Name: mpi cybermotion simulator: implementation of a novel motion simulator to investigate multisensory path integration in three dimensions
Uploaded: 2012-05-10T12:00:00
Duration: 9 min 46 s
Description: Watch this Scientific Journal Video about MPI CyberMotion Simulator: Implementation of a Novel Motion Simulator to Investigate Multisensory Path Integration in Three Dimensions at JoVE.com

MPI Postdoc stipends to MB-C and TM; Korean NRF (R31-2008-000-10008-0) to HHB. Thanks to Karl Beykirch, Michael Kerger &amp; Joachim Tesch for technical assistance and scientific discussion.

1. KUKA Roboter GmbH
<ol>
	<li>The MPI CyberMotion Simulator consists of a six-joint serial robot in a 3-2-1 configuration (Figure 1). It is based on the commercial KUKA Robocoaster (a modified KR-500 industrial robot with a 500 kg payload). The physical modifications and the software control structure needed to have a flexible and safe experimental setup have previously been described, including the motion simulator's velocity and acceleration limitations, and the delays and transfer function of the system 9. Modifications from this previous setup are defined below.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/3436/3436fig1.jpg" /> 
Figure 1. Graphical representation of the current MPI CyberMotion Simulator work space.
<ol start="2">
	<li>Complex motion profiles that combine lateral movements with rotations are possible with the MPI CyberMotion Simulator. Axes 1, 4 and 6 can rotate continuously. 4 pairs of hardware end-stops limit Axis 2, 3 and 5 in both directions. The maximum range of linear movements is strongly dependent on the position from which the movement begins. The current hardware end-stops of the MPI CyberMotion Simulator are shown in Table 1.
	<table border="1">
		<tbody>
			<tr>
				<td>Axis</td>
				<td>Range [deg]</td>
				<td>Max. velocity [deg/s]</td>
			</tr>
			<tr>
				<td>Axis 1</td>
				<td>Continuous</td>
				<td>69</td>
			</tr>
			<tr>
				<td>Axis 2</td>
				<td>-128 to -48</td>
				<td>57</td>
			</tr>
			<tr>
				<td>Axis 3</td>
				<td>-45 to +92</td>
				<td>69</td>
			</tr>
			<tr>
				<td>Axis 4</td>
				<td>Continuous</td>
				<td>76</td>
			</tr>
			<tr>
				<td>Axis 5</td>
				<td>-58 to +58</td>
				<td>76</td>
			</tr>
			<tr>
				<td>Axis 6</td>
				<td>Continuous</td>
				<td>120</td>
			</tr>
		</tbody>
	</table>
	</li>
</ol>
Table 1. Current technical specifications of the MPI CyberMotion Simulator.
<ol start="3">
	<li>Before any experiment is performed on the MPI CyberMotion Simulator, each experimental motion trajectory undergoes a testing phase on a KUKA simulation PC (Office PC). The "Office PC" is a special product sold by KUKA which simulates the real robot arm and includes the identical operating system and control screen layout as the real robot. A schematic overview of the control system of the MPI CyberMotion Simulator for an open-loop configuration is shown in Figure 2.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/3436/3436fig2.jpg" /> 
Figure 2. Schematic overview of the open-loop control system of the MPI CyberMotion Simulator. <a href="https://www.jove.com/files/ftp_upload/3436/3436fig2large.jpg" target="_blank">Click here for larger figure</a>.
<ol start="4">
	<li>The details of the control structure can be found here 9. In brief, for an open-loop configuration such as that used in the current experiment, trajectories are pre-programmed by converting input trajectories in Cartesian coordinates to joint space angles through inverse kinematics (Figure 2).</li>
	<li>The MPI control system reads in these desired joint angle increments and sends these to the KUKA control system to perform axis movements via motor currents. Joint resolver values are sent to the KUKA control system which determines the current joint angle positions at an internal rate of 12ms, which in turn trigger the next joint increment to be read in from file by the MPI control system as well as write the current joint angle positions to disk. Communication between the MPI and KUKA control systems is by an Ethernet connection using the KUKA-RSI protocol.</li>
