The research was supported by grants of the University of Pisa. We thank Mr. Paolo Orsini, Mr. Francesco Montanari, and Mrs. Cristina Pucci for valuable technical assistance, as well as the I.A.C.E.R. S.r.L. company for supporting Dr. Maria Paola Tramonti Fantozzi with a fellowship. Finally, we thank the OCM Projects company for preparing hard pellets and performing hardness and spring constant measurements.

All steps follow the guidelines of the Ethical Committee of the University of Pisa.
1. Participant Recruitment
<ol>
	<li>Recruit a subject population according to the specific goal of the study (i.e., normal subjects and/or patients, males and/or females, young people and/or elders).</li>
</ol>
2. Material Preparation
<ol>
	<li>Prepare a soft pellet; use commercially available chewing gum (Table of Materials; initial hardness = 20 Shore OO).</li>
	<li>Prepare a hard pellet; use silicon rubber pellets (Table of Materials; constant hardness = 60 Shore OO)30.</li>
	<li>Prepare an anti-stress ball for a handgrip task. Use a polyurethane foam-made ball (Table of Materials; constant hardness = 30 Shore OO)30.</li>
	<li>Prepare a tangram puzzle (Table of Materials; number of pieces = seven) for performing the haptic task.</li>
</ol>
3. Flowchart of the Experiment
<ol>
	<li>Flowchart of protocol 1
	<ol>
		<li>Evaluate baseline performance (see section 4.1) in the cognitive (matrices) test (T0, control).</li>
		<li>Evaluate pupil size (see section 4.2) at rest (no activity requested from the subject) (T0, control).</li>
		<li>Evaluate pupil size during a haptic task based on tangram (T0, control).
		<ol>
			<li>Remove one of the pieces from the puzzle and place it in the subject&#39;s hand.</li>
			<li>Ask the subject to put the piece back into the puzzle, without looking at the puzzle.</li>
		</ol>
		</li>
		<li>Ask each subject to perform three specific activities for 2 min or to rest for 2 min, according to steps 3.1.4.1&#8211;3.1.4.4. Ask the subjects to perform these activities in separate sessions occurring on different days (2&#8211;3 days between sessions).
		<ol>
			<li>Ask the subject to chew a self-administered soft pellet for 2 min, letting him/her spontaneously choose both the rate of chewing and side of the mouth on which to chew. After 1 min of chewing, ask him/her to change the chewing side (and the pellet).</li>
			<li>Ask the subject to chew a self-administered hard pellet for 2 min. After 1 min, ask him/her to change the chewing side (but not the pellet).</li>
			<li>Ask the subject to perform a rhythmic squeezing of an anti-stress ball (handgrip exercise) for 2 min at the rate and on the hand of their choosing. After 1 min, ask the subject to switch hands.</li>
			<li>Ask the subject to rest (no activity) for 2 min.</li>
		</ol>
		</li>
		<li>Just after the end of each step (3.1.4.1&#8211;3.1.4.4), evaluate performance in the matrices test and pupil size at rest and during the haptic task (T7). 
		NOTE: The term &#34;at rest&#34; means that the subject during the pupil size measurement is relaxing. The term &#34;during haptic task&#34; means that the subject during the pupil size measurement is performing the task based on tangram.</li>
		<li>Thirty minutes following the end of each step (3.1.4.1&#8211;3.1.4.4), evaluate performance and pupil size at rest and during the haptic task (T37).</li>
	</ol>
	</li>
	<li>Flowchart of protocol 2
	<ol>
		<li>Evaluate pupil size while the subject is resting (T0, control; see section 4.3).</li>
		<li>Evaluate baseline performance in the cognitive (matrices) test while simultaneously testing pupil size (T0, control).</li>
		<li>Ask each subject to perform three specific activities for 2 min or to rest for 2 min, according to steps 3.2.3.1&#8211;3.2.3.4. Ask the subjects to perform these activities in separate sessions occurring on different days (2&#8211;3 days between sessions).
		<ol>
