Name: a high content imaging assay for identification of botulinum neurotoxin inhibitors
Uploaded: 2014-11-14T13:00:00
Description: Watch this Scientific Journal Video about A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors at JoVE.com

Funding was provided by the Joint Science and Technology Office &#8211; Chemical Biological Defense (JSTO-CBD) Defense Threat Reduction Agency (DTRA) under sponsor project number CCAR# CB3675 and National Institutes of Health (1 R21 AI101387-01 and 5 U01AI082051-05).

Plate 20,000 differentiated mouse ES cells (MES) / well in a 96 well Poly-D lysine coated plates and maintain in motor neuron terminal differentiation media for 5-7 days.
1. Compound Administration and Intoxication with BoNT/A
Perform all of the following work in a BSL2 enclosure to maintain compliance with CDC/NIH guidelines.
<ol>
	<li>Prepare 10 mM stock solutions of each library compound in 100% DMSO in polypropylene 96 well plates. Use the 10 mM stock to prepa...

<ol>
	<li>Lamanna, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+most+poisonous+poison.">The most poisonous poison.</a> Science. 130, 763-772 (1959).</li><li>Dover, N., Barash, J. R., Hill, K. K., Xie, G., Arnon, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Characterization+of+a+Novel+Botulinum+Neurotoxin+Type+H+Gene.">Molecular Characterization of a Novel Botulinum Neurotoxin Type H Gene.</a> J. Infect. Dis. , (2013).</li><li>Hakami, R. M., Ruthel, G., Stahl, A. M., Bavari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gaining+ground:+assays+for+therapeutics+against+botulinum+neurotoxin.">Gaining ground: assays for therapeutics against botulinum neurotoxin.</a> Trends in microbiology. 18, 164-172 (2010).</li><li>Yersin, 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=Interactions+between+synaptic+vesicle+fusion+proteins+explored+by+atomic+force+microscopy.">Interactions between synaptic vesicle fusion proteins explored by atomic force microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 100, 8736-8741 (2003).</li><li>Dong, M., Tepp, W. H., Johnson, E. A., Chapman, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+fluorescent+sensors+to+detect+botulinum+neurotoxin+activity+in+vitro+and+in+living+cells.">Using fluorescent sensors to detect botulinum neurotoxin activity in vitro and in living cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 14701-14706 (2004).</li><li>Stahl, A. M., Adler, M., and, C. B. M., Gilfillan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerating+botulism+therapeutic+product+development+in+the+Department+of+Defense.">Accelerating botulism therapeutic product development in the Department of Defense.</a> Drug Development Research. 70, 303-326 (2009).</li><li>Nuss, J. E., 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=Development+of+cell-based+assays+to+measure+botulinum+neurotoxin+serotype+A+activity+using+cleavage-sensitive+antibodies.">Development of cell-based assays to measure botulinum neurotoxin serotype A activity using cleavage-sensitive antibodies.</a> Journal of biomolecular screening. 15, 42-51 (2010).</li><li>Opsenica, I., 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=A+chemotype+that+inhibits+three+unrelated+pathogenic+targets:+the+botulinum+neurotoxin+serotype+A+light+chain,+P.+falciparum+malaria,+and+the+Ebola+filovirus.">A chemotype that inhibits three unrelated pathogenic targets: the botulinum neurotoxin serotype A light chain, P. falciparum malaria, and the Ebola filovirus.</a> Journal of medicinal chemistry. 54, 1157-1169 (2011).</li><li>Opsenica, I., 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+synthesis+of+2,5-bis(4-amidinophenyl)thiophene+derivatives+providing+submicromolar-range+inhibition+of+the+botulinum+neurotoxin+serotype+A+metalloprotease.">The synthesis of 2,5-bis(4-amidinophenyl)thiophene derivatives providing submicromolar-range inhibition of the botulinum neurotoxin serotype A metalloprotease.</a> European journal of medicinal chemistry. 53, 374-379 (2012).</li><li>Nuss, J. E., 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=Pharmacophore+Refinement+Guides+the+Rational+Design+of+Nanomolar-Range+Inhibitors+of+the+Botulinum+Neurotoxin+Serotype+A+Metalloprotease.">Pharmacophore Refinement Guides the Rational Design of Nanomolar-Range Inhibitors of the Botulinum Neurotoxin Serotype A Metalloprotease.</a> ACS medicinal chemistry letters. 1, 301-305 (2010).</li><li>Kiris, E., 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=Embryonic+stem+cell-derived+motoneurons+provide+a+highly+sensitive+cell+culture+model+for+botulinum+neurotoxin+studies,+with+implications+for+high-throughput+drug+discovery.">Embryonic stem cell-derived motoneurons provide a highly sensitive cell culture model for botulinum neurotoxin studies, with implications for high-throughput drug discovery.</a> Stem cell research. 6, 195-205 (2011).</li><li>Pellett, S., 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=Neuronal+targeting,+internalization,+and+biological+activity+of+a+recombinant+atoxic+derivative+of+botulinum+neurotoxin+A.">Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A.</a> Biochemical and biophysical research communications. 405, 673-677 (2011).</li><li>McNutt, P., Celver, J., Hamilton, T., Mesngon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell-derived+neurons+are+a+novel,+highly+sensitive+tissue+culture+platform+for+botulinum+research.">Embryonic stem cell-derived neurons are a novel, highly sensitive tissue culture platform for botulinum research.</a> Biochemical and biophysical research communications. 405, 85-90 (2011).</li><li>Fischer, 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=Bimodal+modulation+of+the+botulinum+neurotoxin+protein-conducting+channel.">Bimodal modulation of the botulinum neurotoxin protein-conducting channel.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 1330-1335 (2009).</li></ol>