	<li>A racecar seat (RECARO Pole Position) equipped with a 5-point safety belt system (Schroth) is attached to a chassis which includes a footrest. The chassis is mounted to the flange of the robot arm (Figure 3a). Experiments are also possible by seating participants within an enclosed cabin (Figure 3b).</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/3436/3436fig3.jpg" /> 
Figure 3. MPI CyberMotion Simulator setup. a) Configuration for current experiment with LCD display. b) Configuration for experiments requiring an enclosed cabin with front projection stereo display. c) Front projection mono display. d) Head mounted display.
<ol start="7">
	<li>As the experiment is performed in darkness, infrared cameras allow visual monitoring from the control room.</li>
</ol>
2. Visualization
<ol>
	<li>Multiple visualization configurations are possible with the MPI CyberMotion Simulator including LCD, stereo or mono front projection, and head mounted displays (Figure 3). For the current experiment visual cues to self-motion are provided by an LCD display (Figure 3a) placed 50 cm in front of the observers who were otherwise tested in the dark.</li>
	<li>The visual presentation was generated using Virtools 4.1 software and consisted of a random, limited life time dot-field. A cuboid extending eight virtual units to the front, right, left, upwards and downwards from the point of view of the participant (i.e., 16 x 16 x 8 units in size) was filled with 200,000 equal size particles consisting of white circles 0.02 units in diameter in front of a black background. The dots were randomly distributed across the space (homogeneous probability distribution within the space). Movement in virtual units was scaled to correspond 1 to 1 with physical motion (1 virtual unit = 1 physical meter).</li>
	<li>Each particle was shown for two seconds before vanishing and immediately showing up again at a random location within the space. Thus half of the dots changed their position within one second. Dots between a distance of 0.085 and 4 units were displayed to the participants (corresponding visual angles: 13&deg; and 0.3&deg;).</li>
	<li>Movement within the dot field was synchronized with physical motion by receiving motion trajectories from the MPI control computer transmitted by an Ethernet connection using the UDP protocol. When moving through the dot-field the average number of dots stayed constant for all movements. This display provided no absolute size scale, but optic flow and motion parallax as dots were spheres with a fixed size; looking smaller according to their distance relative to the observer.</li>
</ol>
3. Experimental Design
<ol>
	<li>16 participants, who were na&iuml;ve to the experiment with the exception of one author (MB-C), wore noise-cancelling headphones equipped with a microphone to allow two-way communication with the experimenter. Additional auditory noise was continuously played through the headphones to further mask noise produced by the robot.</li>
	<li>Participants used a custom built joystick equipped with response buttons with data transmitted by an Ethernet connection using the UDP protocol.</li>
	<li>The angle of the two movement segments was either 45&deg; or 90&deg;. Movements in the horizontal, sagittal and frontal planes consisted of: forward-rightward (FR) or rightward-forward (RF), downward-forward (DF) or forward-downward (FD), and downward-rightward (DR) or rightward-downward (RD) movements respectively (Figure 4a).</li>
</ol>
<img alt="Figure 4" src="/files/ftp_upload/3436/3436fig4.jpg" /> 
Figure 4. Procedure. a) Schematic representation of trajectories used in the experiment. b) Sensory information provided for each trajectory type tested. c) Pointing task used to indicate the origin of where participants thought that they had moved from. <a href="https://www.jove.com/files/ftp_upload/3436/3436fig4large.jpg" target="_blank">Click here for larger figure</a>.
<ol start="4">
	<li>Sensory information was manipulated by providing visual (optic flow, limited lifetime star field), vestibular-kinaesthetic (passive self motion with eyes closed), or visual and vestibular-kinaesthetic motion cues (Figure 4b).</li>
