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 point on the consent form and giving them the opportunity to ask any questions they might have. Ask the participant to complete the form.</li>
</ol>
2. Setting up the Saccadometer
<ol>
	<li>Place the device on the patient&#39;s head, secured by an adjustable elastic strap and resting on the bridge of the nose. Because the stimuli move exactly with the head, no head-restraint is necessary and the device is comfortable to wear.</li>
	<li>Ask the patient to sit at 1.5 m from a flat matte screen.</li>
	<li>Ensure that the ambient lighting is dim so that the stimuli (red light spots) are clearly seen.</li>
</ol>
3. Recording a Saccadic Session
Note: As an example a standard protocol that tests both prosaccades and antisaccades 8 is described here. This protocol consists of five blocks: 60 prosaccades, 40 antisaccades x 3, and 60 prosaccades with a break of 1 min between blocks. The session lasts about 40 min.
<ol>
	<li>Set up the saccadometer so that for each trial the central fixation target is displayed for a random fore-period of 1.0 - 2.0 sec, after which it is extinguished and one of the peripheral targets appears, randomly to right or left 9. 
	Note: The three targets are spots of red light projected onto the screen in front of the participant (see 2.2) by low powered lasers built into the saccadometer. The saccadometer automatically switches the lasers on and off to display/extinguish the targets in the necessary sequence.</li>
	<li>Instruct the participant that the testing consists of five blocks with a one minute break between blocks.</li>
	<li>Prior to the first block instruct the participant to move their eyes as quickly and accurately as possible to follow the red dot jumping from the middle to one side or the other and instruct them to do this 60 times.</li>
	<li>Set the saccadometer to generate a sequence of 60 trials. Press the button on the saccadometer to start the first block of trials (prosaccades).</li>
	<li>After completion of the first block, leave a one minute gap before the second block. Reset the saccadometer to generate a sequence of 40 trials, and towards the end of the one minute gap, instruct the participant for the next block to move their eyes as quickly as possible in the opposite direction to the red dot, and explain that they will be required to do this 40 times. Start the second block of trials (antisaccades).</li>
	<li>After completion of the second block, leave a further 1 min gap, repeat the instruction in step 3.5 and start the third block of trials (antisaccades).</li>
	<li>After completion of the third block, leave a further 1 min gap, repeat the instruction in step 3.5 and start the fourth block of trials (antisaccades).</li>
	<li>After completion of the fourth block, leave a further 1 min gap, and explain that for the final block of tests ask the participants to move their eyes as quickly and accurately as possible to follow the red dot jumping from the middle to one side or the other, exactly as they did in the first block.</li>
	<li>Reset the saccadometer to generate a sequence of 60 trials, and start the final block of trials (prosaccades).</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/53640/53640fig2.jpg" /> 
Figure 2. Eye Movement Tasks. Schematic illustration showing two examples of saccadic tasks. The solid blue spot represents the target and the dotted blue circle represents the area of fixation. LEFT shows a prosaccadic task where the subject is prompted to look towards the target. RIGHT shows an antisaccade where the subject is asked to look away from the visual stimulus. This requires inhibition of the more natural prosaccade response and generation of a saccade in the opposite direction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53640/53640fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Deep Brain Stimulator Settings
Note: For participants with deep brain stimulators conduct the testing so far with stimulator system running as normal, i.e., an &#39;on stimulation&#39; dataset has been obtained. Testing now needs to be repeated with the stimulator system turned off (for healthy control participants without DBS systems this section will not apply).
<ol>
	<li>Switch the DBS system off. Do this by a trained clinical staff. Allow 30 min before testing.</li>
	<li>Repeat the saccadic testing (steps 3.2 - 3.9) to obtain a complete &#39;off stimulation&#39; dataset.</li>
	<li>Switch the DBS system back on (again this should be done by appropriately trained clinical staff).</li>
</ol>
5. Data Analysis
Note: For participants with deep brain stimulators conduct the testing so far with stimulator system running as normal, i.e., an &#39;on stimulation&#39; dataset has been obtained. Testing now needs to be repeated with the stimulator system turned off (for healthy control participants without DBS systems this section will not apply).
<ol>
	<li>Download the raw data from the saccadometer to a computer for analysis.</li>
	<li>Use the saccadometer&#39;s software program to exclude saccades distorted by blinks and head movements, and to calculate variables including saccadic latencies, peak velocities, and amplitudes. 
	Note: Records contaminated by excessive head movements or blinks are automatically removed by the software.
	<ol>
		<li>Remove saccades with latency less than 80 msec or greater than 1,000 msec. 
		Note: Saccadic latency is computed automatically using a saccade-detection algorithm based on velocity and acceleration. The onset of a saccade is identified as the point when the eye velocity exceeds a threshold of 5 degrees/sec.</li>
	</ol>
	</li>
</ol>

