Stimulus characteristics of a novel air-based multiple stimulus aesthesiometer.

PURPOSE: To identify the stimulus airflow characteristics and confirm the consistency of a novel air jet-based aesthesiometer capable of producing and applying multiple stimuli separated either by time and/or by space. METHODS: A novel aesthesiometer (Dolphin Aesthesiometer) was designed around a micro-blower under software management. Two nozzle attachments assisted in airflow control (flexible tube 1.6 mm diameter; brass tube 0.5 mm diameter). Four studies that tested the characteristics of the airflow and stimulus consistency were completed: (i) airflow pattern/trajectory, (ii) airflow surface dispersion, (iii) force of airflow across a range of stimulus strengths and (iv) thermal effects on the ocular surface. RESULTS: Stimulus characteristic studies revealed: (i) airflow is coherent within the expected test distance range for the instrument, and spread rate is constant irrespective of stimulus strength; (ii) airflow dispersion occurs upon encountering a surface and dispersion increases with increasing airflow rate; (iii) a consistent and small force (10-4 N) is applied by the airflow and (iv) repeatable thermal effects occur in relation to the airflow, and the mode of stimulation of the Dolphin aesthesiometer is predominantly thermal in nature. CONCLUSIONS: These studies confirm the repeatability and consistency of the novel instrument. The device is suitable for measuring corneal sensitivity. The availability of additional air jets allows the application of multiple stimuli to facilitate corneal summation investigations.

Sensitivity of Phonation Onset Pressure to Vocal Fold Stiffness Distribution.

Phonation onset is characterized by the unstable growth of vocal fold (VF) vibrations that ultimately results in self-sustained oscillation and the production of modal voice. Motivated by histological studies, much research has focused on the role of the layered structure of the vocal folds in influencing phonation onset, wherein the outer "cover" layer is relatively soft and the inner "body" layer is relatively stiff. Recent research, however, suggests that the body-cover (BC) structure over-simplifies actual stiffness distributions by neglecting important spatial variations, such as inferior-superior (IS) and anterior-posterior gradients and smooth transitions in stiffness from one histological layer to another. Herein, we explore sensitivity of phonation onset to stiffness gradients and smoothness. By assuming no a priori stiffness distribution and considering a second-order Taylor series sensitivity analysis of phonation onset pressure with respect to stiffness, we find two general smooth stiffness distributions most strongly influence onset pressure: a smooth stiffness containing aspects of BC differences and IS gradients in the cover, which plays a role in minimizing onset pressure, and uniform increases in stiffness, which raise onset pressure and frequency. While the smooth stiffness change contains aspects qualitatively similar to layered BC distributions used in computational studies, smooth transitions in stiffness result in higher sensitivity of onset pressure than discrete layering. These two general stiffness distributions also provide a simple, low-dimensional, interpretation of how complex variations in VF stiffness affect onset pressure, enabling refined exploration of the effects of stiffness distributions on phonation onset.

A membrane protein display platform for receptor interactome discovery.

Cell surface receptors are critical for cell signaling and constitute a quarter of all human genes. Despite their importance and abundance, receptor interaction networks remain understudied because of difficulties associated with maintaining membrane proteins in their native conformation and their typically weak interactions. To overcome these challenges, we developed an extracellular vesicle-based method for membrane protein display that enables purification-free and high-throughput detection of receptor-ligand interactions in membranes. We demonstrate that this platform is broadly applicable to a variety of membrane proteins, enabling enhanced detection of extracellular interactions over a wide range of binding affinities. We were able to recapitulate and expand the interactome for prominent members of the B7 family of immunoregulatory proteins such as PD-L1/CD274 and B7-H3/CD276. Moreover, when applied to the orphan cancer-associated fibroblast protein, LRRC15, we identified a membrane-dependent interaction with the tumor stroma marker TEM1/CD248. Furthermore, this platform enabled profiling of cellular receptors for target-expressing as well as endogenous extracellular vesicles. Overall, this study presents a sensitive and easy to use screening platform that bypasses membrane protein purification and enables characterization of interactomes for any cell surface-expressed target of interest in its native state.