	<li>Movement trajectories consisted of two segment lengths (1st: 0.4 m, 2nd: 1 m; &plusmn;0.24 m/s2 peak acceleration; Figure 4b). Trajectories consisted of translation only. No rotations of the participants occurred. To reduce possible interference from motion prior to each trial and ensure that the vestibular system was tested starting from a steady state, a 15 s pause preceded each trajectory.</li>
	<li>Observers pointed back to their origin by moving an arrow that was superimposed on an avatar presented on the screen (Figure 4c). Movement of the arrow was constrained to the trajectory's plane and controlled by the joystick. The avatar was presented from frontal, sagittal and horizontal viewpoints. Observers were allowed to use any or all viewpoints to answer. The starting orientation of the arrow was randomized across trials.</li>
	<li>As the pointing task required participants to mentally transform their pointing perspective from an egocentric to an exocentric representation, participants were given instructions on how to point back to their origin with reference to the avatar prior to practice and experimental trials. Participants were told that pointing should be made as if the avatar were their own body. Participants were then instructed to point to physical targets relative to the self using the exocentric measurement technique. For example, participants were instructed to point to the joystick resting on their lap half-way between themselves and the screen, which required participants to point the arrow forward and down relative to the avatar. All participants were able to perform these tasks without expressing confusion.</li>
	<li>Each experimental condition was repeated 3 times and presented in random order. Signed error and response time were analyzed as dependent variables in two separate 3 (plane) * 2 (angle) * 3 (modality) repeated measures ANOVA. Response times from one extreme outlier participant were removed from analysis.</li>
</ol>
4. Representative Results
Signed error results are collapsed across modalities and angles as no significant main effects were found for these factors. Figure 5a shows the significant main effect of movement plane (F(2,30) = 7.0, p = 0.003) where observers underestimated angle size (average data less than 0&deg;) for movement in the horizontal plane (-8.9&deg;, s.e. 1.8). In the frontal plane observers were more likely on average to overestimate angle size (5.3&deg;, s.e. 2.6), while there was no such bias in the sagittal plane (-0.7&deg;, s.e. 3.7). While main effects of angle and modality were not significant, angle was found to significantly interact with plane (F(2,30) = 11.1, p &lt; 0.001) such that overestimates in the frontal plane were larger for movements through 45&deg; (7.9&deg;, s.e. 2.6) than through 90&deg; (2.8&deg;, s.e. 2.7), while such a discrepancy was absent for the other planes. In addition, modality was found to significantly interact with angle (F(2,30) = 4.7, p = 0.017) such that underestimates from vestibular information alone for movements through 90&deg; were significantly larger (-4.3&deg;, s.e. 2.1) compared to the visual (-2.0&deg;, s.e. 2.4) and vestibular and visual information combined (2.3&deg;, s.e. 2.2) conditions, while such discrepancies were absent for movements through 45&deg;. No significant between subjects effect was for signed error (F(1,15) = 0.7, p = 0.432). Figure 5b shows the response time results. There was a significant main effect of modality (F(2,28) = 22.6, p &lt; 0.001) where observers responded slowest when answering based on vestibular-kinaesthetic information alone (11.0 s, s.e. 1.0) compared to the visual (9.3 s, s.e. 0.8) and combined (9.0 s, s.e. 0.8) conditions. There was also a significant main effect of plane (F(2,28) = 7.5, p = 0.002) where observers responded slowest when moved in the horizontal plane (10.4 s, s.e. 1.0) compared to the sagittal (9.4 s, s.e. 0.8) and the frontal (9.4 s, s.e. 0.9) planes. There was no significant main effect of segment angle or any interactions. A significant between subjects effect was found for response time (F(1,14) = 129.1, p &lt; 0.001).
 
