The authors wish to acknowledge their collaborators on studies that employ eye movement monitoring of memory, with particular thanks to Eyal Reingold, Jiye Shen, Neal J. Cohen, Robert R. Althoff, Deborah Hannula, and Dave Warren.  This work has been supported by Natural Sciences and Engineering Research Council of Canada (NSERC), Canadian Institutes of Health Research (CIHR), the Canada Research Chairs Program and the Canadian Foundation for Innovation (CFI).

Equipment used during data acquisition
Eye tracker
The eye tracker used in the current protocol is an EyeLink II system (SR Research Ltd; Mississauga, Ontario, Canada). This head-mounted, video-based eye tracker records eye position in X, Y-coordinate frame at a sampling rate of either 500 or 250 Hz, with a spatial resolution of &lt; 0.1&deg;. One camera is used to monitor head position by sending infrared markers to sensors placed on the four corners of the display monitor that is viewed by the participants. Two additional cameras are mounted on the headband situated below each of the eyes, and infrared illuminators are used to note the pupil and corneal reflections. Eye position may be based on pupil and corneal reflections, or based on the pupil only. The padded headband of the eye tracker can be adjusted in two planes to comfortably fit the head size of an adult participant. Most eyeglasses and contact lenses can be accommodated by the eye tracker.
Computers
Two PCs are used to support eye movement recording. One computer serves as the display computer, which presents the calibration screens, necessary task instructions and the images used in the experimental paradigm to the participants. The display computer details the data collection parameters that are then governed by the second, host computer. The host computer calculates real-time gaze position and records the eye movement data for later analysis, as well as any button press or keyboard responses made the participant. Participant setup and operations of the eye tracker are performed via the host PC.
Software
In this protocol, the timing and order by which experimental stimuli are to be presented to the participants, and the manner in which eye position is to be collected by the host PC are programmed through Experiment Builder, a software program specifically developed by SR Research Ltd to interface with the eye tracker host computer. However, stimulus presentation can also be conducted through other software programs (e.g., Presentation, Neurobehavioral Systems; Albany, CA). Conversion of the eye movement data to a series of fixation and saccade events that are time-locked to stimulus presentation (or another external event) is achieved through the host computer and can be interrogated with Data Viewer, a software program developed by SR Research Ltd; however, again, other programs can be used to derive the required eye movement measures. Here, the detection of fixations and saccades are dependent on an online parser, which separates raw eye movement samples into meaningful states (saccades, blinks and fixations). If the velocity of two successive eye movement samples exceeds 22 degrees per second over a distance of 0.1&deg;, the samples are labeled as a saccade. If the pupil is missing for 3 or more samples, the eye activity is marked as a blink within the data stream. Non-saccade and non-blink activity are considered fixations.

Eye tracking procedures.
Below, we detail the procedures for obtaining eye movement recordings for each participant.
<ol>
<li>	Consent. Prior to participant setup, participants are shown the eye tracker headband. The experimenter explains to the participants that head and eye positions are monitored through the cameras that are contained on the headband.</li>

<li>	For a given experimental paradigm, participants are seated a fixed distance from the monitor to maintain the same visual angle across participants.</li>

<li>	Eye tracker camera setup. The eye tracker helmet is adjusted so that the helmet is snug and unlikely to move but not uncomfortable around the head. The helmet is further adjusted so that the head camera can send infrared illumination to the external markers on the display monitor. Each of the eye cameras are situated on individual rods that extend from the helmet which allow for adjustment in all axes. The eye cameras are positioned just under and slightly away from each eye without obstructing the participant s view of the display monitor (see Figure 1). The cameras are focused to get a clear and stable image of the pupil and corneal reflections. Status panels on the host computer indicate whether pupil and corneal reflections are being acquired. The illumination threshold can be adjusted to obtain the most stable recording of the pupil and corneal reflections. The experimenter then selects the appropriate experimental paradigm, and designates a filename for the ensuing eye movement recordings. 
<img src="/files/ftp_upload/2108/2108fig1.jpg" alt="Figure 1" /> Figure 1. Example of a head-mounted video-based eye tracker (left) and the display and host PCs (right). Note that the display monitor in front of the participant shows the location of the pupil (blue dot with crosshair) and corneal reflection (yellow dot with crosshair) to aid in camera setup.</li>
<li>	Calibration. To obtain accurate recordings of the eye position on the display monitor, a calibration procedure is initiated following camera setup. During the calibration procedure, the participants are instructed to look at a series of targets that appear at various locations on the display. Typically, nine target locations are used, but calibration can be performed with as few as three target locations. From these nine recorded locations, eye position at any point on the screen can be interpolated.</li>