			<li>Ask the subject to chew a self-administered soft pellet for 2 min, letting him/her spontaneously choose both the rate of chewing and side of the mouth on which to chew. After 1 min of chewing, ask him/her to change the chewing side (and the pellet).</li>
			<li>Ask the subject to chew a self-administered hard pellet for 2 min. After 1 min, ask him/her to change the chewing side (but not the pellet).</li>
			<li>Ask the subject to perform a rhythmic squeezing of an anti-stress ball (handgrip exercise) for 2 min at the rate and on the side of their choosing. After 1 min, ask the subject to switch hands.</li>
			<li>Ask the subject to relax (no activity) for 2 min.</li>
		</ol>
		</li>
		<li>Just after the end of each step (steps 3.2.3.1&#8211;3.2.3.4), evaluate pupil size at rest and both performance and pupil size in the matrices test (T7).</li>
		<li>Thirty minutes following the end of each step (steps 3.2.3.1&#8211;3.2.3.4), evaluate pupil size at rest and both performance and pupil size in the matrices test (T37).</li>
	</ol>
	</li>
</ol>
4. Measured Variables in Protocols 1 and 2
<ol>
	<li>Cognitive performance 
	NOTE: In both protocols 1 and 2, measure cognitive performance using a test based on a modified version of the Spinnler-Tognoni numeric matrices test29.
	<ol>
		<li>Display three numerical matrices (10 x 10) printed on paper to the subject. Then, ask the subject to sequentially scan the matrix lines, while ticking with a pencil as many of the target numbers as possible (60 targets out of 300 total displayed numbers) indicated above each matrix (Figure 1) within 15 s.</li>
		<li>Utilize matrices with different positions of target numbers at T0, T7, and T37 to avoid the introduction of confounders related to learning processes.</li>
		<li>Evaluate offline the performance index (PI), scanning rate (SR), and error rate (ER) as follows: PI = (target numbers underlined in 15 s)/15; SR = (target + non-target numbers scanned in 15 s)/15; ER = (target numbers missed + non-target numbers underlined in 15 s)/15.</li>
	</ol>
	</li>
	<li>Pupil size in protocol 1
	<ol>
		<li>Prepare the subject for the pupil size measurement with a corneal topographer-pupillographer (Table of Materials), which prevents vision of the environment, using one of the following two acquisition procedures.
		<ol>
			<li>Record a single camera shot of the pupil (Figure 2A,B) with a constant illumination level of 40 lux, pressing the specific button on the corneal topographer-pupillographer. Maintain an optimal working distance of 56 mm between the camera and pupil. 
			NOTE: A single measurement is sufficient, due to the low level of variability of pupil size measured at constant illumination.</li>
			<li>Perform a continuous recording of the pupil (sampling rate = 5 Hz; Figure 2C,D) in the continuous acquisition modality. Discard the first 20&#8211;50 measurements (4&#8211;10 s), since during this lapse of time, the pupil diameter is growing (the acquisition starts by switching off pupil illumination at 40 lux). Average the remaining measurements.</li>
		</ol>
		</li>
		<li>Record pupil size of the left and right eyes separately at rest (steps 3.1.2, 3.1.5, and 3.1.6).</li>
		<li>Record pupil size during the haptic task (steps 3.1.3, 3.1.5 and 3.1.6; left and right separately). When using the single shot modality (step 4.2.1.1), acquire the photo during the second of two task repetitions, at the beginning of puzzle surface exploration. In the continuous mode of recording (step 4.2.1.2), start the acquisition when the piece of the puzzle has been placed in the hand of the subject.</li>
		<li>Evaluate offline left and right pupil size at rest and during the haptic task by direct acquisition of the values (in mm) displayed by the software. Calculate the task-related mydriasis by subtracting the pupil size at rest from pupil size during the haptic task, and obtain all average left-right values.</li>
	</ol>
	</li>
	<li>Pupil size in protocol 2
	<ol>
