Name: using saccadometry with deep brain stimulation to study normal and pathological brain function
Uploaded: 2016-07-14T13:00:00
Description: Watch this Scientific Journal Video about Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function at JoVE.com

Dr. Antoniades was supported by the National Institute of Health Research (NIHR) and by the Dementias and Neurodegenerative Diseases Research Network (DENDRON) and by the Wellcome Trust. Dr FitzGerald was supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre.

The local ethics committee approved this study and informed consented was obtained from the participants as detailed below in section 1.
1. Participant Consent
<ol>
	<li>Provide participants with an information sheet that explains in detail what the testing session will include.</li>
	<li>After the participants have the opportunity to read and discuss any questions, concerns or other matters related to their taking part in the study, go through the consent form with them, explaining each poi...

<ol>
	<li>Leigh, R. J., Kennard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+saccades+as+a+research+tool+in+the+clinical+neurosciences.">Using saccades as a research tool in the clinical neurosciences.</a> Brain. 127, 460-477 (2004).</li><li>Carpenter, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neural+control+of+looking.">The neural control of looking.</a> Curr Biol. 10, 291-293 (2000).</li><li>Leigh, R. J., Zee, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Neurology of Eye Movements. , (2006).</li><li>Antoniades, C. A., Xu, Z., Mason, S. L., Carpenter, R. H., Barker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntington's+disease:+changes+in+saccades+and+hand-tapping+over+3+years.">Huntington's disease: changes in saccades and hand-tapping over 3 years.</a> Journal of Neurology. 257, 1890-1898 (2010).</li><li>Blekher, T. M., Yee, R. D., Kirkwood, S. C., Hake, A. M., Stout, J. C., Weaver, M. R., Foroud, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oculomotor+control+in+asymptomatic+and+recently+diagnosed+individuals+with+the+genetic+marker+for+Huntington's+disease.">Oculomotor control in asymptomatic and recently diagnosed individuals with the genetic marker for Huntington's disease.</a> Vision Research. 44, 2729-2736 (2004).</li><li>Chan, F., Armstrong, I. T., Pari, G., Riopelle, R. J., Munoz, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficits+in+saccadic+eye-movement+control+in+Parkinson's+disease.">Deficits in saccadic eye-movement control in Parkinson's disease.</a> Neuropsychologia. 43, 784-796 (2005).</li><li>Antoniades, C. A., Demeyere, N., Kennard, C., Humphreys, G. W., Hu, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antisaccades+and+executive+dysfunction+in+early+drug-naive+Parkinson's+disease:+The+discovery+study.">Antisaccades and executive dysfunction in early drug-naive Parkinson's disease: The discovery study.</a> Mov Disord. , (2015).</li><li>Antoniades, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+internationally+standardised+antisaccade+protocol.">An internationally standardised antisaccade protocol.</a> Vision Res. 84, 1-5 (2013).</li><li>Ober, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hand-Held+system+for+ambulatory+measurement+of+saccadic+durations+of+neurological+patients.+.">Hand-Held system for ambulatory measurement of saccadic durations of neurological patients. .</a> Modelling and Measurement in Medicine. , (2003).</li><li>Temperli, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+parkinsonian+signs+return+after+discontinuation+of+subthalamic+DBS.">How do parkinsonian signs return after discontinuation of subthalamic DBS.</a> Neurology. 60, 78-81 (2003).</li><li>Antoniades, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+brain+stimulation:+eye+movements+reveal+anomalous+effects+of+electrode+placement+and+stimulation.">Deep brain stimulation: eye movements reveal anomalous effects of electrode placement and stimulation.