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<li>Carpenter, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+neural+control+of+looking%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLeigh%2C+RJ+%22The+Neurology+of+Eye+Movements%22">The Neurology of Eye Movements</a>. , Oxford University Press. New York. (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/pubmed?term=((Huntington%27s+disease%3A+changes+in+saccades+and+hand-tapping+over+3+years%5BTitle%5D)+AND+%22Journal+of+Neurology%22%5BJournal%5D)">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/pubmed?term=((Oculomotor+control+in+asymptomatic+and+recently+diagnosed+individuals+with+the+genetic+marker+for+Huntington%27s+disease%5BTitle%5D)+AND+%22Vision+Research%22%5BJournal%5D)">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/pubmed?term=((Deficits+in+saccadic+eye-movement+control+in+Parkinson%27s+disease%5BTitle%5D)+AND+%22Neuropsychologia%22%5BJournal%5D)">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/pubmed?term=((Antisaccades+and+executive+dysfunction+in+early+drug-naive+Parkinson%27s+disease%3A+The+discovery+study%5BTitle%5D)+AND+%22Mov+Disord%22%5BJournal%5D)">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/pubmed?term=((An+internationally+standardised+antisaccade+protocol%5BTitle%5D)+AND+%22Vision+Res%22%5BJournal%5D)">An internationally standardised antisaccade protocol.</a> Vision Res. 84, 1-5 (2013).</li>
<li>Ober, J. K., et al. Hand-Held system for ambulatory measurement of saccadic durations of neurological patients. . <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aOber%2C+JK+%22Modelling+and+Measurement+in+Medicine%22">Modelling and Measurement in Medicine</a>. , (2003).</li>
<li>Temperli, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+do+parkinsonian+signs+return+after+discontinuation+of+subthalamic+DBS%5BTitle%5D)+AND+%22Neurology%22%5BJournal%5D)">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/pubmed?term=((Deep+brain+stimulation%3A+eye+movements+reveal+anomalous+effects+of+electrode+placement+and+stimulation%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">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/pubmed?term=((Effects+of+STN+stimulation+on+the+initiation+and+inhibition+of+saccade+in+Parkinson+disease%5BTitle%5D)+AND+%22Neurology%22%5BJournal%5D)">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/pubmed?term=((New+perspectives+on+the+pathophysiology+of+Parkinson%27s+disease+as+assessed+by+saccade+performance%3A+a+clinical+review%5BTitle%5D)+AND+%22Clin+Neurophysiol%22%5BJournal%5D)">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/pubmed?term=((Saccadic+latency+during+electrical+stimulation+of+the+human+subthalamic+nucleus%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">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/pubmed?term=((Deep+brain+stimulation+abolishes+slowing+of+reactions+to+unlikely+stimuli%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Improvement+of+memory+guided+saccades+in+parkinsonian+patients+by+high+frequency+subthalamic+nucleus+stimulation%5BTitle%5D)+AND+%22J+Neurol+Neurosurg+Psychiatry%22%5BJournal%5D)">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/pubmed?term=((Modulation+of+saccadic+eye+movements+by+predicted+reward+outcome%5BTitle%5D)+AND+%22Experimental+brain+research.+Experimentelle+Hirnforschung%22%5BJournal%5D)">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/pubmed?term=((A+neural+correlate+for+the+gap+effect+on+saccadic+reaction+times+in+monkey%5BTitle%5D)+AND+%22Journal+of+Neurophysiology%22%5BJournal%5D)">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/pubmed?term=((Countermanding+saccades+in+macaque%5BTitle%5D)+AND+%22Visual+Neuroscience%22%5BJournal%5D)">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/pubmed?term=((On+the+gap+effect+for+saccades+evoked+by+electrical+microstimulation+of+frontal+eye+fields+in+monkeys%5BTitle%5D)+AND+%22Experimental+brain+research.+Experimentelle+Hirnforschung%22%5BJournal%5D)">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/pubmed?term=((Gap-overlap+effects+on+latencies+of+saccades%2C+vergence+and+combined+vergence-saccades+in+humans%5BTitle%5D)+AND+%22Vision+Res%22%5BJournal%5D)">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/pubmed?term=((Neuronal+activity+related+to+visually+guided+saccades+in+the+frontal+eye+fields+of+rhesus+monkeys%3A+comparison+with+supplementary+eye+fields%5BTitle%5D)+AND+%22Journal+of+Neurophysiology%22%5BJournal%5D)">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>
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</ol>

The authors have nothing to disclose.