<li>	Validation. Following calibration, a validation procedure is used to check the accuracy of the eye movement recording. The same nine target locations are provided to the participant to fixate, and the difference is computed between the current fixation position and the previously recorded fixation position. If the average accuracy for recording of position for one (or both) eyes exceeds acceptable levels (average error &lt; 0.5&deg; and maximum error of any point &lt; 1.0&deg;), calibration and validation procedures are repeated. Following acceptance of the accuracy levels, the experimenter can determine if eye movement recording should be monocular or binocular. In the case of monocular recording, the experimenter can determine from which eye data is to be recorded, or the host PC can automatically select the eye with the lower average error and the lower maximum error. While calibration and validation can occur with slight head movement, it is advantageous to have participants sit as still as possible. Participants are also instructed not to anticipate the upcoming locations of targets, but rather to move to, and fixate deliberately on, the targets only when they appear and to stay fixated on the target until it disappears.</li>

<li>	Experimental Paradigm. Instructions particular to a given paradigm are presented to the participant (e.g., "Please freely view each of the following images."), and data recording is initiated for the session.</li>

<li>	Drift correction. Prior to the onset of each experimental stimulus, a drift correction can be performed by having the participants look at a central fixation target while pressing a button on a joypad or keyboard to accept the fixation position. When drift correction is employed, a trial will not begin until the participant successfully fixates on the central fixation region within 2.0&deg; of the center target. If the participant consistently fixates greater than 2.0&deg;, the participant must again undergo calibration and validation procedures. Any difference less than 2.0&deg; between the recorded eye position and the initial fixation position obtained during the calibration/validation recordings is then noted as error and accounted for in the data.</li>

<li>	Stimulus presentation. Eye position, including fixation and saccade events, is recorded following drift correction procedures for each stimulus presentation. The experimenter can monitor the accuracy of the recordings online via inspection of the host computer.</li>

<li>	Following the eye movement recording session, measures that characterize the eye movement scanning patterns for different types of images (e.g. novel, repeated) can be derived with a variety of software packages, such as SR Research Ltd s Data Viewer. In addition, viewing to specific regions of a stimulus (e.g. unaltered, manipulated) can be characterized by analyzing eye movements with respect to experimenter-drawn regions of interest that are created for each image either at the paradigm programming phase or after data collection.</li>

<li>	Participants are debriefed regarding the purpose of the experiment at the conclusion of the session.</li>
</ol>

Representative Results
Multiple measures can be derived from the eye movement recordings, including measures that describe overall viewing to the image (including the characteristics of each fixation/saccade), and measures that describe the pattern of viewing that has been directed to a particular region of interest within an image 3. Measures of overall viewing to an image may include (but are not limited to): the number of fixations and the number of saccades made to the image, the average duration of each fixation, and the total amount of viewing time that was spent fixating on the image. Measures that describe the pattern of viewing to a particular region of interest may include (but are not limited to): the number of fixations made to the region of interest, the amount of time spent within a region of interest, and the number of transitions made into/out of a region of interest. Further, measures may be derived from the eye movement recordings that outline the timing by which (i.e., how early) a particular eye movement event has occurred, such as when, following stimulus onset, the eyes fixate on a specific region of interest, when the first saccade is made on an image, and the entropy (constraint/randomness) inherent in the sequence of eye movement patterns.