</a> PLoS ONE. 7, e32830 (2012).</li><li>Yugeta, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+STN+stimulation+on+the+initiation+and+inhibition+of+saccade+in+Parkinson+disease.">Effects of STN stimulation on the initiation and inhibition of saccade in Parkinson disease.</a> Neurology. 74, 743-748 (2010).</li><li>Terao, Y., Fukuda, H., Ugawa, Y., Hikosaka, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+perspectives+on+the+pathophysiology+of+Parkinson's+disease+as+assessed+by+saccade+performance:+a+clinical+review.">New perspectives on the pathophysiology of Parkinson's disease as assessed by saccade performance: a clinical review.</a> Clin Neurophysiol. 124, 1491-1506 (2013).</li><li>Temel, Y., Visser-Vandewalle, V., Carpenter, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saccadic+latency+during+electrical+stimulation+of+the+human+subthalamic+nucleus.">Saccadic latency during electrical stimulation of the human subthalamic nucleus.</a> Curr Biol. 18, 412-414 (2008).</li><li>Antoniades, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+brain+stimulation+abolishes+slowing+of+reactions+to+unlikely+stimuli.">Deep brain stimulation abolishes slowing of reactions to unlikely stimuli.</a> J Neurosci. 34, 10844-10852 (2014).</li><li>Rivaud-Pechoux, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+memory+guided+saccades+in+parkinsonian+patients+by+high+frequency+subthalamic+nucleus+stimulation.">Improvement of memory guided saccades in parkinsonian patients by high frequency subthalamic nucleus stimulation.</a> J Neurol Neurosurg Psychiatry. 68, 381-384 (2000).</li><li>Takikawa, Y., Kawagoe, R., Itoh, H., Nakahara, H., Hikosaka, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+saccadic+eye+movements+by+predicted+reward+outcome.">Modulation of saccadic eye movements by predicted reward outcome.</a> Experimental brain research. Experimentelle Hirnforschung. 142, 284-291 (2002).</li><li>Dorris, M. C., Munoz, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neural+correlate+for+the+gap+effect+on+saccadic+reaction+times+in+monkey.">A neural correlate for the gap effect on saccadic reaction times in monkey.</a> Journal of Neurophysiology. 73, 2558-2562 (1995).</li><li>Hanes, D. P., Schall, 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=Countermanding+saccades+in+macaque.">Countermanding saccades in macaque.</a> Visual Neuroscience. 12, 929-937 (1995).</li><li>Opris, I., Barborica, A., Ferrera, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+gap+effect+for+saccades+evoked+by+electrical+microstimulation+of+frontal+eye+fields+in+monkeys.">On the gap effect for saccades evoked by electrical microstimulation of frontal eye fields in monkeys.</a> Experimental brain research. Experimentelle Hirnforschung. 138, 1-7 (2001).</li><li>Takagi, M., Frohman, E. M., Zee, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap-overlap+effects+on+latencies+of+saccades,+vergence+and+combined+vergence-saccades+in+humans.">Gap-overlap effects on latencies of saccades, vergence and combined vergence-saccades in humans.</a> Vision Res. 35, 3373-3388 (1995).</li><li>Schall, 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=Neuronal+activity+related+to+visually+guided+saccades+in+the+frontal+eye+fields+of+rhesus+monkeys:+comparison+with+supplementary+eye+fields.">Neuronal activity related to visually guided saccades in the frontal eye fields of rhesus monkeys: comparison with supplementary eye fields.</a> Journal of Neurophysiology. 66, 559-579 (1991).</li><li>Pare, M., Hanes, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+movement+processing:+superior+colliculus+activity+associated+with+countermanded+saccades.">Controlled movement processing: superior colliculus activity associated with countermanded saccades.</a> J Neurosci. 23, 6480-6489 (2003).</li></ol>

