Funded by Dutch NWO/ZonMW grants 912-03-037 and 911-02-004.

1. 6DF Motion Platform
Vestibular stimuli were delivered with a motion platform (see Figure 1) capable of generating angular and translational stimuli at a total of six degrees of freedom (FCS-MOOG, Nieuw-Vennep, The Netherlands). The platform is moved by six electro-mechanical actuators connected to a personal computer with dedicated control software. It generates accurate movements with six degrees of freedom. Sensors placed in the actuators continuously monitored the platform motion profile. The device has &lt;0.5 mm precision for linear and &lt;0.05&deg; for angular movements. Vibrations during stimulation were 0.02&deg;. Resonance frequency of the device was &gt;75 Hz. Platform motion profile was reconstructed from the sensor information in the actuators using inverse dynamics and sent to the data collection computer. To synchronize platform and eye movement data, a laser beam was mounted at the backside of the platform and projected onto a small photocell (1 mm, reaction time 10 &mu;sec). The output voltage of the photocell was sampled at a rate of 1 KHz together with the eye movement data and provided a real time indicator of motion onset with 1 msec accuracy. During the offline analysis using Matlab (Mathworks, Natick, MA), the reconstructed motion profile of the platform based on the sensor information of the actuators in the platform was precisely aligned with the onset of platform motion.
2. Subjects
A. Seating
The subjects are seated on a chair mounted at the center of the platform (Figure 2). The subject's body was restrained with a four-point seatbelt as used in racing cars. The seatbelts were anchored to the base of the motion platform. The chair was surrounded by a PVC cubic frame and served as a support for the field coils. The field coil system was adjustable in height, such that the subject's eyes were in the center of the magnetic field.
B. Head fixation
The head is immobilized using an individually moulded dental-impression bite board, which was attached to the cubic frame via a rigid bar. A vacuum pillow folded around the neck and an annulus attached to the chair further ensured fixation of the subject (Figure 1). In addition, to monitor spurious head movements during the stimulation, we attached two 3D sensors (Analog Devices Inc, Norwood, MA) directly to the bite board, one for angular and one for linear accelerations.
3. Coordinate System
Eye rotations are defined in a head-fixed right-handed coordinate system (Figure 3). In this system from the subject's point of view a leftward rotation about the Z-axis (yaw), a downward rotation about the Y-axis (pitch) and rightward rotation about the X-axis (roll) are defined as positive. The planes orthogonal to the X, Y and Z rotation axes are respectively the roll, pitch and yaw planes (Figure 3).
4. Eye Movement Recordings
Eye movements of both eyes were recorded with 3D scleral search coils (Skalar, Delft, The Netherlands) 4 using a standard 25 kHz two field coil system based on the amplitude detection method of Robinson (Model EMP3020, Skalar Medical, Delft, The Netherlands) 5. The coil signals were passed through an analogue low-pass filter with cut-off frequency of 500 Hz and sampled on-line and stored to hard disk at a frequency of 1 kHz with 16 bit precision (CED system running Spike2 v6, Cambridge Electronic Design, Cambridge).
5. Search Coil Calibration
Prior to the experiments, the sensitivity and non-orthogonality of direction and torsion coils was verified in-vitro by mounting the coil on a Fick gimbal system placed in the center of the magnetic field. By rotating the gimbal system about all cardinal axes we verified that all coils used in the experiments were symmetrical for all directions within 2%.
In vivo, the horizontal and vertical signals of both coils were individually calibrated by instructing the subject to successively fixate a series of five targets (central target and a target at 10 degrees left, right, up and down) for five seconds each. Calibration targets were projected onto a translucent screen at 186 cm distance. Post experiment analysis of the calibration data resulted in sensitivity and offset values for the each search coils. These values were then used in the analysis procedures written in Matlab 3.
6 Stimulation
A. Sinusoidal stimulation
The platform delivered whole-body sinusoidal rotations (1 Hz, A = 4&deg;) about the three cardinal axes: The rostral-caudal or vertical axis (yaw), the interaural axis (pitch) and the nasal-occipital axis (roll), and about intermediate horizontal axes incremented in steps of 22.5&deg; between roll and pitch.
