to adult draft horses9, the technician has several methods to choose from based on the individual circumstance. The method demonstrated for a large animal is also appropriate for veterinarians who perform necropsies in the field, since shipping an entire large animal head for rabies testing is cumbersome and costly. Implementing any of these techniques will improve safety by decreasing the potential of aerosol production, reduce the handling of the specimen and save processing time. However, as the field does not have the same advantages as a laboratory set up specific for rabies testing, it is essential that any modifications made to these procedures focus on safety, especially the use of personal protective equipment (PPE).

<table>
	<tbody>
		<tr>
			<td>Chemistry spoon</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Curved, sharp-blunt mayo scissors</td>
			<td>Sklar</td>
			<td>14-2055</td>
			<td>Sklar Operating Scissors 5-1/2 Inch Premium OR Grade Stainless Steel Finger Ring Handle Curved Sharp/Blunt</td>
		</tr>
		<tr>
			<td>Large sharp restaurant-quality carving knife</td>
			<td>Dexter</td>
			<td>P94848</td>
			<td>8&#34; Scalloped Utility Knife, white handle</td>
		</tr>
		<tr>
			<td>Locking tumor-tenacula</td>
			<td>Diamond Scientific and Surgicals</td>
			<td>N/A</td>
			<td>Czerny Tenaculum Forcep</td>
		</tr>
		<tr>
			<td>Modified stiletto knife (6.5 inch long blade carving knife ground to 0.5 inch wide)</td>
			<td>Dexter</td>
			<td>P94848</td>
			<td>Modified 8&#34; Scalloped Utility Knife, white handle</td>
		</tr>
		<tr>
			<td>Orthopedic mallet-hammer</td>
			<td>Mortech</td>
			<td>N/A</td>
			<td>Postmortem hammer with hook</td>
		</tr>
		<tr>
			<td>Sharp councilman orthopedic bone chisel</td>
			<td>Shandon</td>
			<td>60-5</td>
			<td>Councilman&#39;s Chisel Blade: 2 in x 2.25 in standard 7 in</td>
		</tr>
		<tr>
			<td>Sharpened tablespoon or other long handled spoon</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Smooth-tipped tissue dressing forceps without teeth</td>
			<td>Shandon</td>
			<td>63-03</td>
			<td>Shandon Broad Point Dressing Thumb Forceps</td>
		</tr>
		<tr>
			<td>Powder-free non-latex gloves</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Safety glasses, goggles, or faceshield</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgery or N-95 mask</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kraft paper, butcher paper, absorbent pad, etc</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

rabies necropsy techniques in large and small animals

The New York State Department of Health (NYSDOH) Rabies Laboratory receives between 6,000 to 9,000 specimens annually and performs rabies testing for the entire state, with the exception of New York City. The Rabies laboratory necropsies a variety of animals ranging in size from bats to bovids. Most of these specimens are animals exhibiting neurological signs, however, less than 10% actually test positive for rabies; implying trauma, lesions or other infectious agents as the cause of these symptoms. Due to the risk of aerosolizing undiagnosed infectious agents, the Rabies Laboratory does not use power tools or saws. Three necropsy techniques will be presented for animals whose skulls are impenetrable with scissors. The laboratory has implemented these techniques to decrease potential exposure to infectious agents, eliminate unnecessary manipulation of the specimen and reduce processing time. The advantages of a preferred technique opposed to another is subject to the trained individual processing the specimen.

All terrestrial samples submitted with skulls between January 31, 2019 and February 28, 2019 had information regarding the presence of a neck and the method of necropsy collected. During that time, 170 heads were necropsied with 18 species represented. 52% (89/170) were properly decapitated. The remaining had at least one vertebra attached including three whole body specimens. The ventral method was used 75% (128/170) of the time, of those, necks were present on 49. Specimens submitted wi...

Watch this Scientific Journal Video about Rabies Necropsy Techniques in Large and Small Animals at JoVE.com

Rabies Necropsy Techniques in Large and Small Animals

The goal of this protocol is to demonstrate safe necropsy techniques in small and large animals to obtain satisfactory tissue samples for rabies testing.