The most critical factor in obtaining good quality saccadic data is ensuring that the instructions given to the participant are clear and precise. For example, if the instructions for the antisaccadic task are not completely clear, the participant is likely to execute prosaccades instead. Recordings may also be spoiled if the participant cannot clearly see the stimuli or the saccadometer cannot accurately gauge eye position. Thus if the data appear to be of low quality the experimenter should check that the ambient light...

In recent years there has been increasing interest in the use of measurements of reaction times as a quantitative and non-invasive way of gaining information about the high level mechanisms of neural decision making 1. One type of reaction time that has been studied extensively is the time taken to initiate a saccade on presentation of a visual stimulus, known as saccadic latency. Saccades are the fast eye movements that occur when we rapidly shift our gaze from one place to another. They are the commonest type of eye movements we make, occurring at a frequency of typically two or three per second. Each saccade is in effect a decision to look at one cue in the visual world rather than another 2.
The neural pathways controlling eye movements have been studied extensively and are fairly well documented 3. Using sensitive electronic equipment, aspects of oculomotor function can be precisely and objectively quantified. This facilitates the detailed study of eye movements themselves but also allows them to be used as a tool to investigate other areas of neurophysiology and pathophysiology.
Eye movement measurement can provide useful information about disease states. Saccadic eye movements have recently, for example, received much attention as potential biomarkers in neurodegenerative disorders including Huntington&#39;s 4,5 and Parkinson&#39;s diseases 6,7, and it is well established that saccadic reaction times tend to be slower than normal in these conditions. Potential uses of saccadic measurement include aids to diagnosis and disease tracking. Saccadic tasks range from the simple prosaccade (looking as quickly as possible toward a suddenly appearing visual stimulus to left or right) to more complex tasks such as the antisaccade (looking as quickly as possible to the opposite side to a visual stimulus) or memory-guided saccade (looking towards the remembered location of a target that is no longer there).
Deep brain stimulation is an effective treatment for several neurological conditions. It is most commonly used to treat the motor symptoms of Parkinson&#39;s disease including tremor, rigidity, bradykinesia, and dyskinesia. It is also used for other movement disorders including dystonia and essential tremor, and less commonly for neuropathic pain, epilepsy, and psychiatric conditions such as obsessive compulsive disorder. It is the only setting in which scientists have direct electrical access to deep structures of the human brain in vivo and thus offers a precious opportunity for experimental neurology. A variety of targets are stimulated depending on the condition being treated, including several locations in the basal ganglia, many of which are involved in oculomotor pathways. This means that a wide range of studies can be conducted using the DBS system to deliver stimulation to a given brain location and an eye tracking device to record and analyze its effects. Depending on the experimental paradigm, such studies may yield information about the physiology of the region being stimulated, the effects of the disease, or the mechanism by which DBS is working in that particular setting. This article describes a general approach to saccadic eye movement testing in Deep Brain Stimulation patients.
Several different types of eye tracking equipment are available. For the research described in this protocol a portable saccadometer was used to record horizontal saccadic eye movements. Portable saccadometers have the advantage of not requiring head restraint (see Figure 1), which means that sessions are more comfortable for patients with Parkinson&#39;s disease, especially for those suffering with severe dyskinesias. The saccadometer used here is lightweight and approximately 5 cm wide and 10 cm tall. The saccadometer measures eye movements by the use of direct infrared oculography: an infra-red source and sensor positioned in front of the medial canthus use light reflected from the cornea to establish the rotational position of the eyeball at millisecond intervals. In order to acquire good quality data for analysis the saccadometer should sample at a rate of at least 1 kHz with at least a 12 bit resolution. In the saccadometer used here the visual stimuli were three red 13 cd m-2 spots of light produced by built in low power lasers, each spot subtending some 0.1 degrees, with one spot in the midline and the other two at &#177; 10 degrees (i.e., to the right and left).
<img alt="Figure 1" src="/files/ftp_upload/53640/53640fig1.jpg" /> 
Figure 1. The Saccadometer.&#160; Head mounted saccadometer attached to an elastic band and resting on the bridge of the nose. Four miniature lasers project visual targets on to a matte surface, and the participant&#39;s eye movements are measured by differential infrared reflectance transducers on the nasal side of each eye. As the laser targets move with the head, head restraints are not required. <a href="https://www.jove.com/files/ftp_upload/53640/53640fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="88">
	<tbody>
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			<td>Saccadometer device ( Ober Consulting Poland)</td>
		</tr>
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			<td>Computer with Windows environment</td>
		</tr>
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			<td>Software, Latency Meter for downloading the raw data from the saccadometer.</td>
		</tr>
	</tbody>
</table>

using saccadometry with deep brain stimulation to study normal and pathological brain function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

Figure 3 shows an example of saccadic eye movement trajectories, from a Parkinson&#39;s disease patient with a subthalamic nucleus DBS system implanted. The two graphs plot the patient&#39;s prosaccades with the stimulator system switched off (upper graph) and switched on (lower graph). Each trace on the graphs shows the trajectory of a single saccade, i.e., how the eye position in degrees away from the midline (y axis) varies as a function of time (x axis). Both...