Sinusoidal stimuli were delivered in light and darkness. In the light, subjects fixated on a continuously lit visual target (a red LED, 2 mm diameter) located 177 cm in front of the subject at eye level (Figure 1C left panel). Head was positioned such that Reid's line was base (the imaginary line connecting the meatus externa with the lower orbital cantus) was within 6 degrees from earth-horizontal). During sinusoidal stimulation in the dark, the visual target was briefly presented (2 sec) when the platform was stationary during each interval between two consecutive stimuli. To avoid spontaneous eye movements during the stimulation, subjects were instructed to fixate the imaginary location of the space fixed target during sinusoidal stimulation after the target had been switched off just prior to motion onset. We verified that the type of instruction mainly reduced the eye movements made in darkness, and had only a small effect on gain (&lt;10%). This variability occurred in all components (horizontal, vertical and torsion) simultaneously.
B. Impulse stimulation
Short duration whole body impulses were delivered in a dimly lit environment. The only visible stimulus available to the subject was a visual target located at 177 cm in front of the subject at eye level. Each impulse was repeated six times and delivered in random order and with random timing of motion onset (intervals varied between 2.5 and 3.5 sec). The profile of the impulses was a constant acceleration of 100&deg; sec-2 during the first 100 msec of the impulse, followed by a gradual linear decrease in acceleration. This stimulus resulted in a linear increase in velocity reaching a velocity of 10&deg; sec-1 after 100 msec. Aberrant head movements during vestibular stimulation measured by the angular rate and linear acceleration devices were less than 4% of stimulus amplitude. Peak velocity of the eye movements in response to these impulses was 100 times above the noise level of the coil signals.
7. Data Analysis
Coil signals were converted into Fick angles and then expressed as rotation vectors 6,7. From the fixation data of the target straight ahead we determined the misalignment of the coil in the eye relative to the orthogonal primary magnetic field coils. Signals were corrected for this offset misalignment by three-dimensional counter rotation. It was also verified that no coil slippage had occurred during the experiment by verifying the position output during fixation of the target prior to each motion onset.
To express 3D eye movements in the velocity domain, we converted rotation vector data back into angular velocity. Before conversion of rotation vector to angular velocity, we smoothed the data by zero-phase with a forward and reverse digital filter with a 20-point Gaussian window (length 20 msec).
8. Sinusoidal Responses
A Gain. The gain of each component and 3D eye velocity gain was calculated by fitting a sinusoid with a frequency equal to the platform frequency through the horizontal, vertical and torsion angular velocity components. The gain for each component defined as the ratio between eye component peak velocity and platform peak velocity was calculated separately for each eye.
B Misalignment. The misalignment between the 3D eye velocity axis and head velocity axis was calculated using the approach of Aw and colleagues 8,9. From the scalar product of two vectors the misalignment was calculated as the instantaneous angle in three dimensions between the inverse of the eye velocity axis and the head velocity axis. The 3D angular velocity gain and misalignment for each azimuthal orientation were compared to the gain and misalignment predicted from vector summation of the 0&deg; (roll) and 90&deg;(pitch) azimuth components 10. From this vector summation it follows that when velocity gains for roll and pitch are equal, the orientation of the eye rotation axis aligns with the head rotation axis, when the two are different, the maximum deviation between stimulus and eye rotation axis is expected at 45&deg; azimuth.
9. Impulse Responses
Left and right eye data traces of six presentations for each motion direction were separately analysed. Because left and right eye values were almost identical, the data from left and right eye were averaged to determine the gain of eye velocity in response to impulse stimulation. All traces were individually inspected on the computer screen. When the subject made a blink or saccade during the impulse that trace was manually discarded. Angular velocity components (N = 5 to 6) during the first 100 msec after onset of the movement were averaged in time bins of 20 msec (providing an effective low pass filtering) and plotted as function of platform velocity 11,12.

