Quantitative Behavioral Measures Research Articles

Semi-Automated Assessment of Long-Term Olfactory Habituation in Drosophila melanogaster Using the Olfactory Arena.

Long-lasting memories are a core aspect of an animal's life. Such memories are characterized by unique molecular mechanisms and often unique circuitry, neither of which are completely understood in vivo. The deep knowledge of the identity and connectivity of neurons of the fruit fly Drosophila melanogaster, as well as the sophisticated genetic tools that allow in vivo perturbations and physiology monitoring, make it a remarkably useful organism in which to investigate the molecular mechanisms of long-term memories. In this protocol, we focus on habituation, a non-associative form of learning, and describe a reliable, semi-automated technique to induce and assess long-term olfactory habituation (LTH) in Drosophila using the olfactory arena, thus providing a method aligned with recent technological progress in behavioral measurement. Prior work has shown that LTH is induced by a 4-day exposure to an odorant and is characterized by a long-lasting (> 24 h) reduction in behavioral response to the exposed odorant, measured using a manual and skill-intensive Y-maze assay. Here, we present a semi-automated protocol for obtaining quantifiable measures of LTH, at the level of detail required for other investigators in the field. Unlike previously described methods, the protocol presented here provides quantitative and detailed behavioral measurements obtained by video recording that can be shared with the scientific community and allows sophisticated forms of offline analysis. We suggest that this procedure has the potential to advance our understanding of molecular and circuit mechanisms of olfactory habituation, its control via neuromodulation, and its interactions with other forms of memory. Key features • A protocol to induce olfactory aversive long-term habituation in Drosophila melanogaster. • Video recording and analysis of Drosophila in an olfactory arena.

PSX-17 Can Associations Between Qualitative Descriptors and Quantitative Behavioral Measures in Nursery Age Pigs Improve Interpretation of Behavioral studies?

Abstract Qualitative Behavior Assessments (QBA) are a useful way to assess animal welfare by capturing behavioral signals of emotionality not captured by quantitative measures. The objective of this study was to determine if QBA descriptors assigned to nursery age gilts during a social dyad test in a novel arena were associated with differences in quantitative behavioral measures recorded from the test such as latency to make social contact and amount of time spent in proximity. To investigate this, 30 weight-matched purebred Yorkshire gilt pairs underwent a social dyad test at 6 wk-of-age. Dyads were unfamiliar with each other, and one was assigned as the focal gilt. The test began as soon as both gilts were in the novel arena and lasted for 5 minutes. Four independent, untrained observers were asked to perform a QBA by circling all terms that they felt described the focal gilt throughout the test duration from a set list of 20 terms. Overhead video was recorded and used to collect quantitative measures including number of quadrant crosses, percentage of crosses entering an already occupied quadrant, percentage of crosses exiting an already occupied quadrant, latency of the focal pig to touch the other pig, and time spent in an occupied quadrant. For each focal gilt, only terms that were used by at least two observers were kept. To be included in the analysis a given term had to be assigned to at least 6 of the 30 focal gilts such that each group had sufficient sample size to make comparisons. This filtering resulted in 10 terms being kept for analysis including Active (22), Agitated (7), Aimless (10), Calm (15), Indifferent (8), Lively (13), Playful (9), Positively Occupied (16), Sociable (14), and Tense (8). Quantitative measures were analyzed using a t-test to determine if pigs described by each term differed from those that were not. Pigs described as Aimless, Calm, Positively Occupied, Sociable, or Tense did not significantly differ in any of the quantitative measures of behavior. Pigs described as Active, Indifferent, or Lively had significantly more quadrant crosses (P = 0.0003, 0.003, 0.022 respectively). Pigs described as Agitated had a greater proportion of crosses that were entering an occupied quadrant (P = 0.022), while those described as Playful had a significantly lower proportion of crosses that were exiting an occupied quadrant (P = 0.031). Understanding how qualitative descriptors associate with quantitative measures of behavior may assist in better interpretation of QBA results as well as confirming that a chosen metric is capturing the intended behavior.

Biological condensates form percolated networks with molecular motion properties distinctly different from dilute solutions.