Watch this Scientific Journal Video about Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function at JoVE.com

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

The overall goal of this procedure is to measure the effects of deep brain stimulation on saccadic eye movements. The combination of deep brain stimulation with saccade measurement provides a tool to study normal and pathological brain function. This method can help answer key questions about the pathophysiology of diseases relating to the basal ganglia.The main advantage of this technique is that it is quantitative and precise, allowing for the accurate detection of subtle abnormalities. These are some of the types of stimulator implants used in deep brain stimulation. They contain a battery and electronics to generate a small electrical stimulus.The stimulus pulses are delivered to precise target locations in the brain using contacts on the end of a wire like this one. Begin by explaining the details of the study and providing the participant with a consent form to sign. Set up the saccadometer so that for each trial the central fixation target is displayed for a random period of 1.0 to 2.0 seconds, after which it is extinguished, and one of the peripheral red targets appears randomly to the right or left.Next, ask the participant to sit 1.5 meters away from a flat matte screen. Place the saccadometer on the participant's head so that it rests on the bridge of the nose and secure with an elastic adjustable strap. Ensure that the ambient lighting is dim so that the stimuli are clearly seen.Then, instruct the participant that the 40 minute testing session consists of five blocks with a one minute break between each block. Prior to the first block, instruct the participant to move their eyes as quickly and accurately as possible in order to follow the red dot jumping from the middle to one side or the other. Explain that they must do this 60 times.Set the saccadometer to generate a sequence of 60 trials, then press the button on the device to start the first block of trials. After the completion of the first block wait one minute before starting the second block. Reset the saccadometer to generate a sequence of 40 trials.Towards the end of the one minute gap, instruct the participant to move their eyes as quickly as possible in the opposite direction of the red dot. Inform them that they will be required to do this 40 times. Then, start the second block of trials.After completion of the second block, wait one minute, then repeat the instructions from the second block two more times for blocks three and four. Finally, after completing the fourth block, wait one minute, then instruct the participants to move their eyes as quickly and accurately as possible to follow the red dot jumping from the middle to one side to the other, exactly as they did in the first block. Reset the saccadometer to generate a sequence of 60 trials and start the final block of trials.Note for participants with deep brain stimulators, or DBS systems, conduct saccadic testing two times. Once with the DBS system running as normal, and a second time with the DBS system turned off. To conduct the off stimulation session, have a trained clinical staff switch off the DBS system 30 minutes prior to testing.Then, complete saccadic testing. Once the session is complete, switch the DBS system back on. To analyze the data, download the raw data from the saccadometer to a computer.Next, use the saccadometer's software program to exclude saccades that are distorted by blinks and head movements. Calculate variables including saccadic latencies, peak velocities, and amplitudes. Here, prosaccadic latency results are shown for a Parkinson's patient after undergoing DBS of the subthalamic nucleus when the DBS is turned on and off.The distribution of saccades is altered when the stimulator is switched on, showing a reduction in the number of long latency saccades, and a corresponding decrease in the mean latency. Once mastered, this technique can be done in about 70 minutes if it is performed properly. While attempting this procedure, it is important to remember to ensure that the saccadometer is sitting centrally on the bridge of the nose and the room lights are dim.Following this procedure, more advanced saccadic tasks can be used in order to answer additional questions, like how the cognitive control of eye movements is affected by deep brain stimulation. After watching this video, you should have a good understanding of how to use psychodometry in subjects with deep brain stimulator systems. Don't forget that working with lasers can be hazardous, and you should take care to ensure that both the participants and the experimentor don't look directly into the laser beams.

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Intra-Operative Behavioral Tasks in Awake Humans Undergoing Deep Brain Stimulation Surgery