		<li>Prepare the subject for the pupil size measurement using a wearable pupillometer/eye tracker (Figure 3A), endowed with a 3D-printed glass frame structure, using the following procedure.
		<ol>
			<li>Have the subject wear the wearable pupillometer. Adjust the position of the two infrared cameras (Figure 3A-2,3) mounted on bars stemming from the frame (Table of Materials), so that the eyes are within the field of view of the cameras and in focus.</li>
			<li>Acquire images of the pupils (sampling rate = 120 Hz), which are processed online by the software supplied with the wearable pupilometer and provides pupillary diameter (in mm) using a geometrical model of the &#34;average&#34; human ocular. Disregard blink artifacts.</li>
			<li>Record continuously the environmental illumination level using a calibrated logarithmic light sensor mounted on the wearable pupillometer frame. Use a frontal RGB camera mounted on the wearable pupillometer (Figure 3A-1) to record the subject field of view (sampling rate = 30 Hz) useful to study gaze behaviour.</li>
		</ol>
		</li>
		<li>Record simultaneously the size of the two pupils at rest for 20 s (Figure 3B).</li>
		<li>Record the size of the pupils while the subject performs the Spinnler-Tognoni test, so to have pupil size and cognitive performance simultaneously recorded (steps 3.2.2, 3.2.4, and 3.2.5).</li>
		<li>Evaluate offline left and right pupil size at rest and during the Spinnler-Tognoni test, by averaging acquired values (n = 2,400) for each pupil. Calculate the task-related mydriasis by subtracting the pupil size at rest from the pupil size during the matrices test, then all the average left-right values.</li>
	</ol>
	</li>
	<li>Gaze position 
	NOTE: Reconstruct online the fixation point using the images of the two pupils obtained from section 4.3. Process the acquired frames in real-time and estimate the gaze fixation point using a previously calculated transfer function31 specific for each subject wearing the eye tracker.
	<ol>
		<li>If necessary, when performing protocol 2, reconstruct gaze position from the pupil images. To do this, add four computer detectable vision markers (ArUco or AprilTag libraries of the instrument software) to four corners of the matrices sheet used in section 4.1.</li>
		<li>Allow the calibration system (embedded in the eye tracker software as for the pupil headset used) to acquire the data, and evaluate the parameters of the transfer function that map the fixation point, starting from images of the two pupils. As an example, ask the subject to gaze at a predefined sequence of points that are shown in his/her field of view (i.e., the four corners of the matrices sheet and at the center of the sheet itself), which are recorded simultaneously by the additional RGB camera mounted on the frame and facing the field of view.</li>
		<li>Record pupil size during the matrices test.</li>
		<li>Calculate offline gaze position that appears as a mark on every frame of the subject&#39;s field of view. Use the four markers to track gaze position over the matrices across frames.</li>
	</ol>
	</li>
</ol>
5. Statistical Analysis
<ol>
	<li>Analyze pupil size at rest and during the task, task-induced mydriasis, PI, SR, and ER under four conditions (no activity, handgrip, soft pellet, hard pellet) for three times (T0, T7, T37) using repeated measures ANOVA and statistics software package.</li>
	<li>Analyze changes in variables with respect to baseline values (T0) under four conditions, (no activity, handgrip, soft pellet, hard pellet) for two times (T7, T37) using repeated measures ANOVA.</li>
	<li>When running ANOVA, if the software indicates that the data distribution is not spherical, take the p-value corresponding to the Greenhouse-Geisser &#949; correction from the outputted statistic table.</li>
	<li>Correlate the changes in performance (PI, SR, ER) at T7 and T37 with those observed in task-related mydriasis by linear regression analysis.</li>
</ol>