The New York State Department of Health (NYSDOH) Rabies Laboratory receives between 6,000 to 9,000 specimens annually and performs rabies testing for the ...

Tissue collection for rabies testing has both obvious and unapparent dangers. The technician must use the safest methods available and wear proper personal protective equipment. These protocols provide safe necropsy techniques without using power tools or saws, minimizing the production of aerosols.The large animal protocol can also be performed in the field. Using the large animal sample retrieval method in the field eliminates the challenge of transporting an animal head weighing upwards of 45 kilograms to the laboratory. The large animal method is similar to the European protocol for transmissible spongiform encephalopathy sample retrieval and can be used for any investigation using the brainstem and cerebellum.To retrieve the cerebellum and brainstem by the ventral method, position the specimen ventral side up with the nose directed distally toward the back of the biosafety cabinet. Holding an orthopedic mallet in the dominant hand and a councilman chisel in the other hand, position the chisel at a 45 degree angle with the corner point to the chisel in the V shaped opening between the right side of the temporal and occipital bones. Strike the top of the chisel with the mallet until the two bones separate.And make a cut adjacent to the basisphenoid bone. Separate the temporal and occipital bones on the left side of the specimen as just demonstrated. And use the chisel to bend the V region of the skull downward to expose the entire rhombencephalon area of the brain.Then, use scissors and forceps to scoop out the brainstem and cerebellum, removing any remaining tissue pieces from the skull as necessary. If the specimen does not permit ventral retrieval position the sample dorsally, with the nose directed distally toward the back of the biosafety cabinet. And use a tumor tenacula to grasp the left temporal muscle with the teeth of the tenacula.Squeeze the handle to lock the teeth around the tissue and use a sharp carving knife to cut the temporal muscle down to the bone. Rotate the specimen 180 degrees with the tenacula and the knife and repeat the incision on the opposite temporal muscle to expose the skull. Return the head to its original orientation and place the chisel at a 45 degree angle with the corner point to the chisel on the center of the skull at the juncture of the parietal and intraparietal bones.And strike the top of the chisel with the mallet until a horizontal opening is made on the top half of the skull at the parietal bone. Rotate the specimen 180 degrees and make an opening on the opposite side of the skull as just demonstrated. Position the head as needed to insert the point of the chisel into the end of the first cut at a 90 degree angle to the horizontal opening and strike the chisel until the opening reaches the occipital bone.Roll the specimen to make an opening on the opposite occipital bone in the same manner. The openings in the skull should resemble an upside down U.Insert the teeth of the tenacula into the skull at the bottom of the U and pull the bone backward to expose the caudal end of the cerebrum and the cerebellum. Then use scissors as a scoop to pry out the entire cerebellum from within the cavity.And use tissue forceps to tease out the brainstem from the foramen. For large animal cerebellum and brainstem retrieval, position the specimen so that the dorsal part of the skull is in contact with the necropsy surface. With the caudal part of the skull and foramen magnum facing the front of the biosafety cabinet.Grasp the brainstem with forceps. Insert a modified stiletto knife into the foramen magnum between the spinal cord and the spinal meninges as far as possible and scour around the spinal cord to separate the cerebellum and the brainstem from the spinal meninges. Then, pull the stem out of the foramen with forceps.When the knife has been inserted through the foramen magnum, gently angle the blade to follow along the skull as much as possible and insert a metal spatula or knife with a long enough handle to reach under the brain into the space between the neural tissue and the spinal meninges. Probe around the cerebellum to ensure that connections to the spinal meninges have been severed. When the meninges have been completely severed advance the spatula rostrally and dorsally to scoop up the cerebellum.Then scoop out the cerebellum with the spatula. All terrestrial samples submitted with skulls between January 31st, 2019 and February 28th, 2019 had information regarding the presence of a neck and the method of necropsy collected. During that time, 170 heads were necropsied with 18 species represented.52%were properly decapitated. The remaining had at least one vertebrae attached including three whole body specimens. The ventral method was used a 75%of the time.Of those, necks were present on 49. Three large animals were submitted and in two cases, the large animal protocol was used. One squirrel was submitted with a crushed skull and simply cutting away the skin exposed the brain tissue so none of the demonstrated methods were used.The tissues used to make microscopy slides and or extracted from molecular methods when processing multiple animals avoid cross contamination by using new gloves instruments and a clean work area. Be cautious when performing these protocols as the specimens may be infected with rabies virus or other zoonotic diseases. Note that sharp objects may also be present within the specimens.