Deep brain stimulation (DBS) is a surgical procedure that directs chronic, high frequency electrical stimulation to specific targets in the brain through implanted electrodes. Deep brain stimulation was first implemented as a therapeutic modality by Benabid et al. in the late 1980s, when he used this technique to stimulate the ventral intermediate nucleus of the thalamus for the treatment of tremor 1. Currently, the procedure is used to treat patients who fail to respond adequately to medical management for diseases such as Parkinson's, dystonia, and essential tremor. The efficacy of this procedure for the treatment of Parkinson's disease has been demonstrated in well-powered, randomized controlled trials 2. Presently, the U.S. Food and Drug Administration has approved DBS as a treatment for patients with medically refractory essential tremor, Parkinson's disease, and dystonia. Additionally, DBS is currently being evaluated for the treatment of other psychiatric and neurological disorders, such as obsessive compulsive disorder, major depressive disorder, and epilepsy. 
DBS has not only been shown to help people by improving their quality of life, it also provides researchers with the unique opportunity to study and understand the human brain. Microelectrode recordings are routinely performed during DBS surgery in order to enhance the precision of anatomical targeting. Firing patterns of individual neurons can therefore be recorded while the subject performs a behavioral task. Early studies using these data focused on descriptive aspects, including firing and burst rates, and frequency modulation 3. More recent studies have focused on cognitive aspects of behavior in relation to neuronal activity 4,5. This article will provide a description of the intra-operative methods used to perform behavioral tasks and record neuronal data with awake patients during DBS cases. Our exposition of the process of acquiring electrophysiological data will illuminate the current scope and limitations of intra-operative human experiments.

Deep brain stimulation surgery offers a unique opportunity to examine information encoding in the awake human brain. This article will describe intra-operative methods used to perform cognitive and behavioral tasks while simultaneously acquiring physiological data such as EMG, single-unit neuronal activity and/or local field potentials.

Deep brain stimulation (DBS) is a surgical procedure that directs chronic, high frequency electrical stimulation to specific targets in the brain through ...

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are exacerbated when significant brain atrophy and age-related white matter changes (e.g. Leukoaraiosis) are present. Lesion Explorer (LE) is an accurate and reliable neuroimaging pipeline specifically developed to address such issues commonly observed on MRI of Alzheimer's disease and normal elderly. The pipeline is a complex set of semi-automatic procedures which has been previously validated in a series of internal and external reliability tests1,2. However, LE's accuracy and reliability is highly dependent on properly trained manual operators to execute commands, identify distinct anatomical landmarks, and manually edit/verify various computer-generated segmentation outputs.
LE can be divided into 3 main components, each requiring a set of commands and manual operations: 1) Brain-Sizer, 2) SABRE, and 3) Lesion-Seg. Brain-Sizer's manual operations involve editing of the automatic skull-stripped total intracranial vault (TIV) extraction mask, designation of ventricular cerebrospinal fluid (vCSF), and removal of subtentorial structures. The SABRE component requires checking of image alignment along the anterior and posterior commissure (ACPC) plane, and identification of several anatomical landmarks required for regional parcellation. Finally, the Lesion-Seg component involves manual checking of the automatic lesion segmentation of subcortical hyperintensities (SH) for false positive errors.
While on-site training of the LE pipeline is preferable, readily available visual teaching tools with interactive training images are a viable alternative. Developed to ensure a high degree of accuracy and reliability, the following is a step-by-step, video-guided, standardized protocol for LE's manual procedures.

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer&#039;s Disease and Normal Elderly

Lesion Explorer (LE) is a semi-automatic, image-processing pipeline developed to obtain regional brain tissue and subcortical hyperintensity lesion volumetrics from structural MRI of Alzheimer's disease and normal elderly. To ensure a high level of accuracy and reliability, the following is a video-guided, standardized protocol for LE's manual procedures.

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Controlling Parkinson's Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time according to fluctuating disease and medication state. In the present realization of adaptive DBS we record and stimulate from the DBS electrodes implanted in the subthalamic nucleus of patients with Parkinson&#39;s disease in the early post-operative period. Local field potentials are analogue filtered between 3 and 47 Hz before being passed to a data acquisition unit where they are digitally filtered again around the patient specific beta peak, rectified and smoothed to give an online reading of the beta amplitude. A threshold for beta amplitude is set heuristically, which, if crossed, passes a trigger signal to the stimulator. The stimulator then ramps up stimulation to a pre-determined clinically effective voltage over 250 msec and continues to stimulate until the beta amplitude again falls down below threshold. Stimulation continues in this manner with brief episodes of ramped DBS during periods of heightened beta power.
Clinical efficacy is assessed after a minimum period of stabilization (5 min) through the unblinded and blinded video assessment of motor function using a selection of scores from the Unified Parkinson&#39;s Rating Scale (UPDRS). Recent work has demonstrated a reduction in power consumption with aDBS as well as an improvement in clinical scores compared to conventional DBS. Chronic aDBS could now be trialed in Parkinsonism.

