Name: assessing pupil-linked changes in locus coeruleus-mediated arousal elicited by trigeminal stimulation
Uploaded: 2019-11-26T12:30:00
Description: Watch this Scientific Journal Video about Assessing Pupil-linked Changes in Locus Coeruleus-mediated Arousal Elicited by Trigeminal Stimulation at JoVE.com

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>
	<...

<ol>
	<li>Hirano, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+chewing+on+cognitive+processing+speed.">Effects of chewing on cognitive processing speed.</a> Brain and Cognition. 81 (3), 376-381 (2013).</li><li>Hirano, Y., 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=Chewing+and+cognitive+function.">Chewing and cognitive function.</a> Brain and Nerve. 66 (1), 25-32 (2014).</li><li>Allen, A. P., Smith, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+chewing+gum+and+time-on-task+on+alertness+and+attention.">Effects of chewing gum and time-on-task on alertness and attention.</a> Nutritional Neuroscience. 15 (4), 176-185 (2012).</li><li>Johnson, A. 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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...

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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 ...

The locus coeruleus is a deep brain structure that regulates behavioral arousal, attention, and cognitive performance. Its activity can be recorded indirectly by measuring the pupil size. This approach allows the verification of where the stimuli that boost cognitive performance, such as the gemina stimulation, modulate LC activity as the diameter changes in the pupil size.Experimental evidence suggests that neurodegenerative diseases are associated with locus coeruleus dysfunction. The present approach will pave the way for the modulation of the geminal input to the locus coeruleus for therapeutic purposes. Demonstrating the procedure with Tommaso Banfi will be Vincenzo De Cicco, a professional physician and odontologist.To perform the experiment, a commercially available piece of chewing gum, referred to as a soft pellet, and a silicon rubber pellet, referred to as the hard pellet, are needed. A tangram puzzle must also be prepared for the haptic task. For protocol one, provide a paper displaying three 10 by 10 numerical matrices to the subject, and ask the subject to sequentially scan the matrix lines while using a pencil to tick as many target numbers indicated above each matrix within 15 seconds to measure the subject's baseline cognitive performance.Then manually evaluate the performance index, scanning rate, and error rate. To measure the baseline pupil size, seat the subject at an optimal working distance of 56 millimeters from a corneal topographer pupillographer, and acquire single, separate camera shots of the left and right pupils under a constant illumination of 40 lux. To evaluate the pupil size during a tangram haptic task, place one of the pieces from the puzzle into the subject's hand, and ask the subject to return the piece to the tangram while recording the subject's pupil size.To measure the pupil size during the haptic task, acquire the photos while the subject performs the second of two task repetitions at the beginning of the puzzle surface exploration. Evaluating the left and right pupil size by direct acquisition of the values displayed by the software in millimeters. Then subtract the pupil size at rest from the pupil size during the haptic task to calculate the task-related mydriasis and obtain the average value for both the right and left pupils.Next, ask the subject to chew a self-administered soft pellet, letting the subject spontaneously choose both the rate of chewing and the preferred mouth-chewing side. After one minute, provide the subject with a new soft pellet, and ask the subject to switch the chewing side for one more minute of chewing. Immediately after, end the chewing exercise, evaluate the subject's performance in the matrices test as demonstrated while measuring the pupil size both at rest and during the haptic task.30 minutes following the end of the chewing exercise, evaluate the subject performance and pupil size, both at rest and during the haptic task. For protocol two, equip the subject with the wearable pupillometer eye tracker endowed with a 3D-printed glass frame structure, and adjust the position of the two infrared cameras mounted on the frame so that the subject's eyes are in focus with the field of view of the cameras. Using a calibrated logarithmic light sensor mounted on the wearable pupillometer frame to continuously and simultaneously record the environmental illumination level, acquire images of the subject's pupils at rest at a 120 Hertz sampling rate within the wearable pupillometer software for 20 seconds.To obtain the baseline cognitive performance data, record the size of the pupils while the subject performs the Spinnler-Tognoni test. Manually elevate the left and right pupil sizes at rest and during the Spinnler-Tognoni test by averaging the acquired values for each pupil. Then calculate the task-related mydriasis as demonstrated to obtain the average values for both the left and right pupils.Next, ask the subject to chew a self-administered soft pellet, letting the subject spontaneously choose both the rate of chewing and the mouth-chewing side. After one minute, provide the subject with a new soft pellet, and ask the subject to switch the chewing side for an additional minute of chewing. Immediately at the end of the chewing exercise, evaluate the pupil size at rest in both the performance and the pupil size during the matrices test.30 minutes following the end of the chewing exercise, evaluate both the pupil size at rest and both the performance pupil size during the matrices test. In this representative example, the performance index was increased soon after having chewed either a hard or a soft pellet. However, after 30 minutes, the increased performance persisted only for the hard pellet.Two control conditions, such as a lack of activity and the handgrip exercise, had a negative effect on performance, with a tendency to recover after 30 minutes. Qualitatively similar changes were observed for the task-related mydriasis in the same subject displayed in the previous plot. In the continuous acquisition mode of the instrument, final individual samples are representative of the final average value since the pupil reaches a stable size within five seconds from when the light is off.In single subjects, a strong correlation was observed between the performance and the task-related mydriasis after chewing hard and soft pellets. A correlation is also evident when the corresponding changes are normalized for the baseline values. Even stronger evidence of LC involvement in the stimulating effects of chewing on cognitive performance can be obtained by correlating the chewing-induced changes in the performance index with the change in mydriasis observed during the execution of the same matrices test.Using the procedure, it is very important to carefully control environmental lighting as the external light can be a major determinant to the pupil's size, confounding the results. This procedure might find several applications, for example, we are currently studying whether the correction of an occlusal assymetry may boost performance by affecting locus coeruleus activity.

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