Name: real-time video projection in an mri for characterization of neural correlates associated with mirror therapy for phantom limb pain
Uploaded: 2019-04-20T13:00:00
Description: Watch this Scientific Journal Video about Real-time Video Projection in an MRI for Characterization of Neural Correlates Associated with Mirror Therapy for Phantom Limb Pain at JoVE.com

This study was supported by an NIH RO1 grant (1R01HD082302).

1. Preparation of the subject
<ol>
	<li>Prior to participation, have the participant complete a consent form and an MRI safety screening evaluation, the latter carried out by the neuroimaging technician at the scanning facility, to ensure that the participant does not have any known contraindications to being scanned (e.g., metal in their body, a history of claustrophobia, or pregnancy).</li>
	<li>Provide the participant with detailed instructions with regard to the experimental procedure.</li>
	<li>Have the subject li...

This protocol describes a novel, feasible procedure that allows investigators to accurately characterize the neural correlates associated with MT in individuals with PLP.
As previously mentioned, past studies have attempted to investigate the neural correlates associated with MT treatment by incorporating various techniques such as video recording, virtual reality, and prerecorded animations9,33,34. H...

PLP refers to the sensation of pain perceived within the area corresponding to the missing limb postamputation1,2. This condition is a significant chronic health care burden and can have a dramatic impact on an individual&#39;s quality of life3,4. It has been suggested that alterations in the brain structure and function play a fundamental role in the development and neuropathophysiology of PLP5,6. However, the underlying neural correlates of how pain symptoms develop and how they can be alleviated in response to treatment remain unknown. This lack of information is mostly due to technical challenges and limitations associated with performing a given therapeutic approach within the constraints of a neuroimaging environment such as MRI5,7,8.
Results from a number of studies attribute the development of PLP to maladaptive neuroplastic reorganization occurring within sensorimotor cortices, as well as in other areas of the brain. For example, it has been shown that following the amputation of a limb, there is a shift in the corresponding sensorimotor cortical representation of neighboring areas. As a result, neighboring areas apparently start invading the zones that used to correspond to the amputated limb9,10. In order to alleviate pain symptoms associated with PLP, treatments such as MT or motor imagery may be effective9,11,12. It is suggested that the alleviation of symptoms occurs putatively through the cross-modal re-establishment of afferent inputs, provided by the observation of mirror-reflected images from the nonaffected limb12,13,14,15,16,17. Through these images, participants are able to visualize the reflection of the opposite limb instead of the one that has been amputated, thus creating an illusion that both limbs remain. The illusion and immersive effects were previously studied by Diers et al. in healthy subjects in which a comparison of functional activation through functional MRI (fMRI) was evaluated after undergoing a task either with a common mirror box or virtual reality18. However, the neural correlates associated with the reversal of the maladaptive neuroplastic changes and the alleviation of symptoms remain poorly understood. Additionally, the underlying mechanism of PLP remains a topic of research as the clear underlying physiopathologic alteration behind the development of PLP is still incompletely elucidated while controversial findings have been revealed5,19. As stated above, multiple authors attribute the development of pain to deafferentation and cortical reorganization of the affected brain area (area of the amputated limb)6,7,8; however, opposite results were described by Makin and collaborators in which the presence of pain is associated with the preservation of brain structure and pain is attributed to a reduction interregional functional connectivity19. In view of these controversial and opposite findings, we believe that the novel approach presented here will bring additional relevant information to the study of PLP and will allow scientists to evaluate the effects of MT in a live environment with the degree of brain activation while comparing them with the levels of pain assessed in our full protocol19.
Previous literature on this topic has shown that MT is one of the most appropriate behavioral therapies for the treatment of PLP due to its easy implementation and low costs12. In fact, previous studies of this technique have shown evidence of a reversal of maladaptive changes within the primary sensorimotor cortex in amputees with PLP8,20,21. Even though MT is perhaps one the most inexpensive and most effective approach to treat PLP12,22,23,24, more studies are needed to confirm these effects since some patients do not respond to this type of treatment8 and there is a lack of larger randomized clinical trials that provide high-evidence-based results25.
One of the hypotheses by which MT can reduce PLP is related to the fact that the mirror image of the not-amputated body part helps to reorganize and integrate the mismatch between proprioception and visual feedback26. The underlying mechanisms of MT could be associated with the reversion of the maladaptive mapping of somatosensory8,27,28.