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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=Short-Term+Effects+of+Chewing+on+Task+Performance+and+Task-Induced+Mydriasis:+Trigeminal+Influence+on+the+Arousal+Systems.">Short-Term Effects of Chewing on Task Performance and Task-Induced Mydriasis: Trigeminal Influence on the Arousal Systems.</a> Frontiers in Neuroanatomy. 11, 68 (2017).</li><li>Kassner, M., Patera, W., Bulling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pupil:+An+Open+Source+Platform+for+Pervasive+Eye+Tracking+and+Mobile+Gaze-based+Interaction.">Pupil: An Open Source Platform for Pervasive Eye Tracking and Mobile Gaze-based Interaction.</a> arXiv.org. , (2014).</li><li>Gatz, M., 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=Potentially+modifiable+risk+factors+for+dementia+in+identical+twins.">Potentially modifiable risk factors for dementia in identical twins.</a> Alzheimer's &amp; Dementia: The Journal of the Alzheimer's Association. 2 (2), 110-117 (2006).</li><li>Okamoto, N., 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=Relationship+of+tooth+loss+to+mild+memory+impairment+and+cognitive+impairment:+findings+from+the+Fujiwara-kyo+study.">Relationship of tooth loss to mild memory impairment and cognitive impairment: findings from the Fujiwara-kyo study.</a> Behavioral and Brain Functions. 6, 77 (2010).</li><li>Weijenberg, R. A. F., Lobbezoo, F., Knol, D. L., Tomassen, J., Scherder, E. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+masticatory+activity+and+quality+of+life+in+elderly+persons+with+dementia--a+longitudinal+matched+cluster+randomized+single-blind+multicenter+intervention+study.">Increased masticatory activity and quality of life in elderly persons with dementia--a longitudinal matched cluster randomized single-blind multicenter intervention study.</a> BMC Neurology. 13, 26 (2013).</li><li>Kato, T., 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=The+effect+of+the+loss+of+molar+teeth+on+spatial+memory+and+acetylcholine+release+from+the+parietal+cortex+in+aged+rats.">The effect of the loss of molar teeth on spatial memory and acetylcholine release from the parietal cortex in aged rats.</a> Behavioural Brain Research. 83 (1-2), 239-242 (1997).</li><li>Onozuka, M., 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=Impairment+of+spatial+memory+and+changes+in+astroglial+responsiveness+following+loss+of+molar+teeth+in+aged+SAMP8+mice.">Impairment of spatial memory and changes in astroglial responsiveness following loss of molar teeth in aged SAMP8 mice.</a> Behavioural Brain Research. 108 (2), 145-155 (2000).</li><li>Watanabe, 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=The+molarless+condition+in+aged+SAMP8+mice+attenuates+hippocampal+Fos+induction+linked+to+water+maze+performance.">The molarless condition in aged SAMP8 mice attenuates hippocampal Fos induction linked to water maze performance.</a> Behavioural Brain Research. 128 (1), 19-25 (2002).</li><li>Kubo, K. Y., Iwaku, F., Watanabe, K., Fujita, M., Onozuka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molarless-induced+changes+of+spines+in+hippocampal+region+of+SAMP8+mice.">Molarless-induced changes of spines in hippocampal region of SAMP8 mice.</a> Brain Research. 1057 (1-2), 191-195 (2005).</li><li>Oue, H., 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=Tooth+loss+induces+memory+impairment+and+neuronal+cell+loss+in+APP+transgenic+mice.">Tooth loss induces memory impairment and neuronal cell loss in APP transgenic mice.</a> Behavioural Brain Research. 252, 318-325 (2013).</li><li>Mather, M., Harley, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Locus+Coeruleus:+Essential+for+Maintaining+Cognitive+Function+and+the+Aging+Brain.">The Locus Coeruleus: Essential for Maintaining Cognitive Function and the Aging Brain.</a> Trends in Cognitive Sciences. 20 (3), 214-226 (2016).</li></ol>

