We are indebted to Stephan Schwartz and Christian Gutzen of Biobserve GmbH for sponsoring this article and for much technical assistance. We thank Olivia Ochoa, Christian Kaufman, and Zachary Cress for assistance with fish care; we are grateful to the Mexican federal government for permission to collect fish. We are indebted to Glen Vigus, Frederic Parke, and the Visualization Lab at Texas A&amp;M. Athena Mason and Ryan Easterling assisted in preparing this publication. Funding was provided by Texas A&amp;M University and NSF IOS-1045226.

1. Setting up hardware and Viewer system.
The system consists of a testing tank flanked by two monitors. A video camera connected to the BIOBSERVE Viewer system records the movement of the subject in real time. Motion data is passed to the interactive video playback (IVP) program on a separate server, which determines the motion of the animated stimulus on screen.
<ol>
 <li>Place two CRT monitors at opposite ends of an 80-l aquarium filled with clean water; match monitor output.</li>
 <li>Place camera overhead to capture full view of aquarium; connect to BIOBSERVE video card.</li>
 <li>Configure the Viewer system to receive tracking information from the camera.</li>
 <li>Enable plug-in to Viewer that sends the coordinates of the animal's snout, body, and tail to animation server in real time via a specified network IP address.</li>
 <li>Turn on animation server.</li>
</ol>
2. Calibration.
<ol>
 <li>On the animation server, open InteractiveDisplaySetup.txt. Enter screen width and screen height in pixels under "Monitor Info". Under "ProgramType" enter "calibration". Save changes to file.</li>
 <li>Double-click on IVP program icon to start program.</li>
 <li>Enter in the extents of the tank dimensions as set up in the Viewer system in step 2.3. This will ensure correspondence between Viewer data and interactive output from IVP.</li>
 <li>Since the ends of fish tanks are smaller than the monitors, the software allows the user to use the keyboard (a and s for width, z and x for height, and arrow keys for position) to move the viewing area (viewport) of the stimuli to align with the ends of the testing tank. Viewed on the animation server, the pink viewport is echoed on one testing monitor and the blue viewport on the other (figure 1).</li>
 <li>Use z and x on the keyboard to scale the display of the male fish on both viewports until it matches the desired stimulus size.</li>
 <li>Move the boxes displayed so that they align to the edges of the test tank. The user uses the Q or W keys to move the boxes toward or away from the center of the fish.</li>
 <li>Enter experiment information when prompted by the program. The user has the option of selecting among preloaded models (in our demonstration, courting male Xiphophorus birchmanni, X. malinche). The user also specifies the behavior of each fish in each stage (non-interactive or interactive), the side and stage in which each stimulus will appear, and the desired size (standard length) of each stimulus.</li>
 <li>The next question asks if the user wants the fish movements to be mirrored, i.e. that the movement of the model on either side of the aquarium will be exactly the same. This is only used when fish are both in animation mode. The final questions concern the movement of the dorsal fin. The user can select default dorsal; this would make the dorsal only raise during a lateral courtship display. If the dorsal is not set to default, the user can decide if the model raises its dorsal fin according to the distance of the subject from the monitor.</li>
</ol>
3.Starting IVP for mate-choice test.
<ol>
 <li>Open InteractiveDisplaySetup.txt and set "ProgramType" to liveTesting and set 'experimentName' to the same file name that contains the Experiment Data.</li>
 <li>Gently place subject fish in aquarium and wait 10 min.</li>
 <li>Start the Viewer 2.0 and the IVP.</li>
</ol>
4. Mate-choice trial.