For MT, subjects are required to perform several motor and sensory tasks using their intact limb (e.g., flexion and extension) while observing this effect in a mirror located in the midline of the participant&#39;s body, thereby creating a vivid and precise representation of movement within the area of the amputated limb29.
To further develop the scientific understanding of the pathophysiology aspects involved in PLP, it is crucial to better characterize the underlying neuroplastic changes that result from limb amputations, as well as the improvement of pain symptoms provided by MT. In this regard, neuroimaging techniques, such as fMRI, have emerged as powerful tools to help elucidate the pathophysiologic mechanisms associated with cortical reorganization and provide clues toward optimizing the rehabilitation of individuals with PLP in the clinical context30,31. Furthermore, the high spatial resolution afforded by fMRI (as compared to electroencephalography, for example) allows for more accurate mapping of brain responses, such as finger and digit representations, in the sensorimotor cortex along with other regions of the brain32.
To date, the neurophysiology associated with MT remains elusive due in large part to the challenges of carrying out the procedure within the scanner environment (i.e., it is difficult for an individual to perform the therapy while lying in the scanner). Here, we describe a method that allows for an individual to observe their own leg movement in real-time while lying supine within the narrow confines of the scanner bore. An accurate recreation of the vivid and immersive sensation elicited by the therapy can be recreated using a video camera that captures real-time images of the moving leg, and a system of mirrors and a monitor that can be viewed directly by the study participant.
Past studies have attempted to incorporate techniques such as video recording, virtual reality, and prerecorded animations as means to present the visual stimulus and circumvent these technical challenges9,16,33,34. Yet, these techniques have been limited in their effectiveness35,36,37,38,39. In the particular case of using a prerecorded video, there is an often poor synchronization between participants&#39; movements and the ones provided by the video, as well as a lack of timing accuracy, which leads to a poor realistic impression that the individual&#39;s own leg is moving. In order to improve this sense of sensorimotor immersion, other techniques, such as virtual reality and digitized animations, have been attempted. Yet, they have failed to generate visually convincing sensations due to a low image resolution, a limited field of view, unrealistic or nonnatural human-like motions, and presence of motion lag (i.e., desynchronization of movement). Additionally, the lack of an accurate modeling combined with the poor control over other features, such as the effects of friction, momentum, and gravity, hinders the perception of a vivid and immersive feel40. Therefore, for amputees, it is worth to explore strategies to ensure that subjects are engaged in the cognitive task (observation) and immersive on the illusion of amputated limb movement. Finally, the required resources for developing and implementing these complex strategies may be time-consuming and/or cost prohibitive.
We describe a new approach that we believe creates a realistic and vivid sense of immersion whereby the participant can see a live and real-time video of a projected image of their own limb while they perform a session of the MT31. This approach is performed while the individual is lying in the scanner bore and is without substantial costs or extensive technical development.
This protocol is part of a National Institutes of Health (NIH) Research Project Grant (RO1)-sponsored clinical trial that evaluates the effects of the combination of a neuromodulatory technique, namely transcranial direct current stimulation (tDCS), with a behavioral therapy (mirror therapy) in order to relieve phantom limb pain31. We evaluate changes in the visual analog scale (VAS) for pain at baseline, prior, and after each intervention session. fMRI is used as a neurophysiologic tool in order to evaluate structural changes in brain function and its correlation with the relief of PLP. Therefore, an initial fMRI is obtained in order to have a baseline map of the structural organization of the participant&#39;s brain, which will either show that there is cortical maladaptive reorganization5,6,8,11,13,14,18,28 or that there is not19; in the same way, the scientist can observe what areas are activated at baseline with the task of MT in order to understand the areas&#39; activation response to the MT; lastly, it is possible to obtain a second fMRI postintervention to see if changes (modulation) have been generated in the cortical reorganization after the combined therapy with tDCS and MT and to analyze if those changes are correlated or associated with the degree of pain change. Therefore, this protocol allows scientists to evaluate structural reorganization changes in patients with PLPs during MT and also helps them to understand if these changes seen in fMRI are associated with changes in PLP, therefore providing additional details on how MT affects brain structural and functional activity to modify phantom pain.