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 is not too bright and that the saccadometer is sitting properly on the bridge of the nose.
Deep brain stimulation allows the direct modification of neural activity at a various locations in the basal ganglia. Coupled with equipment that can accurately and objectively quantify eye movements, DBS can be used to investigate the normal and abnormal functioning of these brain areas.
Experiments using saccadometry in conjunction with DBS must often contend with more than one unknown simultaneously. The brains under study are diseased to a variable extent, and we do not know for certain the mechanism by which DBS is achieving its effects, with hypotheses including activation of neurons in the targeted area, depolarizing blockade of neurons, stimulation of afferent and/or efferent axons, and more complex effects on network activity.
Subjects with neurodegenerative conditions are frequently on medication and this will be nearly universal for patients with PD patients who have DBS systems. Saccades are affected by antiparkinsonian medication, and in order to make a valid assessment of the effects of DBS on saccadic parameters, medication state must be similar when testing with DBS on and DBS off. This means either having the subject off all medication for the duration of testing (including a withdrawal period beforehand), or else conducting on-DBS and off-DBS tests within a reasonably short period of time. It has been shown that the complete wearing off of the effects of DBS on saccades takes several hours but that most of the change takes place within 30 min 10. The time course suggests multiple mechanisms with different time courses, from very fast (electrical effects) to much more prolonged (e.g., protein synthesis). The 30 min suggested in the protocol will therefore capture most of the DBS induced change, and is much shorter than the typical dosing interval of several hours. It does not give adequate time for the slower mechanisms to settle to steady state, and retesting at longer intervals may be useful in providing insight into these mechanisms. With longer gaps one could not assume that medication levels while testing on and off DBS were approximately similar, thus longer intervals are likely to be easier to interpret when testing off medication.
Most of the literature on the effects of DBS on eye movements has focused on high frequency stimulation of the subthalamic nucleus in PD. Prosaccadic latency is prolonged in PD and it is well established that STN DBS can substantially reduce it toward normal values (exactly how remains open to debate). However when saccadometry is performed in the early postoperative period, within hours to days of lead insertion and before electrical stimulation is switched on, it is found that latencies are actually increased 11. Electrode insertion causes edema and this will lead to temporary functional ablation of the brain immediately surrounding the electrode; this settles within a few weeks and saccadic parameters return to baseline (stimulators are commonly not turned on until a few weeks postoperatively to allow this time to settle). The oppositeness of the effects of electrode insertion and stimulation is of considerable interest, because it illustrates that one of the theories previously advanced to explain how DBS is working, by blockade of the stimulated structure (i.e., functional ablation), cannot comprehensively explain its mechanism of action. Other studies have found similar effects 12-14.
It is possible to construct experiments combining DBS and saccadometry to investigate normal physiology. Great care must be taken with experimental design and interpretation of results, because the experimental subject necessarily has some neurological condition. One approach is to compare the results of saccadic testing with DBS off and on to results from healthy control participants without DBS. If the results in DBS subjects with their stimulators turned off resemble those in controls, then it is reasonable to proceed on the basis that changes in the eye movements seen in response to DBS are likely to be similar to those which would be seen if the experiment could be done in participants without disease. For example, the performance in a prosaccade task which has been set up so that the visual stimulus is more likely to appear on one side than the other, is similar for healthy participants and PD patients with STN DBS systems in place but turned off. Latencies shorten for saccades in the direction where the target is more likely to appear, and lengthen in the less likely direction. When the systems are turned on, the lengthening of latency in the less likely direction disappears, and the interpretation was that the STN is performing a probability normalization function 15. The patients in this study had PD, but that was not directly relevant to the result. Of course this is a significant assumption and it is possible that the disease modulates the response to DBS even though it did not affect the baseline performance. Furthermore, the similarity of off-DBS and control data may be the result of compensatory mechanisms rather than a lack of effect of pathology on the parameters being measured. Results must be interpreted with this in mind.
Once the basic techniques described in the protocol have been mastered the approach can be extended to more complex paradigms. Many different saccadic tasks have been developed of which the probability based task described in the previous paragraph is just one. Further examples include memory guided saccades 16 (looking towards the remembered location of a stimulus that is no longer there) or reward-based saccadic tasks designed to invoke limbic function 17.
Here we have described one of the simplest saccadic tasks, a 10-degree visually guided horizontal step task. Many modifications of the task are possible such as inserting a temporal gap between fixation point disappearance and peripheral target appearance 18-20 or an overlap 21 where fixation point and lateral target are present simultaneously. Paradigms of this sort have been used in order to understand saccadic initiation and neural correlates have been observed in the frontal eye fields 20,22 and the superior colliculus 23. A detailed discussion of the various uses of different saccadic paradigms is beyond the scope of this article; for a review see 1.
Saccadometry has wide application outside its use in DBS. Saccadic pathways involve multiple parts of the basal ganglia and saccadic changes are a potential biomarker for any condition that damages or perturbs the function of these structures. Saccadometry has for example been investigated for use in neurodegenerative conditions including PD, Huntington&#39;s disease, frontotemporal dementia, amyotrophic lateral sclerosis as well as in head injury and in metabolic disorders including hepatic encephalopathy.
In conclusion, saccadometry is a useful quantitative tool by itself, but combining it with deep brain stimulation opens up a range of experimental approaches that may shed new light on brain function in both health and disease.