The overall experimental sequence follows previous studies using non-interactive video stimuli1-4. Females are presented with two different stimuli on monitors at opposite ends of a testing aquarium. Female behavior is tracked in real-time by the Biobserve Viewer system. The response assay is association time, the amount of time that a female spends within 10 cm of one monitor or the other. Association time is automatically computed by the Viewer.
<ol>
 <li>The video stimuli are displayed to subjects in a trial consisting of two, 20-minute stages. A stage consists of four 5-minute segments:
 <ol>
 <li>The first segment acclimatizes the female to the test tank for five minutes by displaying a monochromatic screen on both monitors.</li>
 <li>In the second segment, two different video stimuli are displayed to the female, one on the left monitor and the other on the right for five minutes.</li>
 <li>Immediately following the display of the video stimuli, a monochromatic screen is again displayed on both monitors for five minutes.</li>
 <li>In the fourth and final segment, stimuli from the second segment are presented, but the position of each stimulus is switched. This provides a within-female control for side bias.</li>
 </ol>
 </li>
 <li>The second stage repeats the same pattern, with a different set of simultaneously-presented stimuli.</li>
 <li>Additional trials are run by returning to step 2 and systematically varying side and order of presentation.</li>
</ol>
5. Representative Results.
We evaluated the effectiveness of interactive playback by comparing female response to interactive and non-interactive animations of courting males. The non-interactive stimulus performed a courtship stimulus onscreen, independent of female behavior, as in previous studies1-4.
The interactive stimulus tracked the horizontal position of the female across the screen. The position of either the simulated fish or the subject fish was operationally defined as the midpoint of the line connecting the centroid and the snout. The three rules guiding male behavior were as follows, where the X direction refers to the length of the tank, and the Z direction refers to the width of the tank (figure 2):
Rule 1: The simulated male always follows the subject across the screen, tracking her in the Z direction.
Rule 2: The simulated male's dorsal fin is only raised when it is performing a lateral courtship display.
Rule 3: The simulated male swordtail fish will only perform a lateral courtship display for 50% of the total time it is being displayed to the female. The lateral courtship display is triggered by the male fish being within one-fourth male body length of the female fish in the Z direction. The lateral courtship display is independent of how close the female is to the monitor in the X direction.
The interactive stimulus closely tracked the female's position in real time (figure 3).
Previous work using non-interactive stimuli4,5 showed that female X. birchmanni strongly preferred visual signals of their own species . The non-interactive stimuli created by the IVP were equally effective at eliciting conspecific mate preferences (t = 1.923, N = 9, p = .046). When females were tested on simulated interactive conspecific and heterospecific males immediately before or afterwards in the same trial, however, they failed to show a preference (figure 4).
<img src="/files/ftp_upload/2374/2374fig1.jpg" alt="Figure 1" /> 
 Figure 1. Monitor setup for calibration.
<img src="/files/ftp_upload/2374/2374fig2.jpg" alt="Figure 2" /> 
 Figure 2. Schematic view of tank and monitor setup, indicating axes used to describe position.
<img src="/files/ftp_upload/2374/2374fig3.jpg" alt="Figure 3" /> 
 Figure 3. Horizontal position of animated exemplar and representative female subject over time.
<img src="/files/ftp_upload/2374/2374fig4.jpg" alt="Figure 4" /> 
 Figure 4. Association time (s) of females with interactive and non-interactive conspecific and heterospecific male stimuli. Each plot is created from a five-minute record of the positions output by the prototype. In these plots, time progresses in the vertical Y direction from 0 to 5 minutes.
<img src="/files/ftp_upload/2374/2374fig5.jpg" alt="Figure 5" /> 
 Figure 5. Representative association time data and tracking by two interactive stimuli. Two five-minute segments for two females are shown.