The authors have nothing to disclose.

The protocols presented in this study address the acute effects of sensorimotor trigeminal activity on cognitive performance and the role of the LC in this process. This topic has some relevance, considering that 1) during aging, the deterioration of masticatory activity correlates with cognitive decay32,33,34; people that preserve oral health are less prone to neurodegenerative phenomena; 2) malocclusion and teeth extraction in...

In humans, it is known that chewing quickens cognitive processing1,2 and improves arousal3,4, attention5, learning, and memory6,7. These effects are associated with shortening of the latencies of cortical event-related potentials8 and an increase in the perfusion of several cortical and subcortical structures2,9.
Within cranial nerves, the most relevant information sustaining cortical desynchronization and arousal is carried by trigeminal fibres10, likely due to strong trigeminal connections to the ascending reticular activating system (ARAS)11. Among ARAS structures, the locus coeruleus (LC) receives trigeminal inputs11 and modulates arousal12,13, and its activity covaries with pupil size14,15,16,17,18. Although the relation between LC resting activity and cognitive performance is complex, task-related enhancement of LC activity leads to arousal-associated19 pupil mydriasis20 and enhanced cognitive performance21. There is reliable covariation between LC activity and pupil size, and the latter is currently considered a proxy of central noradrenergic activity22,23,24,25,26.
Asymmetric activation of sensorimotor trigeminal branches induces pupil asymmetries (anisocoria)27,28, confirming the strength of the trigemino-coerulear connection. If the LC participates in the stimulating effects of chewing on cognitive performance, it may affect parallel task-related mydriasis, which is an indicator of LC phasic activation during a task. It may also affect performance, so a correlation can be expected between chewing-induced changes in performance and mydriasis. Moreover, if trigeminal effects are specific, chewing effects should be larger than those elicited by another rhythmic motor task. In order to test these hypotheses, two experimental protocols are hereby presented. They are based on combined measurements of cognitive performance and pupil size, performed before and after a short period of chewing activity. These protocols utilize a test consisting in finding target numbers displayed in numeric attentive matrices29, alongside with non-target numbers. This test verifies attentive and cognitive performance.
The overall goal of these protocols is to illustrate that trigeminal stimulation elicits specific changes in cognitive performance, which cannot be ascribed aspecifically to the generation of motor commands and are related to pupil-linked changes in LC-mediated arousal. Applications of the protocols extend to all behavioral conditions in which performance can be measured and involvement of the LC is suspected.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-stress ball</td>
			<td>Artengo, Decathlon, France</td>
			<td>TB600</td>
			<td></td>
		</tr>
		<tr>
			<td>Chewing gum</td>
			<td>Vigorsol, Perfetti, Italy</td>
			<td>Commercially available product</td>
			<td></td>
		</tr>
		<tr>
			<td>Infrared Camera-Wearable pupillometer</td>
			<td>Pupil Labs, Berlin, Germany</td>
			<td>Pupil Labs headset</td>
			<td></td>
		</tr>
		<tr>
			<td>Pupillographer</td>
			<td>CSO, Florence, Italy</td>
			<td>MOD i02, with chin support</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon rubber</td>
			<td>Prochima, Italy</td>
			<td>gls50</td>
			<td></td>
		</tr>
		<tr>
			<td>Software for pupil detection - wearable pupillometer</td>
			<td>Pupil Labs, Berlin, Germany</td>
			<td>Pupil Labs headset</td>
			<td></td>
		</tr>
		<tr>
			<td>Tangram Puzzle</td>
			<td>Citt&#224; del Sole srl, Milano, Italy</td>
			<td>Tangram Puzzle</td>
			<td></td>
		</tr>
		<tr>
			<td>Wearable pupillometer</td>
			<td>Pupil Labs, Berlin, Germany</td>
			<td>Pupil labs model</td>
			<td>Dimension of the frame: 13.5 x 15.5cm</td>
		</tr>
	</tbody>
</table>

assessing pupil-linked changes in locus coeruleus-mediated arousal elicited by trigeminal stimulation

