Assessment of CB agonist CP55940 in maturity for rat hippocampal neurons using a high-throughput immunocytochemical assay and image digital analysis

Primary neurons in culture are considered to be a highly relevant model in the study of neuronal development and activity. They can be cultivated and differentiated in vitro but are difficult to transfect using conventional methods. To address this problem, a capillary electroporation system called Cellaxess Elektra was developed for efficient and reproducible transfection of primary cortical and hippocampal neurons without significant impact on cell morphology and viability. The cells are transfected in any stage of differentiation and development, directly in cell culture plates. Genetic material is delivered in situ to as many as 384 samples at a time, which enables both high-throughput and high-quality screening for hard-to-transfect primary cells, meaning that gene function can be studied on a genome-wide scale in cells previously inaccessible to genetic manipulation.

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P.146Novel high-throughput digital analysis to quantify the amount of functional sarcolemmal dystrophin and myofibre regeneration in Duchenne muscular dystrophy clinical trial samples (exon 53 skipping with golodirsen)

Neuromuscular Disorders ◽

10.1016/j.nmd.2019.06.202 ◽

2019 ◽

Vol 29 ◽

pp. S91 ◽

Cited By ~ 1

Author(s):

D. Scaglioni ◽

F. Catapano ◽

M. Ellis ◽

S. Torelli ◽

D. Chambers ◽

...

Keyword(s):

Clinical Trial ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

High Throughput ◽

Digital Analysis

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Exo-endocytotic recycling of synaptic vesicles in developing processes of cultured hippocampal neurons

The Journal of Cell Biology ◽

10.1083/jcb.117.4.849 ◽

1992 ◽

Vol 117 (4) ◽

pp. 849-861 ◽

Cited By ~ 210

Author(s):

M Matteoli ◽

K Takei ◽

MS Perin ◽

TC Südhof ◽

P De Camilli

Keyword(s):

Synaptic Vesicles ◽

Hippocampal Neurons ◽

Synapse Formation ◽

Neuronal Processes ◽

Immunocytochemical Assay ◽

Mature Neurons ◽

Cell Processes ◽

Multiple Cycles ◽

Living Neurons ◽

Lumenal Domain

In mature neurons synaptic vesicles (SVs) undergo cycles of exo-endocytosis at synapses. It is currently unknown whether SV exocytosis and recycling occurs also in developing axons prior to synapse formation. To address this question, we have developed an immunocytochemical assay to reveal SV exo-endocytosis in hippocampal neurons developing in culture. In this assay antibodies directed against the lumenal domain of synaptotagmin I (Syt I), an intrinsic membrane protein of SVs, are used to reveal exposure of SV membranes at the cell surface. Addition of antibodies to the culture medium of living neurons for 1 hr at 37 degrees C resulted in their rapid and specific internalization by all neuronal processes and, particularly, by axons. Double immunofluorescence and electron microscopy immunocytochemistry indicated that the antibodies were retained within SVs in cell processes and underwent cycles of exo-endocytosis in parallel with SV membranes. In contrast, another endocytotic marker, wheat germ agglutinin, was rapidly cleared from the processes and transported to the cell body. Antibody-labeled SVs were still present in axons several days after antibody loading and became clustered at presynaptic sites in parallel with synaptogenesis. These results demonstrate that SVs undergo multiple cycles of exo-endocytosis in developing neuronal processes irrespective of the presence of synaptic contacts.

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Vesicular release of glutamate from hippocampal neurons in culture: an immunocytochemical assay

Experimental Brain Research ◽

10.1007/s00221-007-1118-6 ◽

2007 ◽

Vol 184 (4) ◽

pp. 479-492 ◽

Cited By ~ 6

Author(s):

Leif Oltedal ◽

Camilla Haglerød ◽

Tomasz Furmanek ◽

Svend Davanger

Keyword(s):

Hippocampal Neurons ◽

Vesicular Release ◽

Immunocytochemical Assay

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Magnetic Alignment of Microelements Containing Cultured Neuronal Networks for High-Throughput Screening

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057115598609 ◽

2015 ◽

Vol 20 (9) ◽

pp. 1091-1100 ◽

Cited By ~ 2

Author(s):

Kent R. Gordon ◽

Yuli Wang ◽

Nancy L. Allbritton ◽

Anne Marion Taylor

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Hippocampal Neurons ◽

Unmet Need ◽

Cost Effective ◽

Screening Methods ◽

Element Analysis ◽

Fluid Handling ◽

Automated Imaging ◽

Culture Surface

High-throughput screening (HTS) on neurons presents unique difficulties because they are postmitotic, limited in supply, and challenging to harvest from animals or generate from stem cells. These limitations have hindered neurological drug discovery, leaving an unmet need to develop cost-effective technology for HTS using neurons. Traditional screening methods use up to 20,000 neurons per well in 384-well plates. To increase throughput, we use “microraft” arrays, consisting of 1600 square, releasable, paramagnetic, polystyrene microelements (microrafts), each providing a culture surface for 500–700 neurons. These microrafts can be detached from the array and transferred to 384-well plates for HTS; however, they must be centered within wells for automated imaging. Here, we developed a magnet array plate, compatible with HTS fluid-handling systems, to center microrafts within wells. We used finite element analysis to select an effective size of the magnets and confirmed that adjacent magnetic fields do not interfere. We then experimentally tested the plate’s centering ability and found a centering efficiency of 100%, compared with 4.35% using a flat magnet. We concluded that microrafts could be centered after settling randomly within the well, overcoming friction, and confirmed these results by centering microrafts containing hippocampal neurons cultured for 8 days.

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Morphometric analysis of cellular interactions with the endothelium during the development of arterial lesions using Scanning Electron Microscopy and digital image analysis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128870 ◽

1987 ◽

Vol 45 ◽

pp. 918-919

Author(s):

M.E. Rosenfeld ◽

C. Karboski ◽

M.F. Prescott ◽

P. Goodwin ◽

R. Ross

Keyword(s):

Electron Microscopy ◽

Image Analysis ◽

Quantitative Data ◽

Digital Image Analysis ◽

Lower Cost ◽

Matched Control ◽

Digital Analysis ◽

Cellular Interactions ◽

Arterial Lesions ◽

Scanning Electron

Previous research documenting the chronology of the cellular interactions that occur on or below the surface of the endothelium during the initiation and progression of arterial lesions, primarily consisted of descriptive studies. The recent development of lower cost image analysis hardware and software has facilitated the collection of high resolution quantitative data from microscopic images. In this report we present preliminary quantitative data on the sequence of cellular interactions that occur on the endothelium during the initiation of atherosclerosis or vasculitis utilizing digital analysis of images obtained directly from the scanning electron microscope. Segments of both atherosclerotic and normal arteries were obtained from either diet-induced or endogenously (WHHL) hypercholesterolemic rabbits following 1-4 months duration of hypercholesterolemia and age matched control rabbits. Vasculitis was induced in rats following placement of an endotoxin soaked thread adjacent to the adventitial surface of arteries.

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