rabies-necropsy-techniques-in-large-and-small-animals

Research

JoVE Journal

Neuroscience

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal. Simultaneously recording from multiple nerves and muscles gives us precise information about the timing of neural activity. Previous recording methods have worked for two electrodes, but the study of additional nerves or muscles required combining and averaging the recordings of multiple animals, which made it difficult to determine fine details of timing and phasing, because of variability from response to response, and from animal to animal. Implanting four individual electrodes has a very low success rate due to the formation of adhesions that prevent animals from performing normal feeding movements. We developed a new method of electrode fabrication that reduces the bulk of the electrodes inside the animal allowing for normal feeding movements. Using a combination of glues to attach the electrodes results in a more reliable insulation of the electrode which lasts longer, making it possible to record for periods as long as a week. The fabrication technique that we describe could be extended to incorporate several additional electrodes, and would be applicable to vertebrate animals.

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

A technique is described for implanting four in vivo electrodes to monitor the neuromuscular control of feeding behavior in Aplysia californica.

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal.  ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

Assessing Spatial Learning and Memory in Small Squamate Reptiles

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, interest in the cognitive processes of nonmodel species, typically within an ecological context, has also become an emerging field of study. In particular, interest in the cognitive processes in reptiles is growing although experimental studies on reptilian cognition are sparse. The few reptilian studies that have experimentally tested for spatial learning and memory have used rodent paradigms modified for use in reptiles. However, ecologically important aspects of the physiology and behavior of this taxonomic group must be taken into account when testing for spatially based cognition. Here, we describe modifications of the dry land Barnes maze and associated testing protocol that can improve performance when probing for spatial learning and memory ability in small squamate reptiles. The described paradigm and procedures were successfully used with male side-blotched lizards (Uta stansburiana), demonstrating that spatial learning and memory can be assessed in this taxonomic group with an ecologically relevant apparatus and protocol.

This paper describes a modification of the Barnes maze, a standard rodent paradigm used to assess spatial memory and learning, for use in small squamate reptiles.

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, ...

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

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

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

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

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and smaller functional subgroups, it becomes paramount to record from projections and/or genetically-defined subpopulations of neurons. Fiber photometry is an accessible and powerful approach that can overcome these challenges. By combining optical and genetic methodologies, neural activity can be measured in deep brain structures by expressing genetically-encoded calcium indicators, which translate neural activity into an optical signal that can be easily measured. The current protocol details the components of a multi-fiber photometry system, how to access deep brain structures to deliver and collect light, a method to account for motion artifacts, and how to process and analyze fluorescent signals. The protocol details experimental considerations when performing single and dual color imaging, from either single or multiple implanted optic fibers.

This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and ...

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 &#176;C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

Herein, we display an efficient method for the purification of oligodendrocytes and production of oligodendrocyte-conditioned medium that can be used for co-culture experiments.

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...

Real-Time fMRI Brain Mapping in Animals

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a real-time fMRI platform to guide experimenters to monitor fMRI responses instantaneously during acquisition, which can be used to modify the physiology of animals to achieve desired hemodynamic responses in animal brains. The real-time fMRI set-up is based on a 14.1T preclinical MRI system, enabling the real-time mapping of dynamic fMRI responses in the primary forepaw somatosensory cortex (FP-S1) of anesthetized rats. Instead of a retrospective analysis to investigate confounding sources leading to the variability of fMRI signals, the real-time fMRI platform provides a more effective scheme to identify dynamic fMRI responses using customized macro-functions and a common neuroimage analysis software in the MRI system. Also, it provides immediate troubleshooting feasibility and a real-time biofeedback stimulation paradigm for brain functional studies in animals.

Animal brain functional mapping can benefit from the real-time functional magnetic resonance imaging (fMRI) experimental set-up. Using the latest software implemented in the animal MRI system, we established a real-time monitoring platform for small animal fMRI.

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a ...

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...