<ol>
	<li>Fisher, H. S., Mascuch, S., Rosenthal, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multivariate+male+traits+misalign+with+multivariate+female+preferences+in+the+swordtail+fish,+Xiphophorus+birchmanni.">Multivariate male traits misalign with multivariate female preferences in the swordtail fish, Xiphophorus birchmanni.</a> Anim. Behav. 78, 265-269 (2009).</li><li>Fisher, H. S., Rosenthal, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hungry+females+show+stronger+mating+preferences.">Hungry females show stronger mating preferences.</a> Behavioral Ecology. 17, 979-981 (2006).</li><li>Fisher, H. S., Rosenthal, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male+swordtails+court+with+an+audience+in+mind.">Male swordtails court with an audience in mind.</a> Biology Letters. 3, 5-7 (2007).</li><li>Wong, B. B. M., Rosenthal, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Female+disdain+for+swords+in+a+swordtail+fish.">Female disdain for swords in a swordtail fish.</a> American Naturalist. 167, 136-140 (2006).</li><li>Fisher, H. S., Wong, B. B. M., Rosenthal, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+the+chemical+environment+disrupts+communication+in+a+freshwater+fish.">Alteration of the chemical environment disrupts communication in a freshwater fish.</a> Proceedings of the Royal Society B-Biological Sciences. 273, 1187-1193 (2006).</li><li>Rosenthal, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+considerations+and+techniques+for+constructing+video+stimuli.">Design considerations and techniques for constructing video stimuli.</a> Acta Ethol. 3, 49-54 (2000).</li><li>Turnell, E. R., Mann, K. D., Rosenthal, G. G., Gerlach, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mate+choice+in+zebrafish+(Danio+rerio)+analyzed+with+video-stimulus+techniques.">Mate choice in zebrafish (Danio rerio) analyzed with video-stimulus techniques.</a> Biol. Bull. 205, 225-226 (2003).</li><li>Rosenthal, G. G., Ryan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assortative+preferences+for+stripes+in+danios.">Assortative preferences for stripes in danios.</a> Animal Behaviour. 70, 1063-1066 (2005).</li><li>Reynolds, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flocks,+herds+and+schools:+A+distributed+behavioral+model.">Flocks, herds and schools: A distributed behavioral model.</a> , 256 (1985).</li><li>Reynolds, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steering+Behaviors+For+Autonomous+Characters.">Steering Behaviors For Autonomous Characters.</a> , (1999).</li><li>Hoare, D. J., Couzin, I. D., Godin, J. -. G. J., Krause, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context-dependent+group+size+choice+in+fish.">Context-dependent group size choice in fish.</a> Animal Behaviour. 65, 663-669 (2004).</li><li>Hoare, D. J. &. a. m. p. ;. a. m. p., Krause, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+organisation,+shoal+structure+and+information+transfer.">Social organisation, shoal structure and information transfer.</a> Fish and Fisheries. 4, 269-279 (2003).</li></ol>