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			<td height="22" style="height:22px;width:137px;">Scanner</td>
			<td style="width:135px;">Phillips</td>
			<td style="width:129px;">NA</td>
			<td style="width:285px;">3 Tesla Philips Acheiva MRI scanner</td>
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			<td height="22" style="height:22px;width:137px;">Camera</td>
			<td style="width:135px;">Logitech</td>
			<td style="width:129px;">NA</td>
			<td style="width:285px;">HD Pro Webcam C910</td>
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			<td height="42" style="height:43px;width:137px;">Monitor</td>
			<td style="width:135px;">Cambridge Research Systems</td>
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			<td style="width:285px;">&#160;3D BOLD screen for MRI</td>
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			<td height="122" style="height:123px;width:137px;">Mirror</td>
			<td style="width:135px;">TAP Plastics</td>
			<td style="width:129px;">99999</td>
			<td style="width:285px;">Mirrored Acrylic Sheets (Cut&#173;to&#173;Size) &#173; Clear 1/8 (.118)&#34; Thick, 10&#34; Wide, 40&#34; Long</td>
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			<td height="92" style="height:92px;width:137px;">Mirror stand</td>
			<td style="width:135px;"></td>
			<td style="width:129px;">NA</td>
			<td style="width:285px;">Mirror stand was built by the co-investigators from a rectangular piece of wood</td>
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			<td height="112" rowspan="2" style="height:113px;width:137px;">Headphones</td>
			<td rowspan="2" style="width:135px;">Westone Sensimetrics</td>
			<td rowspan="2" style="width:129px;">PN 79245</td>
			<td style="width:285px;">Replacement comply foam tips for universal-fit earphones. Canal size: Standard 6 pieces/ 3 pair&#160;</td>
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			<td height="22" style="height:22px;width:285px;">MR compatible in ear headphones</td>
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			<td height="22" style="height:22px;">MRI Scanner</td>
			<td>Phillips</td>
			<td>3.0 T Philips Achieva System&#160;</td>
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real-time video projection in an mri for characterization of neural correlates associated with mirror therapy for phantom limb pain

Mirror therapy (MT) has been proposed as an effective rehabilitative strategy to alleviate pain symptoms in amputees with phantom limb pain (PLP). However, establishing the neural correlates associated with MT therapy have been challenging given that it is difficult to administer the therapy effectively within a magnetic resonance imaging (MRI) scanner environment. To characterize the functional organization of cortical regions associated with this rehabilitative strategy, we have developed a combined behavioral and functional neuroimaging protocol that can be applied in participants with a leg amputation. This novel approach allows participants to undergo MT within the MRI scanner environment by viewing real-time video images captured by a camera. The images are viewed by the participant through a system of mirrors and a monitor that the participant views while lying on the scanner bed. In this manner, functional changes in cortical areas of interest (e.g., sensorimotor cortex) can be characterized in response to the direct application of MT.

Generating the sensation associated with MT using real-time video projection is feasible. Participants have subjectively reported that the video image perceived is life-like and the sensation is immersive.
Furthermore, the patterns of cortical activation associated with MT (i.e., movement of the leg and viewing the projected mirror image) in the scanner environment are robust. In a pilot study, the cortical responses to MT were ...

Watch this Scientific Journal Video about Real-time Video Projection in an MRI for Characterization of Neural Correlates Associated with Mirror Therapy for Phantom Limb Pain at JoVE.com

Real-time Video Projection in an MRI for Characterization of Neural Correlates Associated with Mirror Therapy for Phantom Limb Pain

We present a novel combined behavioral and neuroimaging protocol employing real-time video projection for the purpose of characterizing the neural correlates associated with mirror therapy within the magnetic resonance imaging scanner environment in leg amputee subjects with phantom limb pain.

Mirror therapy (MT) has been proposed as an effective rehabilitative strategy to alleviate pain symptoms in amputees with phantom limb pain (PLP). ...

