KPM was supported by EU FP7 (RETICIRC).

1. Breeding of fish
Embryos were kept and raised under standard conditions (Brand 2002) and staged according to development in days post fertilization (dpf). Adults and larvae at 5 dpf were utilized for measurements.
2. Experimental procedure
<ol>
	<li>Preparation of instrument</li>
</ol>
Larvae: Fish larvae were embedded in 3% pre-warmed (28&deg;C) methylcellulose to prevent body movements. The embryos were placed dorsal side up in the VisioTracker, facing the projecting screen. Adult fish: Fish were briefly anesthetized in 300 mg/l MS-222, fitted into the immobilizing device and placed in the VisioTracker. Before measurements were initiated, they were left to recover for 1-2 min.
<ol start="2">
	<li>Generation of stimulus patterns</li>
</ol>
Stimulus patterns consisting of vertical black-and-white sine-wave gratings rotating around the fish were created using the proprietary software package. They could be modulated through the software package according to wave form, contrast, intensity, angular speed and spatial frequency. Patterns were projected onto the screen using a digital light projector contained within the VisioTracker. The approximate distance between the fish's eye and the screen was 4.5 cm, and projection size on-screen was 360 deg horizontally and 55 deg vertically. For larval fish, direction of stimulation was altered with a frequency of 0.33 Hz to reduce saccade frequency. Adult fish were stimulated unidirectionally and only the eye stimulated in temporal-to-nasal direction was considered, since nasal-to-temporal eye velocity in general is significantly lower and less constant (refer to Mueller and Neuhauss, 2010).
<ol start="3">
	<li>Recording of eye movements</li>
</ol>
A bright-field image of the fish's head was fed to an infrared video camera. Infrared illumination of fish was effected from below. The camera recorded images at a rate of 5 frames/second (larvae) or 12.5 frames/second (adults), respectively. Images are automatically processed, corrected and smoothed for eye shape. Eye orientation in relation to the horizontal axis was then determined automatically and eye velocity was calculated by the proprietary software package. Small movements of the fish were automatically corrected for by the software. All recording and analysis was achieved in real time.
3. Post-experimental data processing
<ol>
	<li>Raw measurements of eye velocities were filtered for saccades in order to extract slow-phase velocity.</li>
	<li>Saccade-filtered eye velocity curves were smoothed by a running average with a sliding window of 7 frames.</li>
	<li>Eye velocity was averaged over frames with identical stimulus conditions.</li>
	<li>For larval fish, eye velocity was averaged over both eyes.</li>
</ol>
4. Representative results:
In order to assess the capabilities of the VisioTracker for larval and adult fish, experiments were performed with zebrafish larvae at 5 dpf, and adult zebrafish.
For larval Zebrafish, the bumper mutant was chosen. In this mutant, lens epithelial cells hyperproliferate, leading to reduced lens size and ectopic location of the lens. These morphological alterations are reflected by a significant reduction of contrast sensitivity and visual acuity (Schonthaler et al., 2010). Figure 1 demonstrates the difference in contrast sensitivity of bumper mutants versus wild-type siblings. bumper mutants increasingly fail to adjust eye velocity as the stimulus contrast decreases. By analogy, when the stimulus spatial frequency is increased, i.e. the stimulus stripe width is decreased, bumper mutants likewise demonstrate reduced visual acuity (Fig. 2)
The dependence of adult Zebrafish visual performance on environmental conditions was investigated by subjecting the fish to varying concentrations of alcohol in their tank water for 30 minutes and subsequently measuring the optokinetic response under varying stimulus conditions. Adult Zebrafish show a marked reduction in contrast sensitivity when maintained in increasing alcohol concentrations (Fig. 3). A similar dose-dependent reduction of overall eye velocity over a wide range of spatial frequencies could be observed when the fish were treated with increasing alcohol concentrations (Fig. 4). Alcohol treatment furthermore dose-dependently reduces oculomotor performance at more demanding tasks as exemplified by increased stimulus speeds (Fig. 5).
<img alt="Figure 1" src="/files/ftp_upload/3556/3556fig1.jpg" /> 
Figure 1. Zebrafish larval eye velocity is dependent on stimulus contrast. 10 bumper mutants and 10 wild-type siblings were analyzed at 5 dpf under varying stimulus stripe contrast conditions. Graph shows average eye velocity &plusmn; 1 SEM.
<img alt="Figure 2" src="/files/ftp_upload/3556/3556fig2.jpg" /> 
Figure 2. Zebrafish larval eye velocity is dependent on spatial frequency. 10 bumper mutants and 10 wild-type siblings were subjected to different stimulus stripe widths at 5 dpf and analyzed as described. Graph shows average eye velocity &plusmn; 1 SEM.
<img alt="Figure 3" src="/files/ftp_upload/3556/3556fig3.jpg" /> 
Figure 3. Adult Zebrafish show alcohol concentration-dependent reduction in contrast sensitivity. Adult Zebrafish were maintained in varying alcohol concentrations as indicated for 30 minutes and analyzed under varying stimulus stripe contrast conditions. Graph shows the average temporal-to-nasal eye velocity &plusmn; 1 SEM of 9 fish per group (except control group: n=11).
<img alt="Figure 4" src="/files/ftp_upload/3556/3556fig4.jpg" /> 
Figure 4. Adult Zebrafish show alcohol concentration-dependent reduction in overall eye movement over a wide range of stimulus stripe width. Adult Zebrafish were maintained in varying alcohol concentrations as indicated for 30 minutes and analyzed under varying stimulus stripe width conditions. Graph shows the average temporal-to-nasal eye velocity &plusmn; 1 SEM of 9 fish per group (except control group: n=11).
<img alt="Figure 5" src="/files/ftp_upload/3556/3556fig5.jpg" /> 
Figure 5. Adult Zebrafish show alcohol concentration-dependent reduction in overall eye movement over a wide range of stimulus speeds. Adult Zebrafish were maintained in varying alcohol concentrations as indicated for 30 minutes and analyzed under varying stimulus speed conditions. Graph shows the average temporal-to-nasal eye velocity &plusmn; 1 SEM of 9 fish per group (except control group: n=11).