The high potency of Botulinum neurotoxins and the relative ease of their weaponization has resulted in their classification as Category A (highest priority) biothreat agents by the U.S. Centers for Disease Control and Prevention. Unfortunately, there are no FDA approved therapeutics to counter BoNT intoxication after the toxin has been internalized by the motor neurons. Any druggable mechanism that promotes neuronal recovery from BoNT intoxication could lead toward the development of a potential therapy to protect both t...

The bacterium Clostridium botulinum produces Botulinum neurotoxin, one of the most potent biological toxins known to man1. There are 7 distinct BoNT serotypes (BoNT/A-G). BoNT/A-Gall induce paralysis at the neuromuscular junction due to SNARE complex proteolysis 2,3. SNARE proteolysis prevents neurotransmitter vesicle-membrane fusion and therefore blocks neurotransmitter exocytosis4. The specific SNARE target depends upon the particular BoNT serotype involved in the intoxication process. BoNT/A and BoNT/E cleave SNAP-25 whereas BoNT/C cleaves both SNAP-25 and syntaxin5. The remaining serotypes cleave synaptobrevin (also called Vesicle-associated membrane protein (VAMP). BoNT/A was chosen for assay development as it is responsible for a high proportion of naturally occurring botulism and has the longest duration of action6. Development of small molecule therapeutics against BoNT/A is a major goal for our drug discovery program and has utilized traditional target-based methods to identify active site proteolytic inhibitors7,8-10. However, the creation of active site inhibitors with broad spectrum activity against multiple serotypes and post-exposure efficacy will likely be challenging.
We have therefore implemented an innovative, phenotypic drug discovery approach that uses BoNT SNAP-25 cleavage as a functional endpoint to identify small molecules that can block BoNT-mediated motor neuron intoxication. SNAP-25 is required for neurotransmitter release, as degradation of SNAP-25 is predictive of paralysis and lethality in vivo. For example, cell-based screening could lead to discovery of new modulators of cellular factors responsible for toxin inactivation or inhibition of toxin pathways inside targeted cells. An important factor in phenotypic assay development is the selection of physiologically relevant biological models. We and others have described mouse embryonic stem (ES) cell-derived motor neurons that recapitulate the immunologic character of primary motor neurons, including the expression of SNAP-2511-13. Importantly, these cellular systems are highly sensitive to BoNT/A intoxication and demonstrate dose-dependent cleavage of SNAP-25 in response to increasing concentrations of toxin11,12. The differentiated motor neurons are also produced in quantities that are sufficient for high throughput plate based analysis and allowed the design of an array of cellular assays.
The phenotypic assay is an immunofluorescence method utilizing two distinct antibodies to quantify cleavage of endogenously expressed full length SNAP-25 during BoNT/A intoxication of mouse motor neuron culture. A carboxyl terminal BoNT/A cleavage-sensitive (BACS) antibody that recognizes only full length SNAP-25 allows the assessment of BoNT/A mediated proteolysis of SNAP-25 expression in mouse motor neurons10. A schematic diagram of the HCI assay is depicted in Figure 1.