The overall goal of this procedure is to more accurate characterize the neural correlates of mirror therapy in phantom limb pain patients, that is, PLP patients. This is accomplished by the following steps. Step one.Ensure that the participant does not have any known contraindications to MRI scanning and provide a pre-recorded audio to make sure that they are able to understand and follow the instructions provided during the scanning procedure. Step two. Position the patient as comfortably as possible in the scanner bed, where he or she should be lying supine with a single-piece MRI-compatible horizontal mirror between his or her legs.This mirror should be supported by a triangular stand to avoid contact with any part of the patient's body. Step three. Place an MRI-compatible digital camera on an adjustable tripod stand near the patient's intact leg to provide real-time video transmission.Step four, the final step. Begin with an anatomical MRI and adjust the machine settings to each patient and then, while the functional MRI scan is being performed, play the recording to the patient, instructing him or her to complete the specific behavioral tasks. Ultimately, the mirror attached to the MRI coil will allow patients to watch the mirrored leg movements in real time without moving their heads.For this protocol, you will need the following items. An MRI scanner, two MRI-compatible mirrors, a large one to place between the patient's legs, as well as a small one to place on the head coil. Additionally, you'll need sandbags, an MRI-compatible digital camera, a tripod for the camera, a computer-controlled system, and a monitor to place in the back of the scanner bore.Before proceeding to the MRI, it is critical to make sure the patient has no known contraindications to MRI scanning, for example, metal implants, aneurysm clips, or severe claustrophobia. Initially, you will explain to the patients exactly what they should expect during the experimental procedure. The patients will then listen to a recording with instructions to follow during the scan.Mirror.Patients may first practice during a mock scan to become familiar with the tasks as well as the scanner environment. The mock scanner is similar in every way to the real MRI scanner but without the active magnet. Before entering the scanner room, patients must remove their prostheses as well as any metal objects they might be wearing on their heads or bodies, for example, watches or jewelry.The MRI technician will make sure the patients have no metal that might put them at risk. All patients are transported to the scanner room using an MRI-safe wheelchair to avoid falling out. After that, the patients will transfer themselves to the MRI scanner bed.After the patient is lying comfortably in a supine manner on the scanner bed, a single-piece MRI-compatible horizontal mirror is placed between his or her legs. An adjustable arm is then positioned to point the camera at the mirrored leg. The large mirror is positioned between the legs at an angle of about 45 degrees, depending on the patient's height and amputation level.The goal is to cover the stump and make it invisible to the video camera. Sandbags are used to keep the mirror at the correct angle. A smaller mirror is positioned on the head coil, angled at 45 degrees at eye level.This mirror allows the patient to visualize the mirrored leg image directly, without moving their head while lying completely inside the scanner bore. An MRI-compatible digital camera is mounted on a tripod stand near the intact leg. This camera will transmit real-time video images of the mirrored leg movements to a computer control system that then projects the video to a monitor near the patient's head so he or she can view the mirrored leg movements.The patient will undergo a four-minute anatomical scan first followed by four runs of functional acquisitions while he or she performs the tasks. Each run lasts six minutes. During the scans, the patient wears sound-isolating MRI-compliant headphones which emit a series of auditory cues, instructing the patient to perform the given behavioral task.The following commands are used. One, leg, two, mirror, and three, rest. Additionally, the investigator says start and end at the beginning and end of the experimental run.The patient has already been instructed on hearing the word leg to follow the tapping sound presented in the audio. With the eyes closed, he or she will tap the foot at a rate of one tap per two seconds for a total of 10 taps in 20 seconds.Leg. On hearing the second command, mirror, the patient has to continue tapping his or her foot at the same rate, this time, while looking at the display showing the mirrored image of the two legs.Again, this would be at a rate of 10 taps in 20 seconds.Mirror. On hearing the third command, rest, the patient should stop moving his or her foot and lie motionless with both eyes closed.Rest. Data is collected in a single session for each patient and the entire scanning procedure lasts approximately 30 minutes.The investigators take note of any unwanted movements. Between the runs, they can ask the patients to keep the right pace and do the correct movements. After the procedure is finalized, the data is transferred to an encrypted flash drive and stored in a secure location in the facility.A longitudinal analysis design is used, comparing baseline and post-treatment data. The FSL software package and processing stream will be applied. Volumes with motion above 0.9 millimeters in any direction are identified with FSL's motion outlier detection processing stream and mathematically scrubbed from the final analysis.If more than 25%of the volumes are designated for removal, the whole acquisition is excluded from the total data set. A region of interest ROI analysis is used. The primary ROI is defined structurally using Freesurfer's Desikan Atlas of the primary sensory motor cortex and refined with a subject-specific functional activation during the leg versus stress condition at the baseline scan.This ROI is then reflected on the homologous area of the other hemisphere, that is, ipsilateral primary sensory motor representation of the intact lower limb. The secondary ROI is the entire bilateral occipital visual cortex as defined by the anatomical Desikan Atlas. Patients reported that the experience is immersive and the video image is life-like.Therefore, this real-time video projection process can generate the sensations associated with conventional mirror therapy. We expect that the leg condition, that is, the foot-tapping task, will lead to robust activation of the sensory motor cortex representing the intact leg compared to the rest condition. However, we also expect to see a less-pronounced activation of the sensory motor leg area representing the amputated leg.The mirror condition also shows robust contralateral as well as some ipsilateral activation of the cortical leg sensory motor area compared to the rest condition. Additionally, robust cortical activation is seen posteriorly in the visual cortical areas associated with viewing the mirrored leg. The activation pattern is described to represent the baseline condition, that is, prior to beginning therapy.These initial responses serve to define regions of interest, ROIs, and allow for comparisons after the therapeutic protocol is completed in each individual. After watching this video, you should have an adequate and sufficient understanding of the necessary steps required to set up all the equipments to perform your therapy inside the MRI scanner. This protocol describes a novel physical procedure that allows investigators to more accurately characterize the neural correlates associated with mirror therapy in individuals with phantom limb pain.We could answer additional questions regarding brain organization after a limb amputation following the steps of this protocol using other neurophysiological measurements or imaging techniques. A challenge associated with this approach is the risk of generating excessive head motion artifacts, given that the leg must be moved repeatedly inside the scanner. Excessive head motion can compromise image data quality.In this regard, it's important to plan ahead and implement a variety of strategies to mitigate this possibility. These include training the participant in a mock scanner to carry out the task without excessively moving their head, making sure that the head is secure yet comfortably restrained, and implementing motion detection correction strategies during the acquisition and data analysis phases respectively. Given the method to implementing the experimental set-up is relatively simple, this approach may allow the evaluation of the mirror therapy effects not only in limb amputees but also in other conditions, such as stroke or spinal cord injury where mirror therapy is already commonly used in clinical practice.