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.remotexs.ntu.edu.sg/files/ftp_upload/53640/53640fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Saccadometer device</td><td>Ober Consulting Poland</td><td></td><td></td></tr><tr><td>Computer with Windows environment</td><td></td><td></td><td></td></tr><tr><td>Software, Latency Meter for downloading the raw data from the saccadometer</td><td></td><td></td><td></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 leftward and rightward saccades are shown on the graph as deflections to positive degree values. Time zero is the moment that the central target disappears and the peripheral target appears. The interval between this and the onset of the saccade (the moment when the trace deflects from zero degrees) is termed saccadic latency, and the main observation is that the distribution of saccades is altered when the stimulator is switched on, with a reduction in the number of long latency saccades and a corresponding decrease in mean latency.
<img alt="Figure 3" src="/files/ftp_upload/53640/53640fig3.jpg" /> 
Figure 3. Prosaccadic Trajectory Results. This shows the saccadic eye movement latency results from a Parkinson&#39;s patient after undergoing Deep Brain Stimulation of the subthalamic nucleus. UPPER shows the latency profile of the patient when the stimulator is switched off LOWER shows the latency profile of the same patient when the stimulator is switched on. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53640/53640fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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 ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

7.4K Views.  The University of Oxford. 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.

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

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 ...

Combined Invasive Subcortical and Non-invasive Surface Neurophysiological Recordings for the Assessment of Cognitive and Emotional Functions in Humans

In spite of the success in applying non-invasive electroencephalography (EEG), magneto-encephalography (MEG) and functional magnetic resonance imaging (fMRI) for extracting crucial information about the mechanism of the human brain, such methods remain insufficient to provide information about physiological processes reflecting cognitive and emotional functions at the subcortical level. In this respect, modern invasive clinical approaches in humans, such as deep brain stimulation (DBS), offer a tremendous possibility to record subcortical brain activity, namely local field potentials (LFPs) representing coherent activity of neural assemblies from localized basal ganglia or thalamic regions. Notwithstanding the fact that invasive approaches in humans are applied only after medical indication and thus recorded data correspond to altered brain circuits, valuable insight can be gained regarding the presence of intact brain functions in relation to brain oscillatory activity and the pathophysiology of disorders in response to experimental cognitive paradigms. In this direction, a growing number of DBS studies in patients with Parkinson&#39;s disease (PD) target not only motor functions but also higher level processes such as emotions, decision-making, attention, memory and sensory perception. Recent clinical trials also emphasize the role of DBS as an alternative treatment in neuropsychiatric disorders ranging from obsessive compulsive disorder (OCD) to chronic disorders of consciousness (DOC). Consequently, we focus on the use of combined invasive (LFP) and non-invasive (EEG) human brain recordings in assessing the role of cortical-subcortical structures in cognitive and emotional processing trough experimental paradigms (e.g. speech stimuli with emotional connotation or paradigms of cognitive control such as the Flanker task), for patients undergoing DBS treatment.

The present protocol aims at assessing cognitive-emotional functions in the basal ganglia by simultaneous neurophysiological recording of local field potentials and non-invasive brain cortical activity (EEG). The procedure is exemplified by the use of paradigms involving speech stimuli with emotional connotation or the Flanker task involving cognitive control.

In spite of the success in applying non-invasive electroencephalography (EEG), magneto-encephalography (MEG) and functional magnetic resonance imaging ...