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Oliver D.R. Schnaedelbach and Holger D. Russig are employees of TSE Systems GmbH that produces the visual performance tracking system used in this article. Production of this article was sponsored by TSE Systems GmbH. Stephan C.F. Neuhuass is an employee of the University of Zurich which receives remuneration by TSE Systems for each system sold.

The importance of the OKR for the study of visual function has been recognized in the scientific community for a long time (Easter &amp; Nicola 1996, 1997), and attempts to truly quantify the paradigm have started well over a decade ago. Easter and Nicola (1996) developed a system with motorized rotating striped drums, where the video recording of eye movement was analyzed manually. This system suffered from the lack of immobilization of the fish embryo, which required frequent manual repositioning, and could detect the tracking movements of eyes only with great difficulty. A step forward was the use of a video-projected striped drum to allow for more variable computer generated stimulus presentation (Roeser &amp; Baier, 2003; Rinner et al., 2005a). 
The mostly manual, frame-by-frame analysis of videotaped recordings has proven to be extremely laborious, and to a certain degree hampered by observer bias (Beck et al., 2004). Automated analysis in real-time was suggested to allow the use of behavioral feedback learning mechanisms (Major et al., 2004). The use of infrared illumination and frequency-controlled rotating stimuli has been pioneered by Beck et al. (2004). However, the system described there has only been used for larvae, and analysis was carried out off-line. Furthermore, the VisioTracker allows complete control over stimuli, including changing the stimulus during the experiment, thereby allowing greater flexibility and spontaneous influence on the course of the experiment. Also, the digital stimulus creation used by the VisioTracker overcame problems mentioned earlier with acceleration of the inert mass of a striped stimulus drum (Beck et al., 2004).
Larvae restraint by methylcellulose does not significantly interfere with eye movement and does not have any long-term effects on zebrafish well-being. Fish larvae have been successfully maintained embedded in methylcellulose for several days, until oxygen supply through the skin becomes insufficient for the demand with increasing age (Qian et al., 2005).
The adult fish restraining method is equally easy on the animal. The short duration of the experiment, coupled with the option of rapidly exchanging the test animal for a different one, further adds to the positive animal welfare aspects of the system. Since the gills are continuously flushed by water, it is convenient to spike the water with any chemical of choice to study its effect on eye movements and visual performance. Similarly a wash-out experiment can be added without the need to handle the animal between the experiments.
Pixel noise in the video picture was minimized by smoothing algorithms of the proprietary VisioTracker software, enabling highly precise measurements of eye position and angular velocity. Furthermore, to facilitate statistical analysis, the software filtered out saccadic movements which occur at fixed velocity and do not contribute to the experimental statement. An averaging of velocity curves over 7 video frames facilitated later analysis.
The VisioTracker opens a new dimension for many varied research areas. The system and its predecessors have already been used successfully to quantify visual performance in zebrafish larvae, using parameters such as visual acuity, contrast sensitivity and light adaptation (Rinner et al., 2005a, Schonthaler et al., 2010), for functional analysis of cone photoreceptors following manipulation of members of the visual transduction cascade (e.g. Rinner et al., 2005b, Renninger et al., 2011), or the analysis of visual defects in mutant zebrafish larvae (e.g. Schonthaler et al., 2005, 2008; Bahadori et al., 2006). The interdependence of morphological and functional maturation of the visual system has been studied by OKR measurements to show that visual acuity is mainly but not completely limited by photoreceptor spacing at larval stages (Haug et al., 2010).
The VisioTracker is equally suitable to analyze visual function in adult zebrafish and other similar sized fish species (Mueller and Neuhauss (2010), this report).
It is also conceivable to utilize the system in research areas such as toxicology or pharmacology whereby substances to be investigated might be added to the water flow surrounding the adult fish gills. Furthermore, the versatility of VisioTracker enables more thorough analyses for example of ontogenetics of visual function, neural circuit function and development, or sensorimotor control (see review in Huang &amp; Neuhauss, 2008).