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Research

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Neuroscience

Brain Imaging Investigation of the Neural Correlates of Observing Virtual Social Interactions

The ability to gauge social interactions is crucial in the assessment of others&#x2019; intentions. Factors such as facial expressions and body language affect our decisions in personal and professional life alike 1. These "friend or foe" judgements are often based on first impressions, which in turn may affect our decisions to "approach or avoid". Previous studies investigating the neural correlates of social cognition tended to use static facial stimuli 2. Here, we illustrate an experimental design in which whole-body animated characters were used in conjunction with functional magnetic resonance imaging (fMRI) recordings. Fifteen participants were presented with short movie-clips of guest-host interactions in a business setting, while fMRI data were recorded; at the end of each movie, participants also provided ratings of the host behaviour. This design mimics more closely real-life situations, and hence may contribute to better understanding of the neural mechanisms of social interactions in healthy behaviour, and to gaining insight into possible causes of deficits in social behaviour in such clinical conditions as social anxiety and autism 3.

This article demonstrates an experimental design in which whole-body animated characters are used in conjunction with functional magnetic resonance imaging (fMRI) to investigate the neural correlates of observing virtual social interactions.

The ability to gauge social interactions is crucial in the assessment of others&#x2019; intentions. Factors such as facial expressions and body language ...

Brain Imaging Investigation of the Neural Correlates of Emotional Autobiographical Recollection

Recollection of emotional autobiographical memories (AMs) is important to healthy cognitive and affective functioning 1 - remembering positive AMs is associated with increased personal well-being and self-esteem 2, whereas remembering and ruminating on negative AMs may lead to affective disorders 3. Although significant progress has been made in understanding the brain mechanisms underlying AM retrieval in general (reviewed in 4, 5), less is known about the effect of emotion on the subjective re-experience of AMs and the associated neural correlates. This is in part due to the fact that, unlike the investigations of the emotion effect on memory for laboratory-based microevents (reviewed in 6, 7-9), often times AM studies do not have a clear focus on the emotional aspects of remembering personal events (but see 10). Here, we present a protocol that allows investigation of the neural correlates of recollecting emotional AMs using functional magnetic resonance imaging (fMRI). Cues for these memories are collected prior to scanning by means of an autobiographical memory questionnaire (AMQ), therefore allowing for proper selection of emotional AMs based on their phenomenological properties (i.e., intensity, vividness, personal significance). This protocol can be used in healthy and clinical populations alike.