Behavior

A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to screen different neurostimulation patterns in hemiparkinsonian (unilateral lesioned) rats. It consists of creating a unilateral Parkinson&#39;s lesion by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle, implanting chronic stimulation electrodes into the subthalamic nucleus and evaluating motor outcomes at the end of 24 hr periods of cable-bound external neurostimulation. The stimulation was conducted with constant current stimulation. The amplitude was set 20% below the individual threshold for side effects. The motor outcome evaluation was done by the assessment of spontaneous paw use in the cylinder test according to Shallert and by the assessment of skilled reaching in the staircase test according to Montoya. This protocol describes in detail the training in the staircase box, the cylinder test, as well as the use of both in hemiparkinsonian rats. The use of both tests is necessary, because the staircase test seems to be more sensitive for fine motor skill impairment and exhibits greater sensitivity to change during neurostimulation. The combination of the unilateral Parkinson model and the two behavioral tests allows the assessment of different stimulation parameters in a standardized way.

Deep brain stimulation (DBS) is an effective treatment option for Parkinson's disease. We established a study design to screen novel stimulation paradigms in rats. The protocol describes the use of the staircase test and cylinder test for motor outcome assessment in DBS treated hemiparkinsonian rats.

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to ...

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.

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 ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Neuroscience

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 M&#937; was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

Targeting Neuronal Fiber Tracts for Deep Brain Stimulation Therapy Using Interactive, Patient-Specific Models

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for movement disorders and is being applied to a growing number of disorders. Computational modeling has been successfully used to predict the clinical effects of DBS; however, there is a need for novel modeling techniques to keep pace with the growing complexity of DBS devices. These models also need to generate predictions quickly and accurately. The goal of this project is to develop an image processing pipeline to incorporate structural magnetic resonance imaging (MRI) and diffusion weighted imaging (DWI) into an interactive, patient specific model to simulate the effects of DBS. A virtual DBS lead can be placed inside of the patient model, along with active contacts and stimulation settings, where changes in lead position or orientation generate a new finite element mesh and solution of the bioelectric field problem in near real-time, a timespan of approximately 10 seconds. This system also enables the simulation of multiple leads in close proximity to allow for current steering by varying anodes and cathodes on different leads. The techniques presented in this paper reduce the burden of generating and using computational models while providing meaningful feedback about the effects of electrode position, electrode design, and stimulation configurations to researchers or clinicians who may not be modeling experts.

The goal of this project is to develop an interactive, patient-specific modeling pipeline to simulate the effects of deep brain stimulation in near real-time and provide meaningful feedback as to how these devices influence neural activity in the brain.

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for ...

Bioengineering

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for various neurological and psychiatric disorders. HFS in the deep region of the brain, also named deep-brain stimulation (DBS), is becoming increasingly important in clinical trials. Recent progress in the field of high-frequency DBS (HF-DBS) surgery has begun to spread the possibility of utilizing this invasive technique to other situations, such as treatment for major depression disorder (MDD), obsessive-compulsive disorder (OCD), and so on. 
Despite these expanding indications, the underlying mechanisms of the beneficial effects of HF-DBS remain enigmatic. To address this question, one approach is to use implanted electrodes that sparsely activate distributed subpopulations of neurons by HFS. It has been reported that HFS in the anterior nucleus of the thalamus could be used for the treatment of refractory epilepsy in the clinic. The underlying mechanisms might be related to the increased neurogenesis and altered neuronal activity. Therefore, we are interested in exploring the physiological alterations by the detection of neuronal activity as well as neurogenesis in the mouse dentate gyrus (DG) before and after HFS treatment. 
In this manuscript, we describe methodologies for HFS to target the activation of the DG in mice, directly or indirectly and in an acute or chronic manner. In addition, we describe a detailed protocol for the preparation of brain slices for c-fos and Notch1 immunofluorescent staining to monitor the neuronal activity and signaling activation and for bromodeoxyuridine (BrdU) labeling to determine the neurogenesis after the HF-DBS induction. The activation of the neuronal activity and neurogenesis after the HF-DBS treatment provides direct neurobiological evidence and potential therapeutic benefits. Particularly, this methodology can be modified and applied to target other interested brain regions such as the basal ganglia and subthalamic regions for specific brain disorders in the clinic.

This protocol shows how to set up a reliable HFS method in mice. Neurons throughout the hippocampal dentate gyrus are electrically stimulated by HFS directly and indirectly in vivo. Neuronal activity and molecular signaling are examined by c-fos and Notch1 immunofluorescent staining, respectively; neurogenesis is quantified by bromodeoxyuridine labeling assay.

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for ...

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

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