For any given image, eye movement measures can detail where the eyes were fixated, when and for how long. To obtain an index of memory, we can give viewers different types or amounts of exposure to distinct sets of images, and then compare viewing patterns across those sets and across participants. For instance, to probe memory for repetition of an image, scanning patterns can be contrasted between novel images and images that have been viewed multiple times throughout a testing session. Viewing images repeatedly throughout a testing session results in a decrease in overall viewing of the image 2, 4-5, 8. This can be seen in Figure 2. In this representative result from Riggs et al. 11, participants viewed novel pictures of faces once in each of five testing blocks; with increasing exposure, there was a decrease in the amount of fixations that viewers make to the faces. To probe memory for particular details of an image, scanning patterns can be contrasted between images that have been repeatedly viewed in their original, unaltered form (repeated images) and images that have similarly been viewed repeatedly throughout a testing session, but a change has been introduced to some element within a scene during the final exposure (manipulated images). In such cases, scanning patterns are attracted differentially to the region that has been altered within a manipulated image compared to the same, unaltered region of repeated images 2-3, 7-8. However, such eye movement indices of memory are not present in certain populations, such as when healthy older adults and patients with amnesia due to medial temporal lobe damage are assessed for their memory of the spatial relations among objects in scenes, as outlined in Figure 3 2, 8. Therefore, findings from eye movement monitoring can be used to contrast memory among groups of participants with differing neuropsychological status 2-3, 8-10.

 <img src="/files/ftp_upload/2108/2108fig2.jpg" alt="Figure 2" /> 
Figure 2. Eye movements reveal memory for repetition. In this representative result from 	our laboratory [11], participants viewed faces across 5 study blocks; as the number of viewings increased (from 1-5), the number of fixations to the faces decreased.
 <img src="/files/ftp_upload/2108/2108fig3.jpg" alt="Figure 3" /> 
Figure 3. Eye movements reveal memory for changed details. Younger adults [2 (Experiments 1, 2), 8 (Free Viewing Condition)] directed a greater proportion of their total eye fixations to a critical region in a manipulated image that has undergone a change from a prior viewing compared to when the region has not undergone a change, as in novel and repeated images. Such effects of memory were absent in healthy older adults [8 (Free Viewing Condition], and in amnesic patients [2].

<ol>
	<li>Bradfield, A. L., Wells, G. L., Olsen, E. 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+damaging+effect+of+confirming+feedback+on+the+relation+between+eyewitness+certainty+and+identification+accuracy.">The damaging effect of confirming feedback on the relation between eyewitness certainty and identification accuracy.</a> J. Appl. Psychol. 87 (1), 112-120 (2002).</li><li>Ryan, J. D., Althoff, R. R., Whitlow, S., Cohen, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amnesia+is+a+deficit+in+relational+memory.">Amnesia is a deficit in relational memory.</a> Psych. Sci. 11 (6), 454-461 (2000).</li><li>Ryan, J. D., Cohen, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nature+of+change+detection+and+online+representations+of+scenes.">The nature of change detection and online representations of scenes.</a> J. Exp. Psych. Hum. Percept. Perfor. 30, 98-1015 (2004).</li><li>Althoff, R. R., Cohen, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eye-movement-based+memory+effect:+a+re-processing+effect+in+face+perception.">Eye-movement-based memory effect: a re-processing effect in face perception.</a> J. Exp. Psychol. Learn Mem. Cog. 25 (4), 997-1010 (1999).</li><li>Heisz, J. J., Shore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+efficient+scanning+for+familiar+faces.">More efficient scanning for familiar faces.</a> J. Vis. 8 (1), 1-10 (2008).</li><li>Parker, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picture+processing+during+recognition.">Picture processing during recognition.</a> J. Exp. Psych. Hum. Percept. Perfor. 4, 284-293 (1978).</li><li>Hollingworth, A., Williams, C. C., Henderson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+see+and+remember:+Visually+specific+information+is+retained+in+memory+from+previously+attended+objects+in+natural+scenes.">To see and remember: Visually specific information is retained in memory from previously attended objects in natural scenes.</a> Psychon Bull. Rev. 8, 761-768 (2001).</li><li>Ryan, J. D., Leung, G., Turk-Browne, N. B., Hasher, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+age-related+changes+in+inhibition+and+binding+using+eye+movement+monitoring.">Assessment of age-related changes in inhibition and binding using eye movement monitoring.</a> Psychol. Aging. 22 (2), 239-250 (2007).</li><li>Hannula, D. E., Ryan, J. D., Tranel, D., Cohen, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+onset+relational+memory+effects+are+evident+in+eye+movement+behavior,+but+not+in+hippocampal+amnesia.">Rapid onset relational memory effects are evident in eye movement behavior, but not in hippocampal amnesia.</a> J. Cog. Neuro. 19 (10), 1690-1705 (2007).</li><li>Bate, S., Haslam, C., Tree, J. J., Hodgson, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+an+eye+movement-based+memory+effect+in+congenital+prosopsagnosia.">Evidence of an eye movement-based memory effect in congenital prosopsagnosia.</a> Cortex. 44 (7), 806-819 (2008).</li><li>Riggs, L., McQuiggan, D., Chan, J., Anderson, A. K., Ryan, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emotion-+modulated+viewing+of+faces+by+association.">Emotion- modulated viewing of faces by association.</a> , .</li><li>Kramer, A. F., McCarley, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oculomotor+behaviour+as+a+reflection+of+attention+and+memory+processes:+Neural+mechanisms+and+applications+to+human+factors.">Oculomotor behaviour as a reflection of attention and memory processes: Neural mechanisms and applications to human factors.</a> Theor. Issues Ergon. Sci. 4 (1), 21-55 (2003).</li></ol>