The production of this video-article was sponsored by Biobserve Research.

Previous methods for interactive video playback in animal behavior have relied on a human operator to provide responses to behavioral cues from subjects. With IVP, we created a program that applied rules-based interactivity in response to real-time, automated data on subject behavior. We briefly outline the steps involved in creating the program below. The first step was to create digital male exemplars of X. birchmanni and X. malinche. We took an approach similar to previous studies6. We created 3D meshes that are modeled on textures based on photographs of real X. birchmanni and X. malinche. To capture the realistic textures of the real fish, the same photographs used to model the fish shapes were used as textures for the fish. A planar map applied to their UV coordinates aligned the UV map with the photograph texture. Second, the digital fish mesh must deform like a real fish. To accomplish this, a virtual skeleton was created for body and fins and "skinned" to the mesh. The skinning process enables the mesh to be deformed when the joints are rotated. Second, we added motion to the digital fish. Six key movements that a male swordtail fish makes were animated. Three of the movements were used to represent the different speeds at which a fish would swim. The other three movements were the fish remaining still, turning, or exhibiting a lateral courtship display. Since males can raise or lower their dorsal fin in accordance to whether male or female receivers are present3, we decoupled the movement of the dorsal fin from that of the lateral courtship display. The dorsal fin was keyed so that it could be raised or lowered at any point during the cycle. A total of twenty-four animation cycles were used. Each cycle started and ended with the fish in the same posture so that the animation cycles could easily blend together. All of the twenty-four animation cycles were created by rotoscoping7,8 the desired motion from overhead video of a live, courting male X. birchmanni. Third, we enabled interactivity. We used the Biobserve Viewer system to track in real time the position of the snout, body, and tail of the female swordtail and transmit that information in real time to the IVP program. This was done separately for each courting male on each monitor. The male animation followed the subject fish's position. We modeled following using Reynolds arrived steering behavior9,10, which allowed the male to follow the female and decelerate as it approaches the female. To calculate the position of the male swordtail fish at each time step, the system was supplied with the current position of the female, which allowed the program to calculate the forces that drive the male. First, the target-offset vector was calculated by subtracting the position of the male fish from the position of the female fish. Second the distance from the male fish to the female fish was determined by taking the magnitude of the target-offset vector. Third, the desired speed of the male fish was determined by dividing the distance by a constant deceleration value. This allowed the male fish to slow down as it approached the female fish. Last, the desired acceleration was calculated by subtracting the male's current velocity from the desired velocity. Since animations are rendered as discrete frames of video at 60 Hz, calculations were made for each discrete time step, at intervals of 0.016 seconds. Maximum velocity was set to a value of 10 cm/s for these experiments. If the magnitude of the new velocity was greater than the maximum velocity, the velocity was set to the maximum. For this particular simulation, the interactive male fish raised its dorsal fin 50% of the time, and only during courtship interactions. The lateral courtship display behavior was triggered whenever the male stimulus was within 0.25 body lengths of the female swordtail fish in the Z dimension. We were surprised that interactivity abolished the female preference for conspecifics, despite the fact that the non-interactive animations elicited a strong preference and given that females spent the majority of time associating with the interactive stimuli. One possibility is that closely following the female may override visual cues used to evaluate males, such as the sword and dorsal fin. Alternatively, females may be less likely to lose interest in a courting male, and therefore less likely to sample both individuals (figure 5). Nevertheless, our results show that the operating principle of video-game technology namely, software-driven, rules-based agents responding to user input can be successfully applied to interactive playback in studies of animal behavior. This type of rules-based interactive playback should prove useful for studies of shoaling and collective movement11,12. In particular, the ability to manipulate the rules that a virtual exemplar uses for shoaling should give us insight into the processes that animals use to make shoaling decisions.

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<td>Maya 8.0</td>
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<td>C# program using Microsoft&#x2019;s XNA Game Studio 2.0</td>
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<td>BIOBSERVE Viewer 2</td>
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automated interactive video playback for studies of animal communication

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, parameter-based computer animation offers the opportunity to independently manipulate any number of behavioral, morphological, or spectral characteristics in the context of realistic, moving images of animals on screen. A major limitation of conventional playback, however, is that the visual stimulus lacks the ability to interact with the live animal. Borrowing from video-game technology, we have created an automated, interactive system for video playback that controls animations in response to real-time signals from a video tracking system. We demonstrated this method by conducting mate-choice trials on female swordtail fish, Xiphophorus birchmanni. Females were given a simultaneous choice between a courting male conspecific and a courting male heterospecific (X. malinche) on opposite sides of an aquarium. The virtual male stimulus was programmed to track the horizontal position of the female, as courting males do in the wild. Mate-choice trials on wild-caught X. birchmanni females were used to validate the prototype's ability to effectively generate a realistic visual stimulus.

Watch this Scientific Journal Video about Automated Interactive Video Playback for Studies of Animal Communication at JoVE.com

Automated Interactive Video Playback for Studies of Animal Communication

Video playback is a widely used technique in animal behavior. We created and evaluated a program that applies rules-based, interactive playback of 3-D computer animations in response to real-time, automated data on subject behavior.

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

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13.2K Views.  Texas A&M University (TAMU). Video playback is a widely used technique in animal behavior. We created and evaluated a program that applies rules-based, interactive playback of 3-D computer animations in response to real-time, automated data on subject behavior.