Current scientific literature provides evidence that trigeminal sensorimotor activity associated with chewing may affect arousal, attention, and cognitive performance. These effects may be due to widespread connections of the trigeminal system to the ascending reticular activating system (ARAS), to which noradrenergic neurons of the locus coeruleus (LC) belongs. LC neurons contain projections to the whole brain, and it is known that their discharge co-varies with pupil size. LC activation is necessary for eliciting task-related mydriasis. If chewing effects on cognitive performance are mediated by the LC, it is reasonable to expect that changes in cognitive performance are correlated to changes in task-related mydriasis. Two novel protocols are presented here to verify this hypothesis and document that chewing effects are not attributable to aspecific motor activation. In both protocols, performance and pupil size changes observed during specific tasks are recorded before, soon after, and half an hour following a 2 min period of either: a) no activity, b) rhythmic, bilateral handgrip, c) bilateral chewing of soft pellet, and d) bilateral chewing of hard pellet. The first protocol measures level of performance in spotting target numbers displayed within numeric matrices. Since pupil size recordings are recorded by an appropriate pupillometer that impedes vision to ensure constant illumination levels, task-related mydriasis is evaluated during a haptic task. Results from this protocol reveal that 1) chewing-induced changes in performance and task-related mydriasis are correlated and 2) neither performance nor mydriasis are enhanced by handgrip. In the second protocol, use of a wearable pupillometer allows measurement of pupil size changes and performance during the same task, thus allowing even stronger evidence to be obtained regarding LC involvement in the trigeminal effects on cognitive activity. Both protocols have been run in the historical office of Prof. Giuseppe Moruzzi, the discoverer of ARAS, at the University of Pisa.

Figure 4 shows a representative example of the results obtained when protocol 1 was applied to a single subject (46 years old, female). PI was increased soon after having chewed (T7) both a hard (from 1.73 numb/s to 2.27 numb/s) and soft pellet (from 1.67 numb/s to 1.87 numb/s) (Figure 4A). However, 30 min later (T37), the increased performance persisted only for the hard pellet. On the other hand, both a lack of activity and the handgrip exerci...

Watch this Scientific Journal Video about Assessing Pupil-linked Changes in Locus Coeruleus-mediated Arousal Elicited by Trigeminal Stimulation at JoVE.com

Assessing Pupil-linked Changes in Locus Coeruleus-mediated Arousal Elicited by Trigeminal Stimulation

To verify whether trigeminal effects on cognitive performance involve locus coeruleus activity, two protocols are presented that aim to evaluate possible correlations between the performance and task-related pupil size changes induced by chewing. These protocols may be applied to conditions in which locus coeruleus contribution is suspected.

Current scientific literature provides evidence that trigeminal sensorimotor activity associated with chewing may affect arousal, attention, and cognitive ...

assessing-pupil-linked-changes-locus-coeruleus-mediated-arousal

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7.9K Views.  University of Pisa. To verify whether trigeminal effects on cognitive performance involve locus coeruleus activity, two protocols are presented that aim to evaluate possible correlations between the performance and task-related pupil size changes induced by chewing. These protocols may be applied to conditions in which locus coeruleus contribution is suspected.

Video: Assessing Pupil-linked Changes in Locus Coeruleus-mediated Arousal Elicited by Trigeminal Stimulation

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

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 ...

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 ...

Generation of Local CA1 &#947; Oscillations by Tetanic Stimulation

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A protocol is provided to generate a model for studying CA1 &#947; oscillations (20 - 80 Hz). These &#947; oscillations are stable for at least 30 min&#160;and depend upon excitatory and inhibitory synaptic activity in addition to activation of pacemaker currents. Tetanically stimulated oscillations have a number of reproducible and easily quantifiable characteristics including spike count, oscillation duration, latency and frequency that report upon the network state. The advantages of the electrically stimulated oscillations include stability, reproducibility and episodic acquisition enabling robust characterization of network function. This model of CA1 &#947; oscillations can be used to study cellular mechanisms and to systematically investigate how neuronal network activity is altered in disease and by drugs. Disease state pharmacology can be readily incorporated by the use of brain slices from genetically modified or interventional animal models to enable selection of drugs that specifically target disease mechanisms.