<img alt="Figure 5" src="/files/ftp_upload/3436/3436fig5.jpg" /> 
Figure 5. Results. a) Signed error collapsed across modality for the planes tested. b) Response time collapsed across movement planes for the modalities tested. Error bars are +/- 1 s.e.m.

<ol>
	<li>Loomis, J. M., Klatzky, R. L., Golledge, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Navigating+without+vision:+Basic+and+applied+research.">Navigating without vision: Basic and applied research.</a> Optometry and Vision Science. 78, 282-289 (2001).</li><li>Vidal, M., Amorim, M. A., Berthoz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Navigating+in+a+virtual+three-dimensional+maze:+how+do+egocentric+and+allocentric+reference+frames+interact.">Navigating in a virtual three-dimensional maze: how do egocentric and allocentric reference frames interact.</a> Cognitive Brain Research. 19, 244-258 (2004).</li><li>Vidal, M., Amorim, M. A., McIntyre, J., Berthoz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+perception+of+visually+presented+yaw+and+pitch+turns:+Assessing+the+contribution+of+motion,+static,+and+cognitive+cues.">The perception of visually presented yaw and pitch turns: Assessing the contribution of motion, static, and cognitive cues.</a> Perception &#38; Psychophysics. 68, 1338-1350 (2006).</li><li>Loomis, J. M., Klatzky, R. K., Philbeck, J. W., Golledge, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+auditory+distance+perception+using+perceptually+directed+action.">Assessing auditory distance perception using perceptually directed action.</a> Perception &#38; Psychophysics. 60, 966-980 (1998).</li><li>Loomis, J. M., Klatzky, R. L., Golledge, R. G., Cicinelli, J. G., Pellegrino, J. W., Fry, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonvisual+navigation+by+blind+and+sighted:+Assessment+of+path+integration+ability.">Nonvisual navigation by blind and sighted: Assessment of path integration ability.</a> Journal of Experimental Psychology General. 122, 73-91 (1993).</li><li>Bakker, N. H., Werkhoven, P. J., Passenier, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+proprioceptive+and+visual+feedback+on+geographical+orientation+in+virtual+environments.">The effects of proprioceptive and visual feedback on geographical orientation in virtual environments.</a> Presence. 8, 36-53 (1999).</li><li>Kearns, M. J., Warren, W. H., Duchon, A. P., Tarr, 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=Path+integration+from+optic+flow+and+body+senses+in+a+homing+task.">Path integration from optic flow and body senses in a homing task.</a> Perception. 31, 349-374 (2002).</li><li>Pollini, L., Innocenti, M., Petrone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+a+novel+motion+platform+for+flight+simulators+using+an+anthropomorphic+robot.">Study of a novel motion platform for flight simulators using an anthropomorphic robot.</a> , 2006-6360 (2006).</li><li>Teufel, H. J., Nusseck, H. -. G., Beykirch, K. A., Butler, J. S., Kerger, M., Bulthoff, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MPI+motion+simulator:+development+and+analysis+of+a+novel+motion+simulator.">MPI motion simulator: development and analysis of a novel motion simulator.</a> , 2007-6476 (2007).</li><li>Gärling, T., Böök, A., Lindberg, E., Arce, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+elevation+encoded+in+cognitive+maps.">Is elevation encoded in cognitive maps.</a> Journal of Environmental Psychology. 10, 341-351 (1990).</li><li>Montello, D. R., Pick, H. L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+knowledge+of+vertically+aligned+large-scale+spaces.">Integrating knowledge of vertically aligned large-scale spaces.</a> Environment and Behaviour. 25, 457-483 (1993).</li><li>Correia, M. J., Hixson, W. C., Niven, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+predictive+equations+for+subjective+judgments+of+vertical+and+horizon+in+a+force+field.">On predictive equations for subjective judgments of vertical and horizon in a force field.</a> Acta oto-laryngologica Supplementum. 230, 3 (1968).</li><li>Barnett-Cowan, M., Harris, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perceived+timing+of+vestibular+stimulation+relative+to+touch,+light+and+sound.">Perceived timing of vestibular stimulation relative to touch, light and sound.</a> Experimental Brain Research. 198, 221-231 (2009).</li><li>Barnett-Cowan, M., Harris, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+processing+of+active+and+passive+head+movement.">Temporal processing of active and passive head movement.</a> Experimental Brain Research. 214, 27-35 (2011).</li><li>Sanders, M. C., Chang, N. N., Hiss, M. M., Uchanski, R. M., Hullar, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+binding+of+auditory+and+rotational+stimuli.">Temporal binding of auditory and rotational stimuli.</a> Experimental Brain Research. 210, 539-547 (2011).</li><li>Barnett-Cowan, M., Raeder, S. M., Bulthoff, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+perceptual+delay+for+head+movement+onset+relative+to+auditory+stimuli+of+different+duration+and+rise+times.">Persistent perceptual delay for head movement onset relative to auditory stimuli of different duration and rise times.</a> Experimental Brain Research. , (2012).</li></ol>

No conflicts of interest declared.