Controlling Parkinson&#039;s Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) is effective for Parkinson&#8217;s disease, improving symptoms and reducing power consumption compared to conventional deep brain stimulation (cDBS). In aDBS we track a local field potential biomarker (beta oscillatory amplitude) in real time and use this to control the timing of stimulation.

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time ...

A Multimodal Imaging- and Stimulation-based Method of Evaluating Connectivity-related Brain Excitability in Patients with Epilepsy

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations over time in the blood-oxygenation level dependent signal. rs-fcMRI has been applied extensively to identify abnormalities in brain connectivity in different neurologic and psychiatric diseases. However, the relationship among rs-fcMRI connectivity abnormalities, brain electrophysiology and disease state is unknown, in part because the causal significance of alterations in functional connectivity in disease pathophysiology has not been established. Transcranial Magnetic Stimulation (TMS) is a technique that uses electromagnetic induction to noninvasively produce focal changes in cortical activity. When combined with electroencephalography (EEG), TMS can be used to assess the brain's response to external perturbations. Here we provide a protocol for combining rs-fcMRI, TMS and EEG to assess the physiologic significance of alterations in functional connectivity in patients with neuropsychiatric disease. We provide representative results from a previously published study in which rs-fcMRI was used to identify regions with abnormal connectivity in patients with epilepsy due to a malformation of cortical development, periventricular nodular heterotopia (PNH). Stimulation in patients with epilepsy resulted in abnormal TMS-evoked EEG activity relative to stimulation of the same sites in matched healthy control patients, with an abnormal increase in the late component of the TMS-evoked potential, consistent with cortical hyperexcitability. This abnormality was specific to regions with abnormal resting-state functional connectivity. Electrical source analysis in a subject with previously recorded seizures demonstrated that the origin of the abnormal TMS-evoked activity co-localized with the seizure-onset zone, suggesting the presence of an epileptogenic circuit. These results demonstrate how rs-fcMRI, TMS and EEG can be utilized together to identify and understand the physiological significance of abnormal brain connectivity in human diseases.

Resting-state functional-connectivity MRI has identified abnormalities in patients with a wide range of neuropsychiatric disorders, including epilepsy due to malformations of cortical development. Transcranial Magnetic Stimulation in combination with EEG can demonstrate that patients with epilepsy have cortical hyperexcitability in regions with abnormal connectivity.

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations ...

Continuous IV Infusion is the Choice Treatment Route for Arginine-vasopressin Receptor Blocker Conivaptan in Mice to Study Stroke-evoked Brain Edema

Stroke is one of the major causes of morbidity and mortality in the world. Stroke is complicated by brain edema and other pathophysiological events. Among the most important players in the development and evolution of stroke-evoked brain edema is the hormone arginine-vasopressin and its receptors, V1a and V2. Recently, the V1a and V2 receptor blocker conivaptan has been attracting attention as a potential drug to reduce brain edema after stroke. However, animal models which involve conivaptan applications in stroke research need to be modified based on feasible routes of administration. Here the outcomes of 48 hr continuous intravenous (IV) are compared with intraperitoneal (IP) conivaptan treatments after experimental stroke in mice. We developed a protocol in which middle cerebral artery occlusion was combined with catheter installation into the jugular vein for IV treatment of conivaptan (0.2 mg) or vehicle. Different cohorts of animals were treated with 0.2 mg bolus of conivaptan or vehicle IP daily. Experimental stroke-evoked brain edema was evaluated in mice after continuous IV and IP treatments. Comparison of the results revealed that the continuous IV administration of conivaptan alleviates post-ischemic brain edema in mice, unlike the IP administration of conivaptan. We conclude that our model can be used for future studies of conivaptan applications in the context of stroke and brain edema.

Our studies have revealed that the beneficial effects of conivaptan are dependent on the method of delivery after experimental stroke in mice. We have developed a research protocol for delivery of the receptor blocker via IV catheter on stroke-evoked brain edema formation in mice.

Stroke is one of the major causes of morbidity and mortality in the world. Stroke is complicated by brain edema and other pathophysiological events. Among ...