We present a protocol that allows investigation of the neural correlates of recollecting emotional autobiographical memories, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants.

Recollection of emotional autobiographical memories (AMs) is important to healthy cognitive and affective functioning 1 - remembering positive AMs is ...

Brain Imaging Investigation of the Neural Correlates of Emotion Regulation

The ability to control/regulate emotions is an important coping mechanism in the face of emotionally stressful situations. Although significant progress has been made in understanding conscious/deliberate emotion regulation (ER), less is known about non-conscious/automatic ER and the associated neural correlates. This is in part due to the problems inherent in the unitary concepts of automatic and conscious processing1. Here, we present a protocol that allows investigation of the neural correlates of both deliberate and automatic ER using functional magnetic resonance imaging (fMRI). This protocol allows new avenues of inquiry into various aspects of ER. For instance, the experimental design allows manipulation of the goal to regulate emotion (conscious vs. non-conscious), as well as the intensity of the emotional challenge (high vs. low). Moreover, it allows investigation of both immediate (emotion perception) and long-term effects (emotional memory) of ER strategies on emotion processing. Therefore, this protocol may contribute to better understanding of the neural mechanisms of emotion regulation in healthy behaviour, and to gaining insight into possible causes of deficits in depression and anxiety disorders in which emotion dysregulation is often among the core debilitating features.

We present a protocol that allows investigation of the neural correlates of deliberate and automatic emotion regulation, using functional magnetic resonance imaging. This protocol can be used in healthy participants, both young and older, as well as in clinical patients.

The ability to control/regulate emotions is an important coping mechanism in the face of emotionally stressful situations. Although significant progress ...

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 &#181;m away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the ...

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition follows a combinatorial coding scheme, where one OR can be activated by a set of odorants and one odorant can activate a combination of ORs. Through such combinatorial coding, organisms can detect and discriminate between a myriad of volatile odor molecules. Thus, an odor at a given concentration can be described by an activation pattern of ORs, which is specific to each odor. In that sense, cracking the mechanisms that the brain uses to perceive odor requires the understanding odorant-OR interactions. This is why the olfaction community is committed to &#34;de-orphanize&#34;&#160;these receptors. Conventional in vitro systems used to identify odorant-OR interactions have utilized incubating cell media with odorant, which is distinct from the natural detection of odors via vapor odorants dissolution into nasal mucosa before interacting with ORs. Here, we describe a new method that allows for real-time monitoring of OR activation via vapor-phase odorants. Our method relies on measuring cAMP release by luminescence using the Glosensor assay. It bridges current gaps between in vivo and in vitro approaches and provides a basis for a biomimetic volatile chemical sensor.

Physiologically, odorant receptors are activated by odorant molecules inhaled in the vapor phase. However, most in vitro systems utilize liquid phase odorant stimulation. Here, we present a method that allows real-time in vitro monitoring of odorant receptor activation upon odorant stimulation in vapor phase.

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition ...

Real-Time fMRI Brain Mapping in Animals

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a real-time fMRI platform to guide experimenters to monitor fMRI responses instantaneously during acquisition, which can be used to modify the physiology of animals to achieve desired hemodynamic responses in animal brains. The real-time fMRI set-up is based on a 14.1T preclinical MRI system, enabling the real-time mapping of dynamic fMRI responses in the primary forepaw somatosensory cortex (FP-S1) of anesthetized rats. Instead of a retrospective analysis to investigate confounding sources leading to the variability of fMRI signals, the real-time fMRI platform provides a more effective scheme to identify dynamic fMRI responses using customized macro-functions and a common neuroimage analysis software in the MRI system. Also, it provides immediate troubleshooting feasibility and a real-time biofeedback stimulation paradigm for brain functional studies in animals.

Animal brain functional mapping can benefit from the real-time functional magnetic resonance imaging (fMRI) experimental set-up. Using the latest software implemented in the animal MRI system, we established a real-time monitoring platform for small animal fMRI.

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a ...