Path integration has been well established as a means used to resolve where an observer originated but is prone to underestimates of the angle one has moved through 5. Our results show this for translational movement but only within the horizontal plane. In the vertical planes participants are more likely to overestimate the angle moved through or have no bias at all. These results may explain why estimates of elevation traversed-over terrain tend to be exaggerated 10 and also why spatial navigation between different floors of a building is poor 11. These results may also be related to known asymmetries in the relative proportion of saccule to utricule receptors (~0.58) 12. Slower response time based on vestibular-kinaesthetic information alone compared to when visual information is present suggests that there may be additional delays associated with trying to determine one's origin based on inertial cues alone, which may relate to recent studies showing that vestibular perception is slow compared to the other senses 13-16. Overall our results suggest that alternative strategies for determining one's origin may be used when moving vertically which may relate to the fact that humans experience movement mostly within the horizontal plane. Further, while sequential translations are rarely experienced they do occur most often in the sagittal plane - where errors are minimal - such as when we walk toward and move on an escalator. While post-experiment interviews did not reflect different strategies among the planes, experiments should explore this possibility. Experiments with trajectories using additional degrees of freedom, longer paths, with the body differently orientated relative to gravity, as well as using larger fields of view which are now possible with the MPI CyberMotion Simulator are planned to further investigate path integration performance in three dimensions.

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Dell 24" 1920x1200 lcd display (effective field of view masked to 1200 x 1200)
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Custom built joystick with UDP communication

mpi cybermotion simulator: implementation of a novel motion simulator to investigate multisensory path integration in three dimensions

Path integration is a process in which self-motion is integrated over time to obtain an estimate of one's current position relative to a starting point 1. Humans can do path integration based exclusively on visual 2-3, auditory 4, or inertial cues 5. However, with multiple cues present, inertial cues - particularly kinaesthetic - seem to dominate 6-7. In the absence of vision, humans tend to overestimate short distances (&lt;5 m) and turning angles (&lt;30&deg;), but underestimate longer ones 5. Movement through physical space therefore does not seem to be accurately represented by the brain.
Extensive work has been done on evaluating path integration in the horizontal plane, but little is known about vertical movement (see 3 for virtual movement from vision alone). One reason for this is that traditional motion simulators have a small range of motion restricted mainly to the horizontal plane. Here we take advantage of a motion simulator 8-9 with a large range of motion to assess whether path integration is similar between horizontal and vertical planes. The relative contributions of inertial and visual cues for path navigation were also assessed.
16 observers sat upright in a seat mounted to the flange of a modified KUKA anthropomorphic robot arm. Sensory information was manipulated by providing visual (optic flow, limited lifetime star field), vestibular-kinaesthetic (passive self motion with eyes closed), or visual and vestibular-kinaesthetic motion cues. Movement trajectories in the horizontal, sagittal and frontal planes consisted of two segment lengths (1st: 0.4 m, 2nd: 1 m; &plusmn;0.24 m/s2 peak acceleration). The angle of the two segments was either 45&deg; or 90&deg;. Observers pointed back to their origin by moving an arrow that was superimposed on an avatar presented on the screen.
Observers were more likely to underestimate angle size for movement in the horizontal plane compared to the vertical planes. In the frontal plane observers were more likely to overestimate angle size while there was no such bias in the sagittal plane. Finally, observers responded slower when answering based on vestibular-kinaesthetic information alone. Human path integration based on vestibular-kinaesthetic information alone thus takes longer than when visual information is present. That pointing is consistent with underestimating and overestimating the angle one has moved through in the horizontal and vertical planes respectively, suggests that the neural representation of self-motion through space is non-symmetrical which may relate to the fact that humans experience movement mostly within the horizontal plane.

Watch this Scientific Journal Video about MPI CyberMotion Simulator: Implementation of a Novel Motion Simulator to Investigate Multisensory Path Integration in Three Dimensions at JoVE.com

MPI CyberMotion Simulator: Implementation of a Novel Motion Simulator to Investigate Multisensory Path Integration in Three Dimensions

An efficient way to gain insight into how humans navigate themselves in three dimensions is described. The method takes advantage of a motion simulator capable of moving observers in ways unattainable by traditional simulators. Results confirm that movement in the horizontal plane is underestimated, while vertical movement is overestimated.