Symmetric Bihemispheric Postmortem Brain Cutting to Study Healthy and Pathological Brain Conditions in Humans

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena described in those patients for whom a brain autopsy has been requested. Although the advancements of biomedical sciences and neuroimaging have revolutionized the neuropsychiatric field, they have also generated the misleading idea that brain autopsies have only a confirmatory value. This false idea created a drastic reduction of autopsy rates and, consequently, a reduced possibility to perform more detailed and extensive neuropathological investigations, which are necessary to comprehend numerous normal and pathological aspects yet unknown of the human brain. The traditional inferential method of correlation between observed neuropsychiatric phenomena and corresponding localization/characterization of their possible neurohistological correlates continues to have an undeniable value. In the context of neuropsychiatric diseases, the traditional clinicopathological method is still the best possible methodology (and often the only available) to link unique neuropsychiatric features to their corresponding neuropathological substrates, since it relies specifically upon the direct physical assessment of brain tissues. The assessment of postmortem brains is based on brain cutting procedures that vary across different neuropathology centers. Brain cuttings are performed in a relatively extensive and systematic way based on the various clinical and academic contingencies present in each institution. A more anatomically inclusive and symmetric bi-hemispheric brain cutting methodology should at least be used for research purposes in human neuropathology to coherently investigate, in depth, normal and pathological conditions with the peculiarities of the human brain (i.e., hemispheric specialization and lateralization for specific functions). Such a method would provide a more comprehensive collection of neuropathologically well-characterized brains available for current and future biotechnological and neuroimaging techniques. We describe a symmetric bi-hemispheric brain cutting procedure for the investigation of hemispheric differences in human brain pathologies and for use with current as well as future biomolecular/neuroimaging techniques.

Organized brain cutting procedures are necessary to correlate specific neuropsychiatric phenomena with definitive neuropathologic diagnoses. Brain cuttings are performed differently based on various clinico-academic contingencies. This protocol describes a symmetric bihemispheric brain cutting procedure to investigate hemispheric differences in human brain pathologies and to maximize current and future biomolecular/neuroimaging techniques.

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena ...

Updated Technique for Reliable, Easy, and Tolerated Transcranial Electrical Stimulation Including Transcranial Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a noninvasive method of neuromodulation using low-intensity direct electrical currents. This method of brain stimulation presents several potential advantages compared to other techniques, as it is noninvasive, cost-effective, broadly deployable, and well-tolerated provided proper equipment and protocols are administered. Even though tDCS is apparently simple to perform, correct administration of the tDCS session, especially the electrode positioning and preparation, is vital for ensuring reproducibility and tolerability. The electrode positioning and preparation steps are traditionally also the most time consuming and error-prone. To address these challenges, modern tDCS techniques, using fixed-position headgear and pre-assembled sponge electrodes, reduce complexity and setup time while also ensuring that the electrodes are consistently placed as intended. These modern tDCS methods present advantages for research, clinic, and remote-supervised (at home) settings. This article provides a comprehensive step-by-step guide for administering a tDCS session using fixed-position headgear and pre-assembled sponge electrodes. This guide demonstrates tDCS using commonly applied montages intended for motor cortex and dorsolateral prefrontal cortex (DLPFC) stimulation. As described, selection of the head size and montage-specific headgear automates electrode positioning. Fully assembled pre-saturated snap-electrodes are simply affixed to the set position snap-connectors on the headgear. The modern tDCS method is shown to reduce setup time and reduce errors for both novice and expert operators. The methods outlined in this article can be adapted to different applications of tDCS as well as other forms of transcranial electrical stimulation (tES) such as transcranial alternating current stimulation (tACS) and transcranial random noise stimulation (tRNS). However, since tES is application specific, as appropriate, any methods recipe is customized to accommodate subject, indication, environment, and outcome specific features.

When administering transcranial direct current stimulation (tDCS), reproducible electrode preparation and placement are vital for a tolerated and effective session. The purpose of this article is to demonstrate updated modern setup procedures for the administration of tDCS and related transcranial electrical stimulation techniques, such as transcranial alternating current stimulation (tACS).

Transcranial direct current stimulation (tDCS) is a noninvasive method of neuromodulation using low-intensity direct electrical currents. This method of ...