Asymmetric triangular body-cover model of the vocal folds with bilateral intrinsic muscle activation.

Many voice disorders are linked to imbalanced muscle activity and known to exhibit asymmetric vocal fold vibration. However, the relation between imbalanced muscle activation and asymmetric vocal fold vibration is not well understood. This study introduces an asymmetric triangular body-cover model of the vocal folds, controlled by the activation of bilateral intrinsic laryngeal muscles, to investigate the effects of muscle imbalance on vocal fold oscillation. Various scenarios were considered, encompassing imbalance in individual muscles and muscle pairs, as well as accounting for asymmetry in lumped element parameters. Measurements of amplitude and phase asymmetries were employed to match the oscillatory behavior of two pathological cases: unilateral paralysis and muscle tension dysphonia. The resulting simulations exhibit muscle imbalance consistent with expectations in the composition of these voice disorders, yielding asymmetries exceeding 30% for paralysis and below 5% for dysphonia. This underscores the relevance of muscle imbalance in representing phonatory scenarios and its potential for characterizing asymmetry in vocal fold vibration.

Dipole- and vortex sheet-based models of fish swimming.

Elucidating the hydrodynamics of fish swimming is critical to identifying the processes underlying fish orientation and schooling. Due to their mathematical tractability, models based on potential flow are preferred in the study of bidirectional interactions of fish with their surroundings. Dipole-based models that assimilate fish to pairs of vortices are particularly enticing, but yet to be thoroughly validated. Here, we embark on a computational fluid dynamics (CFD) campaign informed by experimental data to validate the accuracy of dipole-based models. The locomotory patterns of a fish undergoing carangiform swimming are reconstructed from existing experimental data, which are used as inputs to CFD simulations of a fish swimming in a channel flow. We demonstrate that dipole-based models are accurate in capturing key features of the fluid flow, but cannot predict the elongated flow streamlines around the fish that are evident in CFD. To address this issue, we propose an alternative model that replaces each vortex in the pair with a sheet along the fish length. Using a pair of vortex sheets that span approximately 80% of the fish body length with a separation distance of approximately 50% of the body width, the model is successful in predicting the fluid flow around the swimming fish for a range of background flow speeds and channel widths. The proposed model shows improved accuracy at the cost of a mildly increased computational effort, thereby constituting an ideal basis for research on fish hydrodynamics.

Effect of nodule size and stiffness on phonation threshold and collision pressures in a synthetic hemilaryngeal vocal fold model.

Synthetic vocal fold (VF) replicas were used to explore the role of nodule size and stiffness on kinematic, aerodynamic, and acoustic measures of voiced speech production. Emphasis was placed on determining how changes in collision pressure may contribute to the development of phonotrauma. This was performed by adding spherical beads with different sizes and moduli of elasticity at the middle of the medial surface of synthetic silicone VF models, representing nodules of varying size and stiffness. The VF models were incorporated into a hemilaryngeal flow facility. For each case, self-sustained oscillations were investigated at the phonation threshold pressure. It was found that increasing the nodule diameter increased the open quotient, phonation threshold pressure, and phonation threshold flow rate. However, these values did not change considerably as a function of the modulus of elasticity of the nodule. Nevertheless, the ratio of collision pressure to subglottal pressure increased significantly for both increasing nodule size and stiffness. This suggests that over time, both growth in size and fibrosis of nodules will lead to an increasing cycle of compensatory vocal hyperfunction that accelerates phonotrauma.

Stability of patch-turnover relationships under equilibrium and nonequilibrium metapopulation dynamics driven by biogeography.