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			<td height="21" style="height:21px;width:255px;">Name of Reagent/ Equipment</td>
			<td style="width:180px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:280px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Botulinum neurotoxin A&#160;</td>
			<td style="width:180px;">Metabiologics</td>
			<td style="width:119px;">NA</td>
			<td style="width:280px;">No catalog number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microtitre plates</td>
			<td style="width:180px;">Greiner&#160;</td>
			<td style="width:119px;">655946</td>
			<td style="width:280px;">Poly-D-Lysine 96-well plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">BACs antibody</td>
			<td style="width:180px;">Lampire Biological</td>
			<td style="width:119px;">NA</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microchem</td>
			<td style="width:180px;">National&#160;</td>
			<td style="width:119px;">0255</td>
			<td style="width:280px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Methanol</td>
			<td style="width:180px;">Thermo Scientific</td>
			<td style="width:119px;">A412-20</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Formaldehyde</td>
			<td style="width:180px;">Thermo Scientific</td>
			<td style="width:119px;">28908</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Horse serum</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">16050</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">PE JANUS MDT Mini Automated Workstation</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td style="width:119px;">AJMDT01</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Opera</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td>OP-QEHS-01</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Triton X 100</td>
			<td style="width:180px;">Sigma-Aldrich</td>
			<td style="width:119px;">9002-93-1</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">BIII tubulin antibody</td>
			<td style="width:180px;">R&#38;D Systems</td>
			<td style="width:119px;">BAM1195</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tween 20</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:119px;">P1379-1L</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342dye&#160;</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">3570</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Antimouse IgG</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">A21236</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Anti rabbit IgG</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">A10042</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Columbus Image analysis software</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td style="width:119px;">Ver 2.4</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Spotfire</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td style="width:119px;">Ver 5.5</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Clorox bleach</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:119px;">18-861-284</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">PlateStack</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:280px;"></td>
		</tr>
	</tbody>
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a high content imaging assay for identification of botulinum neurotoxin inhibitors

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.

Data from high and low controls created two distinct populations with the difference of two medians exceeding 3 standard deviations (Figure 7A). The goal of the screening process is to find the compounds within the sample population with values closer to positive control population, assuming a normal distribution within the sample population (Figure 7B, (i)). Data points that are 3 standard deviations beyond the mean are considered statistically different from the noise and classified as...

Watch this Scientific Journal Video about A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors at JoVE.com

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic ...

a-high-content-imaging-assay-for-identification-botulinum-neurotoxin

Research

JoVE Journal

Neuroscience

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

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

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

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

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

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

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

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

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay&#39;s use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT&#39;s proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.

The BoTest Matrix botulinum neurotoxin (BoNT) detection assays rapidly purify and quantify BoNT from a range of sample matrices.&#160;Here, we present a protocol for the detection and quantification of BoNT from both solid and liquid matrices and demonstrate the assay with BOTOX, tomatoes, and milk.

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample ...

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

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

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

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

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

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

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

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

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

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

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

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 ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.