Video: Automated Interactive Video Playback for Studies of Animal Communication

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to investigate the effects of locally manipulated neurotransmitter and signaling pathways in awake, behaving animals. Moreover, the stereotaxic implantation of electrodes for electrophysiological stimulation and recording studies has been instrumental to our current understanding of neuroplasticity and brain networks in behaving animals. Ever-increasing knowledge about optimizing surgical techniques in rodents1-4, public awareness concerning animal welfare issues and stringent legislation (e.g., the 2010 European Union Directive on the use of laboratory animals5) prompted us to refine these surgical procedures, particularly with respect to implementing new procedures for oxygen supplementation and the continuous monitoring of blood oxygenation and heart rate levels during the surgery as well as introducing a standardized protocol for post-surgical care. Our observations indicate that these modifications resulted in an increased survival rate and an improvement in the general condition of the animals after surgery (e.g. less weight loss and a more active animal). This video presentation will show the general procedures involved in this type of stereotaxic surgery with special attention to our several modifications. We will illustrate these surgical procedures in rats, but it is also possible to perform this type of surgery in mice or other small laboratory animals by using special adaptors for the stereotaxic apparatus6.

Stereotaxic surgery on rodents allows for targeted administration of drugs or electrical stimulation and recordings in awake, behaving animals. In this video presentation we will demonstrate recent procedural refinements to this long-standing procedure that successfully improved survival rate and reduced post-surgical weight loss.

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to ...

Whole Animal Perfusion Fixation for Rodents

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small pieces of tissue, larger specimens like the intact brain pose a problem for immersion fixation because the fixative does not reach all regions of the tissue at the same rate 5,7. Often, changes in response to hypoxia begin before the tissue can be preserved 12. The advantage of directly perfusing fixative through the circulatory system is that the chemical can quickly reach every corner of the organism using the natural vascular network. In order to utilize the circulatory system most effectively, care must be taken to match physiological pressures 3. It is important to note that physiological pressures are dependent on the species used. Techniques for perfusion fixation vary depending on the tissue to be fixed and how the tissue will be processed following fixation. In this video, we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain for immunohistochemistry. The main advantage of this technique (vs. gravity-fed systems) is that the circulatory system is utilized most effectively.

Here we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain.

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small ...

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. Few studies have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem, which contains second order neurons of the auditory pathway, is a potential site for gene transfer. In this protocol, a technique for direct and maximal exposure of the murine DCN via a posterior fossa approach is demonstrated. This approach allows for either acute or survival surgery. Following direct visualization of the DCN, a host of experiments are possible, including injection of opsins into the cochlear nucleus and subsequent stimulation by an optical fiber coupled to a blue light laser.&#160;Other neurophysiology experiments, such as electrical stimulation and neural injector tracings are also feasible.&#160;The level of visualization and the duration of stimulation achievable make this approach applicable to a wide range of experiments.

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

Assessment and Communication for People with Disorders of Consciousness

In this experiment, we demonstrate a suite of hybrid Brain-Computer Interface (BCI)-based paradigms that are designed for two applications: assessing the level of consciousness of people unable to provide motor response and, in a second stage, establishing a communication channel for these people that enables them to answer questions with either &#39;yes&#39; or &#39;no&#39;. The suite of paradigms is designed to test basic responses in the first step and to continue to more comprehensive tasks if the first tests are successful. The latter tasks require more cognitive functions, but they could provide communication, which is not possible with the basic tests. All assessment tests produce accuracy plots that show whether the algorithms were able to detect the patient&#39;s brain&#39;s response to the given tasks. If the accuracy level is beyond the significance level, we assume that the subject understood the task and was able to follow the sequence of commands presented via earphones to the subject. The tasks require users to concentrate on certain stimuli or to imagine moving either the left or right hand. All tasks are designed around the assumption that the user is unable to use the visual modality, and thus, all stimuli presented to the user (including instructions, cues, and feedback) are auditory or tactile.

With this experiment, one might be able to detect consciousness in people with disorders of consciousness. Furthermore, the approach can create a simple communication channel that enables people to give simple YES/NO answers to questions.

In this experiment, we demonstrate a suite of hybrid Brain-Computer Interface (BCI)-based paradigms that are designed for two applications: assessing the ...

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...