Two controversial tenets of metapopulation biology are whether patch quality and the surrounding matrix are more important to turnover (colonisation and extinction) than biogeography (patch area and isolation) and whether factors governing turnover during equilibrium also dominate nonequilibrium dynamics. We tested both tenets using 18 years of surveys for two secretive wetland birds, black and Virginia rails, during (1) a period of equilibrium with stable occupancy and (2) after drought and arrival of West Nile Virus (WNV), which resulted in WNV infections in rails, increased extinction and decreased colonisation probabilities modified by WNV, nonequilibrium dynamics for both species and occupancy decline for black rails. Area (primarily) and isolation (secondarily) drove turnover during both stable and unstable metapopulation dynamics, greatly exceeding the effects of patch quality and matrix conditions. Moreover, slopes between turnover and patch characteristics changed little between equilibrium and nonequilibrium, confirming the overriding influences of biogeographic factors on turnover.

Modeling the influence of COVID-19 protective measures on the mechanics of phonation.

In an effort to mitigate the 2019 novel coronavirus disease pandemic, mask wearing and social distancing have become standard practices. While effective in fighting the spread of the virus, these protective measures have been shown to deteriorate speech perception and sound intensity, which necessitates speaking louder to compensate. The goal of this paper is to investigate via numerical simulations how compensating for mask wearing and social distancing affects measures associated with vocal health. A three-mass body-cover model of the vocal folds (VFs) coupled with the sub- and supraglottal acoustic tracts is modified to incorporate mask and distance dependent acoustic pressure models. The results indicate that sustaining target levels of intelligibility and/or sound intensity while using these protective measures may necessitate increased subglottal pressure, leading to higher VF collision and, thus, potentially inducing a state of vocal hyperfunction, a progenitor to voice pathologies.

Triangular body-cover model of the vocal folds with coordinated activation of the five intrinsic laryngeal muscles.

Poor laryngeal muscle coordination that results in abnormal glottal posturing is believed to be a primary etiologic factor in common voice disorders such as non-phonotraumatic vocal hyperfunction. Abnormal activity of antagonistic laryngeal muscles is hypothesized to play a key role in the alteration of normal vocal fold biomechanics that results in the dysphonia associated with such disorders. Current low-order models of the vocal folds are unsatisfactory to test this hypothesis since they do not capture the co-contraction of antagonist laryngeal muscle pairs. To address this limitation, a self-sustained triangular body-cover model with full intrinsic muscle control is introduced. The proposed scheme shows good agreement with prior studies using finite element models, excised larynges, and clinical studies in sustained and time-varying vocal gestures. Simulations of vocal fold posturing obtained with distinct antagonistic muscle activation yield clear differences in kinematic, aerodynamic, and acoustic measures. The proposed tool is deemed sufficiently accurate and flexible for future comprehensive investigations of non-phonotraumatic vocal hyperfunction and other laryngeal motor control disorders.

Population trends in Vermivora warblers are linked to strong migratory connectivity.

Migratory species can experience limiting factors at different locations and during different periods of their annual cycle. In migratory birds, these factors may even occur in different hemispheres. Therefore, identifying the distribution of populations throughout their annual cycle (i.e., migratory connectivity) can reveal the complex ecological and evolutionary relationships that link species and ecosystems across the globe and illuminate where and how limiting factors influence population trends. A growing body of literature continues to identify species that exhibit weak connectivity wherein individuals from distinct breeding areas co-occur during the nonbreeding period. A detailed account of a broadly distributed species exhibiting strong migratory connectivity in which nonbreeding isolation of populations is associated with differential population trends remains undescribed. Here, we present a range-wide assessment of the nonbreeding distribution and migratory connectivity of two broadly dispersed Nearctic-Neotropical migratory songbirds. We used geolocators to track the movements of 70 Vermivora warblers from sites spanning their breeding distribution in eastern North America and identified links between breeding populations and nonbreeding areas. Unlike blue-winged warblers (Vermivora cyanoptera), breeding populations of golden-winged warblers (Vermivora chrysoptera) exhibited strong migratory connectivity, which was associated with historical trends in breeding populations: stable for populations that winter in Central America and declining for those that winter in northern South America.