Path integration is a process in which self-motion is integrated over time to obtain an estimate of one's current position relative to a starting point 1. ...

mpi-cybermotion-simulator-implementation-novel-motion-simulator-to

Research

JoVE Journal

Neuroscience

Using a Comparative Species Approach to Investigate the Neurobiology of Paternal Responses

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors. Using animal models to accomplish this goal, many methodological strategies require invasive techniques to manipulate the intensity of the behavior of interest (e.g., lesion methods, pharmacological manipulations, microdialysis techniques, genetically-engineered animal models). The utilization of a comparative species approach allows researchers to take advantage of naturally occurring differences in response strategies existing in closely related species. In our lab, we use two species of the Peromyscus genus that differ in paternal responses. The male California deer mouse (Peromyscus californicus) exhibits the same parental responses as the female whereas its cousin, the common deer mouse (Peromyscus maniculatus) exhibits virtually no nurturing/parental responses in the presence of pups. Of specific interest in this article is an exploration of the neurobiological factors associated with the affiliative social responses exhibited by the paternal California deer mouse. Because the behavioral neuroscience approach is multifaceted, the following key components of the study will be briefly addressed: the identification of appropriate species for this type of research; data collection for behavioral analysis; preparation and sectioning of the brains; basic steps involved in immunocytochemistry for the quantification of vasopressin-immunoreactivity; the use of neuroimaging software to quantify the brain tissue; the use of a microsequencing video analysis to score behavior and, finally, the appropriate statistical analyses to provide the most informed interpretations of the research findings.

The comparative species approach allows behavioral neuroscientists to explore various neurobiological factors associated with specific behaviors viewed as characteristic of a specific animal model. Taking advantage of naturally occurring differences in behavior between closely related species, this technique doesn&#x2019;t require invasive techniques to manipulate the expression of the behavior.

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors.  Using animal models to accomplish ...

Three Dimensional Vestibular Ocular Reflex Testing Using a Six Degrees of Freedom Motion Platform

The vestibular organ is a sensor that measures angular and linear accelerations with six degrees of freedom (6DF). Complete or partial defects in the vestibular organ results in mild to severe equilibrium problems, such as vertigo, dizziness, oscillopsia, gait unsteadiness nausea and/or vomiting. A good and frequently used measure to quantify gaze stabilization is the gain, which is defined as the magnitude of compensatory eye movements with respect to imposed head movements. To test vestibular function more fully one has to realize that 3D VOR ideally generates compensatory ocular rotations not only with a magnitude (gain) equal and opposite to the head rotation but also about an axis that is co-linear with the head rotation axis (alignment). Abnormal vestibular function thus results in changes in gain and changes in alignment of the 3D VOR response.
Here we describe a method to measure 3D VOR using whole body rotation on a 6DF motion platform. Although the method also allows testing translation VOR responses 1, we limit ourselves to a discussion of the method to measure 3D angular VOR. In addition, we restrict ourselves here to description of data collected in healthy subjects in response to angular sinusoidal and impulse stimulation.
Subjects are sitting upright and receive whole-body small amplitude sinusoidal and constant acceleration impulses. Sinusoidal stimuli (f = 1 Hz, A = 4&deg;) were delivered about the vertical axis and about axes in the horizontal plane varying between roll and pitch at increments of 22.5&deg; in azimuth. Impulses were delivered in yaw, roll and pitch and in the vertical canal planes. Eye movements were measured using the scleral search coil technique 2. Search coil signals were sampled at a frequency of 1 kHz.
The input-output ratio (gain) and misalignment (co-linearity) of the 3D VOR were calculated from the eye coil signals 3.
Gain and co-linearity of 3D VOR depended on the orientation of the stimulus axis. Systematic deviations were found in particular during horizontal axis stimulation. In the light the eye rotation axis was properly aligned with the stimulus axis at orientations 0&deg; and 90&deg; azimuth, but gradually deviated more and more towards 45&deg; azimuth.
The systematic deviations in misalignment for intermediate axes can be explained by a low gain for torsion (X-axis or roll-axis rotation) and a high gain for vertical eye movements (Y-axis or pitch-axis rotation (see Figure 2). Because intermediate axis stimulation leads a compensatory response based on vector summation of the individual eye rotation components, the net response axis will deviate because the gain for X- and Y-axis are different.
In darkness the gain of all eye rotation components had lower values. The result was that the misalignment in darkness and for impulses had different peaks and troughs than in the light: its minimum value was reached for pitch axis stimulation and its maximum for roll axis stimulation.
Case Presentation
Nine subjects participated in the experiment. All subjects gave their informed consent. The experimental procedure was approved by the Medical Ethics Committee of Erasmus University Medical Center and adhered to the Declaration of Helsinki for research involving human subjects.
Six subjects served as controls. Three subjects had a unilateral vestibular impairment due to a vestibular schwannoma. The age of control subjects (six males and three females) ranged from 22 to 55 years. None of the controls had visual or vestibular complaints due to neurological, cardio vascular and ophthalmic disorders.
The age of the patients with schwannoma varied between 44 and 64 years (two males and one female). All schwannoma subjects were under medical surveillance and/or had received treatment by a multidisciplinary team consisting of an othorhinolaryngologist and a neurosurgeon of the Erasmus University Medical Center. Tested patients all had a right side vestibular schwannoma and underwent a wait and watch policy (Table 1; subjects N1-N3) after being diagnosed with vestibular schwannoma. Their tumors had been stabile for over 8-10 years on magnetic resonance imaging.