<table border="1">
<tbody>
<tr>
<th>Name of the reagent</th>
<th>Company</th>
<th>Catalogue number</th>
</tr>
<tr>
<td>Methylcellulose</td>
<td>Sigma-Aldrich</td>
<td>M0387</td>
</tr>
<tr>
<td>Ethyl 3-aminobenzoate methanesulfonate (MS-222)</td>
<td>Sigma-Aldrich</td>
<td>E10521</td>
</tr>
<tr>
<td>35 mm cell culture dish</td>
<td>Corning</td>
<td>430165</td>
</tr>
<tr>
<td>Serum pipette</td>
<td>Greiner bio-one</td>
<td>612 361</td>
</tr>
<tr>
<td>VisioTracker</td>
<td>TSE Systems</td>
<td>302060</td>
</tr>
</tbody>
</table>

visiotracker, an innovative automated approach to oculomotor analysis

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, robust, and simple to manipulate. A suitable model has been found in the optokinetic response (OKR), a reflexive behavior present in all vertebrates due to its high selection value. The OKR involves slow stimulus-following movements of eyes alternated with rapid resetting saccades. The measurement of this behavior is easily carried out in zebrafish larvae, due to its early and stable onset (fully developed after 96 hours post fertilization (hpf)), and benefitting from the thorough knowledge about zebrafish genetics, for decades one of the favored model organisms in this field. Meanwhile the analysis of similar mechanisms in adult fish has gained importance, particularly for pharmacological and toxicological applications.
Here we describe VisioTracker, a fully automated, high-throughput system for quantitative analysis of visual performance. The system is based on research carried out in the group of Prof. Stephan Neuhauss and was re-designed by TSE Systems. It consists of an immobilizing device for small fish monitored by a high-quality video camera equipped with a high-resolution zoom lens. The fish container is surrounded by a drum screen, upon which computer-generated stimulus patterns can be projected. Eye movements are recorded and automatically analyzed by the VisioTracker software package in real time.
Data analysis enables immediate recognition of parameters such as slow and fast phase duration, movement cycle frequency, slow-phase gain, visual acuity, and contrast sensitivity.
Typical results allow for example the rapid identification of visual system mutants that show no apparent alteration in wild type morphology, or the determination of quantitative effects of pharmacological or toxic and mutagenic agents on visual system performance.

Watch this Scientific Journal Video about VisioTracker, an Innovative Automated Approach to Oculomotor Analysis at JoVE.com

VisioTracker, an Innovative Automated Approach to Oculomotor Analysis

The VisioTracker is an automated system for the quantitative analysis of visual performance of larval and small adult fish based on the recording of eye movements. It features full control over visual stimulus properties and real-time analysis, enabling high-throughput research in fields such as visual system development and function, pharmacology, neural circuit studies and sensorimotor integration.

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, ...

visiotracker-an-innovative-automated-approach-to-oculomotor-analysis

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JoVE Journal

Neuroscience

11.0K Views.  University of Zurich. VisioTracker представляет собой автоматизированную систему для количественного анализа визуального представления личинок и взрослых рыб небольших основан на регистрации движений глаз. К услугам гостей полный контроль над визуальными свойствами стимулов и анал?...