Formation of membraneless organelles or biological condensates via phase separation and related processes hugely expands the cellular organelle repertoire. Biological condensates are dense and viscoelastic soft matters instead of canonical dilute solutions. To date, numerous different biological condensates have been discovered, but mechanistic understanding of biological condensates remains scarce. In this study, we developed an adaptive single-molecule imaging method that allows simultaneous tracking of individual molecules and their motion trajectories in both condensed and dilute phases of various biological condensates. The method enables quantitative measurements of concentrations, phase boundary, motion behavior, and speed of molecules in both condensed and dilute phases, as well as the scale and speed of molecular exchanges between the two phases. Notably, molecules in the condensed phase do not undergo uniform Brownian motion, but instead constantly switch between a (class of) confined state(s) and a random diffusion-like motion state. Transient confinement is consistent with strong interactions associated with large molecular networks (i.e., percolation) in the condensed phase. In this way, molecules in biological condensates behave distinctly different from those in dilute solutions. The methods and findings described herein should be generally applicable for deciphering the molecular mechanisms underlying the assembly, dynamics, and consequently functional implications of biological condensates.

Long-term changes in oral feeding behaviors of growing rats.

Oral feeding is critical for survival in both humans and animals. However, few studies have reported quantitative behavioral measures associated with the development of oral feeding behaviors. Therefore, the present study investigated developmental changes in the oral feeding behaviors of rats by quantitatively assessing pasta eating and licking behaviors. In the pasta eating test, the time to finish pasta sticks of three different thicknesses (Φ = 0.9, 1.4, and 1.9mm, 4cm long) was recorded between postnatal day 29 (P29) and P49, because all rats were able to finish eating these pasta sticks on P29. A developmental decrease in the time to finish pasta sticks of all thicknesses was observed during the initial period of recordings and plateaued before P35. The extent of this decrease was dependent on the thickness of pasta sticks. In the licking test, the number of licks per 10s and the total intake volume during the test were recorded between P19 and P49, because all rats were able to access and lick the solution on P19. The time courses of developmental increases in the number of licks and the total intake volume were similar to the results obtained in the pasta eating test. Collectively, these results suggest that developmental changes in pasta eating and licking behaviors markedly differed between the weanling and periadolescent periods. The present study also demonstrated the applicability of the pasta eating and licking tests to the quantification of developmental changes in the oral feeding behaviors of rats.

Drosophila functional screening of de novo variants in autism uncovers damaging variants and facilitates discovery of rare neurodevelopmental diseases.

SUMMARYIndividuals with autism spectrum disorder (ASD) exhibit an increased burden of de novo mutations (DNMs) in a broadening range of genes. While these studies have implicated hundreds of genes in ASD pathogenesis, which DNMs cause functional consequences in vivo remains unclear. We functionally test the effects of ASD missense DNMs using Drosophila through “humanization” rescue and overexpression-based strategies. We examine 79 ASD variants in 74 genes identified in the Simons Simplex Collection and find 38% of them to cause functional alterations. Moreover, we identify GLRA2 as the cause of a spectrum of neurodevelopmental phenotypes beyond ASD in 13 previously undiagnosed subjects. Functional characterization of variants in ASD candidate genes points to conserved neurobiological mechanisms and facilitates gene discovery for rare neurodevelopmental diseases.

The Extent of Engagement With Telehealth Approaches by Patients With Advanced Cancer: Systematic Review.