Generation of Local CA1 &amp;#947; Oscillations by Tetanic Stimulation

Oscillations are fundamental network properties and are modulated by disease and drugs. Studying brain-slice oscillations allows characterization of isolated networks under controlled conditions. Protocols are provided for the preparation of acute brain slices for evoking CA1 &#947; oscillations.

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A ...

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when compared to warm-blooded vertebrates. Because of this, in vitro experiments in turtles can be performed for extended periods of time to investigate the neural signals and control of their target actions. Using an isolated head preparation, we measured the kinematics of eye movements in turtles, and their modulation by electrical signals carried by cranial nerves. After the brain was removed from the skull, leaving the cranial nerves intact, the dissected head was placed in a gimbal to calibrate eye movements. Glass electrodes were attached to cranial nerves (oculomotor, trochlear, and abducens) and stimulated with currents to evoke eye movements. We monitored eye movements with an infrared video tracking system and quantified rotations of the eyes. Current pulses with a range of amplitudes, frequencies, and train durations were used to observe effects on responses. Because the preparation is separated from the brain, the efferent pathway going to muscle targets can be examined in isolation to investigate neural signaling in the absence of centrally processed sensory information.

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

This protocol describes how to use an in vitro isolated turtle head preparation to measure the kinematics of their eye movements. After removal of the brain from the cranium, cranial nerves can be stimulated with currents to quantify rotations of the eye and changes in pupil sizes.

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when ...

Localization of the Locus Coeruleus in the Mouse Brain

The locus coeruleus (LC) is a major hub of norepinephrine producing neurons that modulate a number of physiological functions. Structural or functional abnormalities of LC impact several brain regions including cortex, hippocampus, and cerebellum and may contribute to depression, bipolar disorder, anxiety, as well as Parkinson disease and Alzheimer disease. These disorders are often associated with metal misbalance, but the role of metals in LC is only partially understood. Morphologic and functional studies of LC are needed to better understand the human pathologies and contribution of metals. Mice are a widely used experimental model, but the mouse LC is small (~0.3 mm diameter) and hard to identify for a non-expert. Here, we describe a step-by-step immunohistochemistry-based protocol to localize the LC in the mouse brain. Dopamine-&#946;-hydroxylase (DBH), and alternatively, tyrosine hydroxylase (TH), both enzymes highly expressed in the LC, are used as immunohistochemical markers in brain slices. Sections adjacent to LC-containing sections can be used for further analysis, including histology for morphological studies, metabolic testing, as well as metal imaging by X-ray fluorescence microscopy (XFM).

The locus coeruleus is a small cluster of neurons involved in a variety of physiological processes. Here, we describe a protocol to prepare mouse brain sections for studies of proteins and metals in this nucleus.

The locus coeruleus (LC) is a major hub of norepinephrine producing neurons that modulate a number of physiological functions. Structural or functional ...

Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided here is a protocol for successfully establishing a rat model of acute pontine infarction. Rats weighing about 250 g are used, and a probe with an insulated sheath is injected into the pons using a stereotaxic apparatus. A lesion is produced by the electrical stimulation with a single pulse. The Longa score, Berderson score, and beam balance test are used to assess neurological deficits. Additionally, the adhesive-removal somatosensory test is used to determine sensorimotor function, and the limb placement test is used to evaluate proprioception. MRI scans are then used to assess the infarction in vivo, and TTC staining is used to confirm the infarction in vitro. Here, a successful infarction is identified that is located in the anterolateral basis of the rostral pons. In conclusion, a new method is described to establish an acute pontine infarction rat model.

Presented here is a protocol for establishing acute pontine infarction in a rat model via electrical stimulation with a single pulse.

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided ...