Video: VisioTracker, an Innovative Automated Approach to Oculomotor Analysis

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

VisualEyes: A Modular Software System for Oculomotor Experimentation

Eye movement studies have provided a strong foundation forming an understanding of how the brain acquires visual information in both the normal and dysfunctional brain.1 However, development of a platform to stimulate and store eye movements can require substantial programming, time and costs. Many systems do not offer the flexibility to program numerous stimuli for a variety of experimental needs. However, the VisualEyes System has a flexible architecture, allowing the operator to choose any background and foreground stimulus, program one or two screens for tandem or opposing eye movements and stimulate the left and right eye independently. This system can significantly reduce the programming development time needed to conduct an oculomotor study. The VisualEyes System will be discussed in three parts: 1) the oculomotor recording device to acquire eye movement responses, 2) the VisualEyes software written in LabView, to generate an array of stimuli and store responses as text files and 3) offline data analysis. Eye movements can be recorded by several types of instrumentation such as: a limbus tracking system, a sclera search coil, or a video image system. Typical eye movement stimuli such as saccadic steps, vergent ramps and vergent steps with the corresponding responses will be shown. In this video report, we demonstrate the flexibility of a system to create numerous visual stimuli and record eye movements that can be utilized by basic scientists and clinicians to study healthy as well as clinical populations.

Neural control and cognitive processes can be studied through eye movements. The VisualEyes software allows an operator to program stimuli on two computer screens independently using a simple, custom scripting language. The system can stimulate tandem eye movements (saccades and smooth pursuit) or opposing eye movements (vergence) or any combination.

Eye movement studies have provided a strong foundation forming an understanding of how the brain acquires visual information in both the normal and ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has proven to be an invaluable model organism in helping to tackle this challenge, from inspiring technological approaches,&#160;building the human brain connectome, to actually shedding light on the specific molecular drivers of basic functional patterns. The bulk of the behavioral studies in C. elegans have been performed on solid substrates. In liquid, animals exhibit behavioral patterns that include movement at a range of speeds in 3D, as well as partial body movements, such as a posterior curl without anterior shape change, which introduce new challenges for quantitation. The steps of a simple procedure, and use of a software that enables high-resolution analysis of C. elegans swim behavior, are presented here. The software, named CeleST, uses a specialized computer program that tracks multiple animals simultaneously and provides novel measures of C. elegans locomotion in liquid (swimming). The measures are mostly grounded in animal posture and based on mathematics used in computer vision and pattern recognition, without computational requirements for threshold cut-offs. The software tool can be used to both assess overall swimming prowess in hundreds of animals from combined small batch trials and to reveal novel phenotypes even in well-characterized genetic mutants. The preparation of specimens for analysis with CeleST is simple and low-tech, enabling wide adaptation by the scientific community. Use of the computational approach described here should therefore contribute to the greater understanding of behavior and behavioral circuits in the C. elegans model.

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

An efficient and simple methodology for computer-based analysis of nematode swimming behavior in liquid is described. The method requires little to no investment for C. elegans laboratories. The hardware used is standard, and the computer software for the behavioral analysis (CeleST) is an open source one.

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has ...

Automated Gait Analysis in Mice with Chronic Constriction Injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

An Unbiased Approach of Sampling TEM Sections in Neuroscience

Investigations of the ultrastructural features of neurons and their synapses are only possible with electron microscopy. Especially for comparative studies of the changes in densities and distributions of such features, an unbiased sampling protocol is vital for reliable results. Here, we present a workflow for the image acquisition of brain samples. The workflow allows systematic uniform random sampling within a defined brain region, and the images can be analyzed using a disector. This technique is much faster than extensive examination of serial sections but still presents a feasible approach to estimate the densities and distributions of ultrastructure features. Before embedding, stained vibratome sections were used as a reference to identify the brain region under investigation, which helped speed up the overall specimen preparation process. This approach was used for comparative studies investigating the effect of an enriched-housing environment on several ultrastructural parameters in the mouse brain. Based on the successful use of the workflow, we adapted it for the purpose of elemental analysis of brain samples. We optimized the protocol in terms of the time of user-interaction. Automating all the time-consuming steps by compiling a script for the open source software SerialEM helps the user to focus on the main work of acquiring the elemental maps. As in the original workflow, we paid attention to the unbiased sampling approach to guarantee reliable results.

We introduce a novel workflow for electron microscopy investigations of brain tissue. The method allows the user to examine neuronal features in an unbiased fashion. For elemental analysis, we also present a script that automatizes most of the workflow for randomized sampling.

Investigations of the ultrastructural features of neurons and their synapses are only possible with electron microscopy. Especially for comparative ...