BackgroundTelehealth approaches are increasingly being used to support patients with advanced diseases, including cancer. Evidence suggests that telehealth is acceptable to most patients; however, the extent of and factors influencing patient engagement remain unclear.ObjectiveThe aim of this review is to characterize the extent of engagement with telehealth interventions in patients with advanced, incurable cancer reported in the international literature.MethodsThis systematic review was registered with PROSPERO (International Prospective Register of Systematic Reviews) and is reported in line with PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) 2020 guidelines. A comprehensive search of databases was undertaken for telehealth interventions (communication between a patient with advanced cancer and their health professional via telehealth technologies), including MEDLINE, Embase, CINAHL, PsycINFO, Cochrane Library, Sociological s, and Web of Science, from the inception of each electronic database up until December 31, 2020. A narrative synthesis was conducted to outline the design, population, and context of the studies. A conceptual framework of digital engagement comprising quantitative behavioral measures (frequency, amount, duration, and depth of use) framed the analysis of engagement with telehealth approaches. Frequency data were transformed to a percentage (actual patient engagement as a proportion of intended engagement), and the interventions were characterized by intensity (high, medium, and low intended engagement) and mode of delivery for standardized comparisons across studies.ResultsOf the 19,676 identified papers, 40 (0.2%) papers covering 39 different studies were eligible for inclusion, dominated by US studies (22/39, 56%), with most being research studies (26/39, 67%). The most commonly reported measure of engagement was frequency (36/39, 92%), with substantial heterogeneity in the way in which it was measured. A standardized percentage of actual patient engagement was derived from 17 studies (17/39, 44%; n=1255), ranging from 51% to 100% with a weighted average of 75.4% (SD 15.8%). A directly proportional relationship was found between intervention intensity and actual patient engagement. Higher engagement occurred when a tablet, computer, or smartphone app was the mode of delivery.ConclusionsUnderstanding engagement for people with advanced cancer can guide the development of telehealth approaches from their design to monitoring as part of routine care. With increasing telehealth use, the development of meaningful and context- and condition-appropriate measures of telehealth engagement is needed to address the current heterogeneity in reporting while improving the understanding of optimal implementation of telehealth for oncology and palliative care.Trial RegistrationPROSPERO (International Prospective Register of Systematic Reviews) CRD42018117232; https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42018117232

Fractional modeling of flexural behavior of toenail plates: First step for clinical purposes

This work presents an identification procedure of flexural behavior of toenail plates in twenty subjects with no history of feet or nail injury as of in-vivo measurements. In particular, four different mechanical models are considered to describe such properties, ranging from the pure elastic to viscoelastic behavior, the latter from the classical and fractional points of view. The quality of the adjustment of each model is examined by a group of performance indices. Experimental data show that the best identification is achieved by the fractional order viscoelastic model for all subjects. These novel results in modeling flexural behavior of toenails are consistent with the published literature suggesting that viscoelastic materials may be successfully modeled with derivatives of fractional order. This could contribute, together with additional variables, to help health professionals, and more especially podiatrists, to have reliable and quantitative measures of the nail flexural behavior which can be susceptible of treatment or for prevention.

The influence of expertise and experimental paradigms on the visual behavior of tennis athletes in returning a serve

To return a serve, one must pick up information from the server’s kinematics and anticipate the ball trajectory. Although the perceptual requirements are important, the literature diverges in terms of the differences between experts and novices as well as the importance of the experimental paradigm (in-situ vs. video-based) for the results. This study aimed to address both concerns. We compared experts’ (n=7, 20.6±1.1 year of age) and novices’ (n=7, 20.0±0.4 years of age) visual pattern when returning a serve (Experiment 1) and the influence of the experimental paradigm in experts (Experiment 2). Experts fixated more and longer the upper body and ball, while novices showed a more distributed pattern and with longer fixations outside of the server’s body. Also, the pattern was different when comparing in-situ and laboratory settings, differing mainly in fixation frequency. The influence of expertise was observed in qualitative (relative) and quantitative (absolute) measures of visual behavior with the setting having an important influence. Thus, studies should be as close to the actual situation if trying to understand experts’ behavior.

Using an exploratory sequential mixed methods design to adapt an Illness Perception Questionnaire for African Americans with diabetes: the mixed data integration process

ABSTRACT Background Although qualitative methods have been used to develop quantitative behavioral health measurements, studies rarely report on the exact development process of these questionnaires. In this methodological paper, we highlight the procedure of a mixed data integration process in using qualitative data to create quantitative questionnaire items. Methods We used an exploratory sequential mixed methods study design to culturally adapt the Illness Perception Questionnaire-Revised (IPQ-R) and address the sociocultural contexts of African Americans with type 2 diabetes. Forty African Americans with type 2 diabetes taking oral diabetes medication completed the qualitative focus groups and 170 participants completed the quantitative phase (surveys). Using the ‘building approach’ to integration, qualitative themes from the focus groups were matched to survey domains based on the self-regulatory model. Qualitative themes assessing perceptions of diabetes among African Americans were used to develop new survey items for a culturally adapted IPQ-R, as well as adapt original survey items. Results Important themes included the effect on friend/family relationships, lifestyle changes, food experiences (consequences domain), importance of medications (treatment control), comparisons with family members (illness coherence), fear, future worries, and anger (emotional representations). A new domain, ‘sociocultural influences’ was added to the adapted questionnaire based on qualitative themes of race and racism on provider roles, personal control, and community influences. Merging and integration of the qualitative and quantitative phases, (reported via a joint display) showed evidence of congruence between the illness perceptions from the qualitative focus groups and scores on the survey items. Conclusion The use of mixed methods allowed for the development of a robust and patient-centered questionnaire. Future research should consider psychometric testing of the adapted IPQ-R, so that it may be used in addressing illness perceptions among African Americans.