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We have nothing to disclose.

This paper describes a method to accurately measure 3D angular VOR in response to whole body rotations in humans. The advantage of the method is that it gives quantitative information about gain and misalignment of 3D angular VOR in all three dimensions. The method is useful for fundamental research and has also potential clinical value e.g. for testing patients with vertical canal problems or patients with ill-understood central vestibular problems. Another advantage of the device is the ability to test transla...

<table border="1">
 <tbody>
		<tr>
			<td>Electric Motion Base MB-E-6DOF/24/1800KG * (Formerly E-CUE 624-1800)</td>
			<td>FCS-MOOG, Nieuw-Vennep, The Netherlands</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic field with detector, Model EMP3020</td>
			<td>Skalar Medical, Delft, The Netherlands</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CED power 1401, running Spike2 v6</td>
			<td>Cambridge Electronic Design, Cambridge</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electromagnetic search coils</td>
			<td>Chronos Vision, Berlin, Germany</td>
			<td></td>
			<td></td>
		</tr>
 </tbody>
	</table>

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.

Sinusoidal stimulation light
Figure 4 (top panel) shows for the control group the mean gain of the horizontal, vertical and torsion angular velocity components for all tested sinusoidal stimulations in the horizontal plane in the light. Torsion was maximal at 0&deg; azimuth, whereas vertical had its maximum at 90&deg;. Figure 5 shows the 3D eye velocity gain in the light. Gain varied between 0.99 &plusmn; 0.12 (pitch) and 0.54 &plusmn; 0.16 (roll). The measured d...

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

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 ...

three-dimensional-vestibular-ocular-reflex-testing-using-six-degrees

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Neuroscience

15.8K Aufrufe.  Erasmus MC. Es wird ein Verfahren beschrieben, um dreidimensionale vestibulo okulären Reflexe (3D VOR) beim Menschen mit Hilfe eines sechs Freiheitsgraden (6DF) Bewegungssimulator messen. Die Verstärkung und Versatz des 3D-Winkelstellung VOR stellen ein direktes Maß für die Qualität der Vestibularfunktion. Repräsentative Daten auf gesunden Probanden vorgesehen

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

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.

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. ...

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 ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed as a water tank with narrow platforms on the bottom. The model rats were put into the tank and stood on the platforms for 14 h (18:00 - 8:00) per day for a consecutive 21 days, with a blank control group set for contrast. At the end of modeling, rats in the model group showed an obvious fatigued appearance. To assess the model, we performed several behavioral tests, including the open field test (OFT), the elevated plus maze (EPM) test, and the exhaustive swimming (ES) test. The results showed that anxiety, spatial cognition impairment, poor muscle performance, and declined voluntary activity presented in model rats confirm the diagnosis of&#160;central fatigue. Changes of the central neurotransmitters also verified the result. In conclusion, the model successfully simulated central fatigue, and future study with the model may help reveal the pathological mechanism of the disease.

Here, we present a protocol to introduce a rat model of central fatigue using the modified multiple platform method (MMPM).

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Three-Dimensional Shape Modeling and Analysis of Brain Structures

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. We have developed a software package for accurate and robust shape modeling and group-wise analysis. Here, we introduce an pipeline for the shape analysis, from individual 3D shape modeling to quantitative group shape analysis. We also describe the pre-processing and segmentation steps using open software packages. This practical guide would help researchers save time and effort in 3D shape analysis on brain structures.

We introduce a semi-automatic protocol for shape analysis on brain structures, including image segmentation using open software, and further group-wise shape analysis using an automated modeling package. Here, we demonstrate each step of the 3D shape analysis protocol with hippocampal segmentation from brain MR images.

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. ...

In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the regulation of cerebral blood flow (CBF) is incompletely understood, especially at the capillary level. Two-photon microscopy is a powerful tool widely used to study CBF and its regulation. Currently, this field is limited by the lack of in vivo two-photon microscopy studies examining (1) CBF responses in three-dimensions, (2) conducted vascular responses, and (3) localized interventions within the vascular network. Here, we describe a 3D in vivo method using two-photon microscopy to study conducted vascular responses elicited by&#160;local ejection of ATP with a glass micro-pipette. Our method uses fast and repetitive hyperstack two-photon imaging providing precise diameter measurements by maximal intensity projection of the obtained images. Furthermore, we show that this method can also be used to study 3D astrocytic calcium responses. We also discuss the advantages and limitations of glass micro-pipette insertion and two-photon hyperstack imaging.

We present an optimized local ejection procedure using a glass micro-pipette and a fast two-photon hyperstack imaging method, which allows precise measurement of capillary diameter changes and investigation of its regulation in three dimensions.

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the ...