No conflicts of interest declared.

Eye movement monitoring is an efficient, useful tool with which to assess memory function in a variety of populations. This protocol describes the use of a head-mounted video-based eye tracker, but the protocol can be easily adapted to the use of remote eye tracking devices, as remote eye trackers remove the need for helmet adjustment and simplify the camera adjustments. However, with a remote eye tracker, head movement must be constrained to maintain accuracy of eye recordings. Accurate calibration of the eye movemen...

A correction was made to <a href="http://www.jove.com/index/details.stp?id=2108">Eye Movement Monitoring of Memory</a>. There was an error in the author, Douglas A. McQuiggan's, name. The author's name has been corrected to:
Douglas A. McQuiggan
instead of:
Doug McQuiggan

A correction to an author's name has been made for the article Eye Movement Monitoring of Memory.

Erratum: Eye Movement Monitoring of Memory

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<td>Eye Tracker</td>
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eye movement monitoring of memory

Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank yesterday."), however such reports can often be unreliable or sensitive to response bias 1, and may be unobtainable in some participant populations. Furthermore, explicit reports only reveal when information has reached consciousness and cannot comment on when memories were accessed during processing, regardless of whether the information is subsequently accessed in a conscious manner. Eye movement monitoring (eye tracking) provides a tool by which memory can be probed without asking participants to comment on the contents of their memories, and access of such memories can be revealed on-line 2,3. Video-based eye trackers (either head-mounted or remote) use a system of cameras and infrared markers to examine the pupil and corneal reflection in each eye as the participant views a display monitor. For head-mounted eye trackers, infrared markers are also used to determine head position to allow for head movement and more precise localization of eye position. Here, we demonstrate the use of a head-mounted eye tracking system to investigate memory performance in neurologically-intact and neurologically-impaired adults. Eye movement monitoring procedures begin with the placement of the eye tracker on the participant, and setup of the head and eye cameras. Calibration and validation procedures are conducted to ensure accuracy of eye position recording. Real-time recordings of X,Y-coordinate positions on the display monitor are then converted and used to describe periods of time in which the eye is static (i.e. fixations) versus in motion (i.e., saccades). Fixations and saccades are time-locked with respect to the onset/offset of a visual display or another external event (e.g. button press). Experimental manipulations are constructed to examine how and when patterns of fixations and saccades are altered through different types of prior experience. The influence of memory is revealed in the extent to which scanning patterns to new images differ from scanning patterns to images that have been previously studied 2, 4-5. Memory can also be interrogated for its specificity; for instance, eye movement patterns that differ between an identical and an altered version of a previously studied image reveal the storage of the altered detail in memory 2-3, 6-8. These indices of memory can be compared across participant populations, thereby providing a powerful tool by which to examine the organization of memory in healthy individuals, and the specific changes that occur to memory with neurological insult or decline 2-3, 8-10.