Strategy for screening cognitive impairment in Chinese patients with amyotrophic lateral sclerosis

BackgroundIt is an economical strategy to design a screening method to decide which patients with amyotrophic lateral sclerosis/ motor neuron disease (ALS/MND) should enter into the stage for further comprehensive neuropsychological investigation. Methods59 patients (including 8 with frank dementia) were recruited. They underwent the extensive neuropsychological evaluation and short screening batteries, namely the Mini-Mental State Examination (MMSE), the Peking Union Medical College Hospital version of Montreal Cognitive Assessment (MoCA-P) and the Frontal Assessment Battery (FAB). Results of the extensive neuropsychological evaluation were set as the gold standard to diagnose cognitive impairment, and the effectiveness of screening tests were measured against them. ResultsBy comparing the sensitivity and specificity, we found that the combination of FAB plus both or either of the other 2 short batteries provided a satisfactorily high sensitivity, but none of these screening batteries was significantly associated with quantitative behavioral measurements among non-demented subjects, the Frontal Behavioral Inventory-ALS version (FBI-ALS). ConclusionsThe combination of the FBI-ALS, the FAB, and the MMSE or the MoCA-P or both could effectively screen comorbid dementia, cognitive and behavioral impairment in ALS but this implicates a limited specificity. And the FAB needed to be validated in large Chinese sample.

Behavioral-genetic associations in the Human Connectome Project.

The Human Connectome Project (HCP) provides a rich dataset of quantitative and domain-specific behavioral measures from twins and extensive family structures. This makes the dataset a unique and a valuable resource to investigate heritability and determine individual differences. Using a set of measures of behavioral domains (motor, emotion, personality, sensory, and cognition), we estimated the intraclass correlations (ICCs) and heritability of 56 behavioral measures for 4 genetically identified groups of participants: monozygotic (MZ) twins, dizygotic (DZ) twins, non-twin siblings (SB), and unrelated individuals (NR). The ICCs range varied among behavioral domains but systematically so among the four genetic groups. We found the same rank order of ICCs, from the highest values for MZ twins, statistically significantly smaller for the DZ twins and sibling group (compared to MZ), and close to zero for NR. The mean heritability values of the five behavioral domains were: cognition h2 = 0.405, emotion h2 = 0.316, motor h2 = 0.138, personality h2 = 0.444, and sensory h2 = 0.193. These domains share overlapping brain networks. The heritability of motor domain was significantly smaller than cognitive, personality, and emotion domains. These findings provide new insight into the effect of genetics on the various diverse behavioral measures.

African American Child and Adolescent Academic Help-Seeking: A Scoping Review

To influence school achievement, researchers investigate student academic help-seeking. However, a growing population of students of color combined with the risk of poorer academic performance yields attention toward African American student academic help-seeking. In this review, 13 studies analyzed sampling characteristics, theoretical frameworks, and designing methodologies. Results illustrated that samples varied in terms of data collection, settings, participant characteristics, and research purpose. Also, frameworks differed with the majority of the studies referencing culture or motivation-informed theory. The methodology focused on quantitative measures of help-seeking behavior, attitude, and influencing variables. Qualitative measures showed African American students experienced help-seeking challenges, family involvement, and assumed responsibility for seeking help. Implications for research and practice are provided.

The Stockman's Scorecard: Validity and Reliability as an Instrument for Measuring Stockmanship

The quality of beef cattle stockmanship typically is evaluated through quantitative and qualitative measurements of animal behavior. The Stockman's Scorecard is an observation instrument that has been developed to directly measure the actions of beef cattle stockmen. This article documents a pilot project for determining the content validity, internal consistency, and intrarater reliability of the scorecard as an evaluation instrument. Our results show that the scorecard is a valid and reliable instrument for measuring the actions of stockmen. The instrument can be a valuable tool for Extension educators in evaluating their stockmanship programming impacts.