A method is described to measure three-dimensional vestibulo ocular reflexes (3D VOR) in humans using a six degrees of freedom (6DF) motion simulator. The gain and misalignment of the 3D angular VOR provide a direct measure of the quality of vestibular function. Representative data on healthy subjects are provided

The vestibular organ is a sensor that measures angular and linear accelerations with six degrees of freedom (6DF). Complete or partial defects in the ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris)

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from flowering plants. Only a few studies have described the gustatory responses of bees toward toxins in food, and these experiments have mainly used the proboscis extension response on restrained honey bees. Here, a new behavioral assay is presented for measuring the feeding responses of freely-moving, individual worker bumble bees to nutrients and toxins. This assay measures the amount of solution ingested by each bumble bee and identifies how tastants in food influence the microstructure of the feeding behavior.
The solutions are presented in a microcapillary tube to individual bumble bees that have been previously starved for 2-4 hr. The behavior is captured on digital video. The fine structure of the feeding behavior is analyzed by continuously scoring the position of the proboscis (mouthparts) from video recordings using event logging software. The position of the proboscis is defined by three different behavioral categories: (1) proboscis is extended and in contact with the solution, (2) proboscis is extended but not in contact with the solution and (3) proboscis is stowed under the head. Furthermore the speed of the proboscis retracting away from the solution is also estimated.
In the present assay the volume of solution consumed, the number of feeding bouts, the duration of the feeding bouts and the speed of the proboscis retraction after the first contact is used to evaluate the phagostimulatory or the deterrent activity of the compounds tested.
This new taste assay will allow researchers to measure how compounds found in nectar influence the feeding behavior of bees and will also be useful to pollination biologists, toxicologists and neuroethologists studying the bumble bee&#39;s taste system.

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees Bombus terrestris

A novel behavioral assay is described for investigating the short term gustatory responses of the mouthparts of freely-moving bumble bees (Bombus terrestris) toward nutrients and toxins in solution.

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from ...

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic Brain Injury (mTBI). The mechanisms responsible for its complex pathological changes remain largely elusive, despite a recent consensus to define the neuropathological criteria. Here, we describe a novel method to develop a model of CTE in Drosophila melanogaster&#160;(Drosophila&#160;)&#160;in an attempt to identify the key genes and pathways that lead to the characteristic hyperphosphorylated tau accumulation and neuronal death in the brain. Adjustable-strength impacts to inflict mild closed injury are delivered directly to the fly head, subjecting the head to rapid acceleration and deceleration. Our method eliminates the potential problems inherent with other Drosophila mTBI models (e.g.,animal death might be induced by damage to other parts of the body or to internal organs). The less labor- and cost-intensive animal care, short life span, and extensive genetic tools make the fruit fly ideal to study CTE pathogenesis and make it possible to perform large-scale, genome-wide forward genetic and pharmacological screens. We anticipate that the ongoing characterization of the model will generate important mechanistic insights on disease prevention and therapeutic approaches.

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Here, we describe a new approach to inflict closed-head traumatic brain injury in Drosophila melanogaster. Our method has the advantage of directly delivering repetitive impacts with adjustable strength to the head alone. Further exploration of the invertebrate system will help to illuminate the pathogenesis of chronic traumatic encephalopathy.

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic ...

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 ...