Three-Dimensional Motor Nerve Organoid Generation

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and neurodegenerative diseases. Although numerous studies of axons have been conducted, our understanding of formation and dysfunction of axon fascicles is still limited due to the lack of robust three-dimensional in vitro models. Here, we describe a step-by-step protocol for the rapid generation of a motor nerve organoid (MNO) from human induced pluripotent stem (iPS) cells in a microfluidic-based tissue culture chip. First, fabrication of chips used for the method is described. From human iPS cells, a motor neuron spheroid (MNS) is formed. Next, the differentiated MNS is transferred into the chip. Thereafter, axons spontaneously grow out of the spheroid and assemble into a fascicle within a microchannel equipped in the chip, which generates an MNO tissue carrying a bundle of axons extended from the spheroid. For the downstream analysis, MNOs can be taken out of the chip to be fixed for morphological analyses or dissected for biochemical analyses, as well as calcium imaging and multi-electrode array recordings. MNOs generated with this protocol can facilitate drug testing and screening and can contribute to understanding of mechanisms underlying development and diseases of axon fascicles.

This protocol provides a comprehensive procedure to fabricate human iPS cell-derived motor nerve organoid through spontaneous assembly of a robust bundle of axons extended from a spheroid in a tissue culture chip.

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and ...

Measuring the Influence of Magnetic Vestibular Stimulation on Nystagmus, Self-Motion Perception, and Cognitive Performance in a 7T MRT

Strong magnetic fields induce dizziness, vertigo, and nystagmus due to Lorentz forces acting on the cupula in the semi-circular canals, an effect called magnetic vestibular stimulation (MVS). In this article, we present an experimental setup in a 7T MRT scanner (MRI scanner) that allows the investigation of the influence of strong magnetic fields on nystagmus as well as perceptual and cognitive responses. The strength of MVS is manipulated by altering the head positions of the participants. The orientation of the participants&#39; semicircular canals with respect to the static magnetic field is assessed by combining a 3D magnetometer and 3D constructive interference in steady-state (3D-CISS) images. This approach allows to account for intra- and inter-individual differences in participants&#8217; responses to MVS. In the future, MVS can be useful for clinical research, for example, in the investigation of compensatory processes in vestibular disorders. Furthermore, it could foster insights into the interplay between vestibular information and cognitive processes in terms of spatial cognition and the emergence of self-motion percepts under conflicting sensory information. In fMRI studies, MVS can elicit a possible confounding effect, especially in tasks influenced by vestibular information or in studies comparing vestibular patients with healthy controls.

In this article, we describe the experimental setup, material, and procedures to assess reflexive eye movements, self-motion perception, and cognitive tasks under magnetic vestibular stimulation, as well as the anatomical orientation of the vestibular organs, in a 7 Tesla Magnetic resonance tomography (7T-MRT) scanner.

Strong magnetic fields induce dizziness, vertigo, and nystagmus due to Lorentz forces acting on the cupula in the semi-circular canals, an effect called ...

Production of Human Neurogenin 2-Inducible Neurons in a Three-Dimensional Suspension Bioreactor

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first advent, protocols have been continuously optimized and are now widely used in research and drug development. However, the very long duration of these conventional differentiation and maturation protocols and the increasing demand for high-quality hiPSCs and their neural derivatives raise the need for the adoption, optimization, and standardization of these protocols to large-scale production. This work presents a fast and efficient protocol for the differentiation of genetically modified, doxycycline-inducible neurogenin 2 (iNGN2)-expressing hiPSCs into neurons using a benchtop three-dimensional (3D) suspension bioreactor.
In brief, single-cell suspensions of iNGN2-hiPSCs were allowed to form aggregates within 24 h, and neuronal lineage commitment was induced by the addition of doxycycline. Aggregates were dissociated after 2 days of induction and cells were either cryopreserved or replated for terminal maturation. The generated iNGN2 neurons expressed classical neuronal markers early on and formed complex neuritic networks within 1 week after replating, indicating an increasing maturity of neuronal cultures. In summary, a detailed step-by-step protocol for the fast generation of hiPSC-derived neurons in a 3D environment is provided that holds great potential as a starting point for disease modeling, phenotypic high-throughput drug screenings, and large-scale toxicity testing.

This article describes a protocol for the generation of human induced pluripotent stem cell-derived neurons in a benchtop 3D suspension bioreactor.

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first ...