Electrocorticographic Recording of Cerebral Cortex Areas Manipulated Using an Adeno-Associated Virus Targeting Cofilin in Mice

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. Adeno-associated viruses (AAVs) are increasingly used to improve understanding of brain circuits and their functions. The AAV-mediated manipulation of specific cell populations and/or of precise molecular components has been tremendously useful to identify new sleep regulatory circuits/molecules and key proteins contributing to the adverse effects of sleep loss. For instance, inhibiting activity of the filamentous actin-severing protein&#160;cofilin&#160;using AAV prevents sleep deprivation-induced memory impairment. Here, a protocol is&#160;described that combines the manipulation of cofilin function in a cerebral cortex area with the recording of ECoG activity to examine whether cortical cofilin modulates the wakefulness and sleep ECoG signals. AAV injection is performed during the same surgical procedure as the implantation of ECoG and electromyographic (EMG) electrodes in adult male and female mice. Mice are anesthetized, and their heads are shaved. After skin cleaning and incision, stereotaxic coordinates of the motor cortex are determined, and the skull is pierced at this location. A cannula prefilled with an AAV expressing cofilinS3D, an inactive form of cofilin, is slowly positioned in the cortical tissue. After AAV infusion, gold-covered screws (ECoG electrodes) are screwed through the skull and cemented to the skull with gold wires inserted in the neck muscles (EMG electrodes). The animals are allowed three weeks to recover and to ensure sufficient expression of cofilinS3D. The infected area and cell type are verified using immunohistochemistry, and the ECoG is analyzed using visual identification of vigilance states and spectral analysis. In summary, this combined methodological approach allows the investigation of the precise contribution of molecular components regulating neuronal morphology and connectivity to the regulation of synchronized cerebral cortex activity during wakefulness and sleep.

This article describes a protocol for the manipulation of molecular targets in the cerebral cortex using adeno-associated viruses and for monitoring the effects of this manipulation during wakefulness and sleep using electrocorticographic recordings.

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. ...

Open-Source Real-Time Closed-Loop Electrical Threshold Tracking for Translational Pain Research

Nociceptors are a class of primary afferent neurons that signal potentially harmful noxious stimuli. An increase in nociceptor excitability occurs in acute and chronic pain conditions. This produces abnormal ongoing activity or reduced activation thresholds to noxious stimuli. Identifying the cause of this increased excitability is required for the development and validation of mechanism-based treatments. Single-neuron electrical threshold tracking can quantify nociceptor excitability. Therefore, we have developed an application to allow such measurements and demonstrate its use in humans and rodents. APTrack provides real-time data visualization and action potential identification using a temporal raster plot. Algorithms detect action potentials by threshold crossing and monitor their latency after electrical stimulation. The plugin then modulates the electrical stimulation amplitude using an up-down method to estimate the electrical threshold of the nociceptors. The software was built upon the Open Ephys system (V0.54) and coded in C++ using the JUCE framework. It runs on Windows, Linux, and Mac operating systems. The open-source code is available (<a data-saferedirecturl="https://www.google.com/url?q=https://github.com/Microneurography/APTrack&amp;source=gmail&amp;ust=1680789829274000&amp;usg=AOvVaw0nMk9J_EI2jUn8SlmC5JJm" href="https://github.com/Microneurography/APTrack" target="_blank">https://github.com/Microneurography/APTrack</a>). The electrophysiological recordings were taken from nociceptors in both a mouse skin-nerve preparation using the teased fiber method in the saphenous nerve and in healthy human volunteers using microneurography in the superficial peroneal nerve. Nociceptors were classified by their response to thermal and mechanical stimuli, as well as by monitoring the activity-dependent slowing of the conduction velocity. The software facilitated the experiment by simplifying the action potential identification through the temporal raster plot. We demonstrate real-time closed-loop electrical threshold tracking of single-neuron action potentials during&#160;in vivo&#160;human microneurography, for the first time, and during&#160;ex vivo&#160;mouse electrophysiological recordings of C-fibers and A&#948;-fibers. We establish proof of principle by showing that the electrical threshold of a human heat-sensitive C-fiber nociceptor is reduced by heating the receptive field. This plugin enables the electrical threshold tracking of single-neuron action potentials and allows the quantification of changes in nociceptor excitability.

APTrack is a software plugin developed for the Open Ephys platform that enables real-time data visualization and the closed-loop electrical threshold tracking of neuronal action potentials. We have successfully used this in microneurography for human C-fiber nociceptors and mouse C-fiber and A&#948;-fiber nociceptors.

Nociceptors are a class of primary afferent neurons that signal potentially harmful noxious stimuli. An increase in nociceptor excitability occurs in ...