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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Neuroscience

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 ...

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

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.

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 ...