In vivo Positron Emission Tomography to Reveal Activity Patterns Induced by Deep Brain Stimulation in Rats

Deep brain stimulation (DBS) is an invasive neurosurgical technique based on the application of electrical pulses to brain structures involved in the patient&#39;s pathophysiology. Despite the long history of DBS, its mechanism of action and appropriate protocols remain unclear, highlighting the need for research aiming to solve these enigmas. In this sense, evaluating the in vivo effects of DBS using functional imaging techniques represents a powerful strategy to determine the impact of stimulation on brain dynamics. Here, an experimental protocol for preclinical models (Wistar rats), combined with a longitudinal study [18F]-fluorodeoxyclucose positron emission tomography (FDG-PET), to assess the acute consequences of DBS on brain metabolism is described. First, animals underwent stereotactic surgery for bilateral implantation of electrodes into the prefrontal cortex. A post-surgical computerized tomography (CT) scan of each animal was acquired to verify electrode placement. After one week of recovery, a first static FDG-PET of each operated animal without stimulation (D1) was acquired, and two days later (D2), a second FDG-PET was acquired while animals were stimulated. For that, the electrodes were connected to an isolated stimulator after administering FDG to the animals. Thus, animals were stimulated during the FDG uptake period (45 min), recording the acute effects of DBS on brain metabolism. Given the exploratory nature of this study, FDG-PET images were analyzed by a voxel-wise approach based on a paired T-test between D1 and D2 studies. Overall, the combination of DBS and imaging studies allows describing the neuromodulation consequences on neural networks, ultimately helping to unravel the conundrums surrounding DBS.

In vivo Positron Emission Tomography to Reveal Activity Patterns Induced by Deep Brain Stimulation in Rats

We describe a preclinical experimental method to evaluate metabolic neuromodulation induced by acute deep brain stimulation with in vivo FDG-PET. This manuscript includes all experimental steps, from stereotaxic surgery to the application of the stimulation treatment and the acquisition, processing, and analysis of PET images.

Deep brain stimulation (DBS) is an invasive neurosurgical technique based on the application of electrical pulses to brain structures involved in the ...

Assessing Changes in Synaptic Plasticity Using an Awake Closed-Head Injury Model of Mild Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a prevalent health issue in North America. There is increasing pressure to utilize ecologically valid models of closed-head mTBI in the preclinical setting to increase translatability to the clinical population. The awake closed-headed injury (ACHI) model uses a modified controlled cortical impactor to deliver closed-headed injury, inducing clinically relevant behavioral deficits without the need for a craniotomy or the use of an anesthetic.
This technique does not normally induce fatalities, skull fractures, or brain bleeds, and is more consistent with being a mild injury. Indeed, the mild nature of the ACHI procedure makes it ideal for studies investigating repetitive mTBI (r-mTBI). Growing evidence indicates that r-mTBI can result in a cumulative injury that produces behavioral symptoms, neuropathological changes, and neurodegeneration. r-mTBI is common in youths playing sports, and these injuries occur during a period of robust synaptic reorganization and myelination, making the younger population particularly vulnerable to the long-term influences of r-mTBI.
Further, r-mTBI occurs in cases of intimate partner violence, a condition for which there are few objective screening measures. In these experiments, synaptic function was assessed in the hippocampus in juvenile rats that had experienced r-mTBI using the ACHI model. Following the injuries, a tissue slicer was utilized to make hippocampal slices to evaluate bidirectional synaptic plasticity in the hippocampus at either 1 or 7 days following the r-mTBI. Overall, the ACHI model provides researchers with an ecologically valid model to study changes in synaptic plasticity following mTBI and r-mTBI.

Here, it is demonstrated how an awake closed-head injury model can be used for examining the effects of repeated mild traumatic brain injury (r-mTBI) on synaptic plasticity in the hippocampus. The model replicates important features of r-mTBI in patients and is used in conjunction with in vitro electrophysiology.

Mild traumatic brain injuries (mTBIs) are a prevalent health issue in North America. There is increasing pressure to utilize ecologically valid models of ...