The role of RDoC in future classification of mental disorders .

The Research Domain Criteria (RDoC) project constitutes a translational framework for psychopathology research, initiated by the National Institute of Mental Health in an attempt to provide new avenues for research to circumvent problems emerging from the use of symptom-based diagnostic categories in diagnosing disorders. The RDoC alternative is a focus on psychopathology based on dimensions simultaneously defined by observable behavior (including quantitative measures of cognitive or affective behavior) and neurobiological measures. Key features of the RDoC framework include an emphasis on functional dimensions that range from normal to abnormal, integration of multiple measures in study designs (which can foster computational approaches), and high priority on studies of neurodevelopment and environmental influences (and their interaction) that can contribute to advances in understanding the etiology of disorders throughout the lifespan. The paper highlights key implications for ways in which RDoC can contribute to future ideas about classification, as well as some of the considerations involved in translating basic behavioral and neuroscience data to psychopathology.

Assessing the Autonomic and Behavioral Effects of Passive Motion in Rats using Elevator Vertical Motion and Ferris-Wheel Rotation

The overall goal of this study is to assess the autonomic and behavioral effects of passive motion in rodents using the elevator vertical motion and Ferris-wheel rotation devices. These assays can help confirm the integrity and normal functioning of the autonomic nervous system. They are coupled to quantitative measures based on defecation counting, open-field examination, and balance beam crossing. The advantages of these assays are their simplicity, reproducibility, and quantitative behavioral measures. The limitations of these assays are that the autonomic reactions could be epiphenomena of non-vestibular disorders and that a functioning vestibular system is required. Examination of disorders such as motion sickness will be greatly aided by the detailed procedures of these assays.

DeepPoseKit, a software toolkit for fast and robust animal pose estimation using deep learning.

Quantitative behavioral measurements are important for answering questions across scientific disciplines-from neuroscience to ecology. State-of-the-art deep-learning methods offer major advances in data quality and detail by allowing researchers to automatically estimate locations of an animal's body parts directly from images or videos. However, currently available animal pose estimation methods have limitations in speed and robustness. Here, we introduce a new easy-to-use software toolkit, DeepPoseKit, that addresses these problems using an efficient multi-scale deep-learning model, called Stacked DenseNet, and a fast GPU-based peak-detection algorithm for estimating keypoint locations with subpixel precision. These advances improve processing speed >2x with no loss in accuracy compared to currently available methods. We demonstrate the versatility of our methods with multiple challenging animal pose estimation tasks in laboratory and field settings-including groups of interacting individuals. Our work reduces barriers to using advanced tools for measuring behavior and has broad applicability across the behavioral sciences.

Visually Guided Behavior and Optogenetically Induced Learning in Head-Fixed Flies Exploring a Virtual Landscape

Studying the intertwined roles of sensation, experience, and directed action in navigation has been facilitated by the development of virtual reality (VR) environments for head-fixed animals, allowing for quantitative measurements of behavior in well-controlled conditions. VR has long featured in studies of Drosophila melanogaster, but these experiments have typically allowed the fly to change only its heading in a visual scene and not its position. Here we explore how flies move in two dimensions (2D) using a visual VR environment that more closely captures an animal's experience during free behavior. We show that flies' 2D interaction with landmarks cannot be automatically derived from their orienting behavior under simpler one-dimensional (1D) conditions. Using novel paradigms, we then demonstrate that flies in 2D VR adapt their behavior in response to optogenetically delivered appetitive and aversive stimuli. Much like free-walking flies after encounters with food, head-fixed flies exploring a 2D VR respond to optogenetic activation of sugar-sensing neurons by initiating a local search, which appears not to rely on visual landmarks. Visual landmarks can, however, help flies to avoid areas in VR where they experience an aversive, optogenetically generated heat stimulus. By coupling aversive virtual heat to the flies' presence near visual landmarks of specific shapes, we elicit selective learned avoidance of those landmarks. Thus, we demonstrate that head-fixed flies adaptively navigate in 2D virtual environments, but their reliance on visual landmarks is context dependent. These behavioral paradigms set the stage for interrogation of the fly brain circuitry underlying flexible navigation in complex multisensory environments.