Watch this Scientific Journal Video about Eye Movement Monitoring of Memory at JoVE.com

Eye Movement Monitoring of Memory

Eye movement monitoring (or eye tracking) reveals where in space the eyes linger, when and for how long. Here, we demonstrate how eye tracking can be used to investigate the integrity of memory in multiple participant populations, without requiring verbal, or otherwise explicit, reports.

Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank ...

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14.5K Views.  Rotman Research Institute. Eye movement monitoring (or eye tracking) reveals where in space the eyes linger, when and for how long. Here, we demonstrate how eye tracking can be used to investigate the integrity of memory in multiple participant populations, without requiring verbal, or otherwise explicit, reports.

Video: Eye Movement Monitoring of Memory

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and focused on psychological aspects. Until now, objective observation of neurophysiological parameters has not been carried out - undoubtedly because the technical and methodological means have not been available -, investigations into the neurophysiological effects of weightlessness are in their infancy (Schneider et al. 2008).
While imaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI) would be hardly applicable in space, the non-invasive near-infrared spectroscopy (NIRS) technique represents a method of mapping hemodynamic processes in the brain in real time that is both relatively inexpensive and that can be employed even under extreme conditions. The combination with electroencephalography (EEG) opens up the possibility of following the electrocortical processes under changing gravity conditions with a finer temporal resolution as well as with deeper localization, for instance with electrotomography (LORETA).
Previous studies showed an increase of beta frequency activity under normal gravity conditions and a decrease under weightlessness conditions during a parabolic flight (Schneider et al. 2008a+b). Tilt studies revealed different changes in brain function, which let suggest, that changes in parabolic flight might reflect emotional processes rather than hemodynamic changes. However, it is still unclear whether these are effects of changed gravity or hemodynamic changes within the brain. Combining EEG/LORETA and NIRS should for the first time make it possible to map the effect of weightlessness and reduced gravity on both hemodynamic and electrophysiological processes in the brain. Initially, this is to be done as part of a feasibility study during a parabolic flight. Afterwards, it is also planned to use both techniques during medium- and long-term space flight.
It can be assumed that the long-term redistribution of the blood volume and the associated increase in the supply of oxygen to the brain will lead to changes in the central nervous system that are also responsible for anaemic processes, and which can in turn reduce performance (De Santo et al. 2005), which means that they could be crucial for the success and safety of a mission (Genik et al. 2005, Ellis 2000).
Depending on these results, it will be necessary to develop and employ extensive countermeasures. Initial results for the MARS500 study suggest that, in addition to their significance in the context of the cardiovascular and locomotor systems, sport and physical activity can play a part in improving neurocognitive parameters. Before this can be fully established, however, it seems necessary to learn more about the influence of changing gravity conditions on neurophysiological processes and associated neurocognitive impairment.

The effect of weightlessness and hypergravity on both hemodynamic and electrophysiological processes in the brain is going to be followed during parabolic flight by EEG and NIRS techniques. 
A feasibility study of a more complex experiment, which is planned to carry out during medium- and long-term space flight.

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and ...

Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4. Rod photoreceptors are responsible for vision in dim light, cones in bright light. Phototransduction takes place in the outer segment of the photoreceptor cell, a specialized compartment that contains a high concentration of visual pigment, the primary light detector. The visual pigment is composed of a chromophore, 11-cis retinal, attached to a protein, opsin. A photon absorbed by the visual pigment isomerizes the chromophore from 11-cis to all-trans. This photoisomerization brings about a conformational change in the visual pigment that 
initiates a cascade of reactions culminating in a change in membrane potential, and bringing about the transduction of the light stimulus to an electrical signal. The 
recovery of the cell from light stimulation involves the deactivation of the intermediates activated by light, and the reestablishment of the membrane potential. Ca2+ modulates the activity of several of the enzymes involved in phototransduction, and its concentration is reduced upon light stimulation. In this way, Ca2+ plays an important role in the recovery of the cell from light stimulation and its adaptation to background light.
Another essential part of the recovery process is the regeneration of the visual pigment that has been destroyed during light-detection by the 
photoisomerization of its 11-cis chromophore to all-trans5-7. This regeneration begins with the release of all-trans retinal by 
the photoactivated pigment, leaving behind the apo-protein opsin. The released all-trans retinal is rapidly reduced in a reaction utilizing NADPH to all-
trans retinol, and opsin combines with fresh 11-cis retinal brought into the outer segment to reform the visual pigment. All-trans retinol is 
then transferred out of the outer segment and into neighboring cells by the specialized carrier Interphotoreceptor Retinoid Binding Protein (IRBP).
Fluorescence imaging of single photoreceptor cells can be used to study their physiology and cell biology. Ca2+-sensitive fluorescent dyes can be used to examine in detail the interplay between outer 
segment Ca2+ changes and response to light8-12 as well as the role of inner segment Ca2+ stores in Ca2+ homeostasis13,14. 
Fluorescent dyes can also be used for measuring Mg2+ concentration15, pH, and as tracers of aqueous and membrane compartments16. 
Finally, the intrinsic fluorescence of all-trans retinol (vitamin A) can be used to monitor the kinetics of its formation and removal in single 
photoreceptor cells17-19.

A method is described for the preparation of single living photoreceptor cells from different vertebrate species for fluorescence imaging. The method can be used to image the fluorescence of endogenous fluorophores, such as NADH or vitamin A, or that of exogenously added fluorescent dyes sensitive to Ca2+ or other factors.

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4.  ...

Using Affordable LED Arrays for Photo-Stimulation of Neurons

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single cells in response to electrical or pharmacological manipulations1,2. With the invention of methods to optically control genetically targeted neurons (optogenetics), researchers now have an unprecedented level of control over specific groups of neurons in the standard slice preparation. In particular, photosensitive channelrhodopsin-2 (ChR2) allows researchers to activate neurons with light3,4. By combining careful calibration of LED-based photostimulation of ChR2 with standard slice electrophysiology, we are able to probe with greater detail the role of adult-born interneurons in the olfactory bulb, the first central relay of the olfactory system. Using viral expression of ChR2-YFP specifically in adult-born neurons, we can selectively control young adult-born neurons in a milieu of older and mature neurons. Our optical control uses a simple and inexpensive LED system, and we show how this system can be calibrated to understand how much light is needed to evoke spiking activity in single neurons. Hence, brief flashes of blue light can remotely control the firing pattern of ChR2-transduced newborn cells.

Adult-born neurons expressing ChR2 can be manipulated in slice electrophysiological preparations in order to examine their contribution towards the function of olfactory neural circuits.

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker bees spend the first half of their life inside the dark hive, where they are nursing the larvae or building the regular hexagonal combs for food (e.g. pollen or nectar) and brood3. The antennae are extraordinary multisensory feelers and play a pivotal role in various tactile mediated tasks4, including hive building5 and pattern recognition6. Later in life, each single bee leaves the hive to forage for food. Then a bee has to learn to discriminate profitable food sources, memorize their location, and communicate it to its nest mates7. Bees use different floral signals like colors or odors7, 8, but also tactile cues from the petal surface9 to form multisensory memories of the food source. Under laboratory conditions, bees can be trained in an appetitive learning paradigm to discriminate tactile object features, such as edges or grooves with their antennae10, 11, 12, 13. This learning paradigm is closely related to the classical olfactory conditioning of the proboscis extension response (PER) in harnessed bees14. The advantage of the tactile learning paradigm in the laboratory is the possibility of combining behavioral experiments on learning with various physiological measurements, including the analysis of the antennal movement pattern.

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L.

In this protocol we show how to condition harnessed honey bees to tactile stimuli and introduce a 2D motion capture technique for analyzing the kinematics of fine-scale antennal sampling pattern.

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker ...

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report1, the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...