Priming, enabling and assessment of curiosity

In the information age, where all answers are just a click away, curiosity, the intrinsic drive to learn, becomes of paramount importance. How easy is it to prime for curiosity and what are its effects? What simple interventions can be used to enable curiosity-driven behaviors? We have conducted a large-scale study to address these questions, using a novel curiosity-based application (app) on university applicants. Using the same app, we addressed the issue of curiosity assessment, which in recent years has mainly been performed via self-reporting questionnaires. The curiosity assessment tool was developed in order to assess curiosity via an objective, quantitative and digital way. The tool measured several behavioral aspects during a free and task-less interaction with the tablet app. From the recorded activity logs we calculated quantitative behavioral measures related to their exploration patterns. We show that a single word can prime the participants and induce better learning of their self-explored knowledge. We also show that by simply enabling more time to explore, without the ability to stop at will, induces more exploration and more learning. Finally, we show that our behavioral measures, obtained with the digital quantitative assessment tool, are significant predictors of the participants’ self-reported curiosity and Psychometric Entrance Test scores. These results suggest that simple priming for curiosity and enabling enough time to explore improve self-paced learning, and that a relatively simple and short interaction with a digital app can greatly improve state-of-the-art curiosity assessment.

What can the quantitative and qualitative behavioural assessment of videos of sheep moving through an autonomous data capture system tell us about welfare?

Sheep can be exposed to a variety of challenges and failure to adapt to these challenges can compromise their health and wellbeing. Regular monitoring of stock on large-scale or extensive systems may not always be possible, although recent technological advancements in automated data capture, such as walk-over-weighing (WoW), can make monitoring easier. The potential benefit of including behavioural assessment in such a system has yet to be tested. We investigated whether quantitative and qualitative behavioural assessment (QBA) methods could be applied to short video footage collected automatically within a WoW setup, to differentiate between sheep that were, presumably, in different (positive and negative) welfare states. Video footage was collected from 36 Merino sheep within the four treatment groups; Control (n = 12), Habituated to the WoW setup and human interaction (n = 8), Lame (n = 8) and Inappetent (n = 8). Habituated sheep were exposed to a low-stress handling regimen for six consecutive days prior to filming. At the same time, feeding behaviour was recorded by means of radio-frequency identification (RFID) technology to identify sheep suffering inappetence. Lame sheep were identified using a 6-point scoring system, and Control animals were selected ensuring that they were not Lame, Inappetent or Habituated. For QBA, the footage was presented, in a random order, to 18 observers. There was a significant (P < 0.001) consensus among the observers in their assessment of the behavioural expression of the sheep. Observers described the Habituated and Lame sheep as significantly more focused/collected/assured compared to the Control sheep (P < 0.05). There was no difference in observer scores between the Inappetent sheep compared to the Controls. A number of associations were found between the QBA scores and the quantitative behavioural measures recorded. Sheep that baulked more frequently at the entrance to the WoW system (Rs = −0.70; P < 0.001) or had a greater number of circling events (Rs = −0.68; P < 0.001) were described as more reluctant/tense/wary, while those that recorded faster walking speeds (Rs = 0.65; P < 0.001) or spent less time standing stationary (Rs= −0.48; P < 0.01) were described as more focused/collected/assured. We conclude that qualitative and quantitative behavioural measures can be used to identify differences in animal behaviour, presumably related to their welfare state, when applied to short video clips automatically collected in a WoW setup. These findings suggest that behavioural measures could be collected, practically, within automated biometric data capture systems to provide additional information to aid in the assessment of sheep welfare in extensive systems.

S65. High Temporal Resolution Event-Related Functional Spectroscopy Reveals Grossly Abnormal Neurotransmitter Responses in Pontine Reticular Nuclei in Autism Spectrum Disorder

Atypical excitation:inhibition has been posited as a core deficit of autism spectrum disorder (ASD). To test this in the amygdala, we previously adapted functional magnetic resonance spectroscopy to an event-related design with high temporal resolution (ht-fMRS). We found an inverted or delayed glutamate response and a relation between higher glutamate:GABA and more severe social impairments. In parallel, our previous work mapped autistic social impairments onto quantitative measures of motor behavior. The microstructural abnormality in the medial pons mediated this relationship. We designed the current study to investigate excitation:inhibition in the pons with a motor-specific ht-fMRS paradigm.