Vocal fold dynamics in a synthetic self-oscillating model: Contact pressure and dissipated-energy dose.

The energy dissipated during vocal fold (VF) contact is a predictor of phonotrauma. Difficulty measuring contact pressure has forced prior energy dissipation estimates to rely upon generalized approximations of the contact dynamics. To address this shortcoming, contact pressure was measured in a self-oscillating synthetic VF model with high spatiotemporal resolution using a hemilaryngeal configuration. The approach yields a temporal resolution of less than 0.26 ms and a spatial resolution of 0.254 mm in the inferior-superior direction. The average contact pressure was found to be 32% of the peak contact pressure, 60% higher than the ratio estimated in prior studies. It was found that 52% of the total power was dissipated due to collision. The power dissipated during contact was an order of magnitude higher than the power dissipated due to internal friction during the non-contact phase of oscillation. Both the contact pressure magnitude and dissipated power were found to be maximums at the mid anterior-posterior position, supporting the idea that collision is responsible for the formation of benign lesions, which normally appear at the middle third of the VF.

Physics of phonation offset: Towards understanding relative fundamental frequency observations.

Relative fundamental frequency (RFF) is a promising assessment technique for vocal pathologies. Herein, we explore the underlying laryngeal factors dictating RFF behaviours during phonation offset. To gain physical insights, we analyze a simple impact oscillator model and follow that with a numerical study using the well-established body-cover model of the vocal folds (VFs). Study of the impact oscillator suggests that the observed decrease in fundamental frequency during offset is due, at least in part, to the increase in the neutral gap between the VFs during abduction and the concomitant decrease in collision forces. Moreover, the impact oscillator elucidates a correlation between sharper drops in RFF and increased stiffness of the VFs, supporting experimental RFF studies. The body-cover model study further emphasizes the correlation between the drops in RFF and collision forces. The numerical analysis also illustrates the sensitivity of RFF to abduction initiation time relative to the phase of the phonation cycle, and the abduction period length. In addition, the numerical simulations display the potential role of the cricothyroid muscle to mitigate the RFF reduction. Last, simplified models of phonotraumatic vocal hyperfunction are explored, demonstrating that the observed sharper drops in RFF are associated with increased pre-offset collision forces.

Vocal fold dynamics in a synthetic self-oscillating model: Intraglottal aerodynamic pressure and energy.

Self-sustained oscillations of the vocal folds (VFs) during phonation are the result of the energy exchange between the airflow and VF tissue. Understanding this mechanism requires accurate investigation of the aerodynamic pressures acting on the VF surface during oscillation. A self-oscillating silicone VF model was used in a hemilaryngeal flow facility to measure the time-varying pressure distribution along the inferior-superior thickness of the VF and at four discrete locations in the anterior-posterior direction. It was found that the intraglottal pressures during the opening and closing phases of the glottis are highly dependent on three-dimensional and unsteady flow behaviors. The measured aerodynamic pressures and estimates of the medial surface velocity were used to compute the intraglottal energy transfer from the airflow to the VFs. The energy was greatest at the anterior-posterior midline and decreased significantly toward the anterior/posterior endpoints. The findings provide insight into the dynamics of the VF oscillation and potential causes of some VF disorders.

Continuous forearm cooling attenuates gastrointestinal temperature increase during cycling.

Hot environmental conditions can challenge thermoregulation resulting in exacerbated heat strain. This study evaluated the influence of continuous inner forearm cooling on gastrointestinal temperature (TGI) and physiological responses to exercise in hot (30°C) and humid (relative humidity: 70%) conditions. Eleven trained cyclists (seven male age: 37±12 years; four female age: 41±15 years; mean±standard deviation) performed two experimental trials, cycling at 66% of their self-reported functional threshold power (average work rate over an hour of maximum effort cycling; 175±34W) for 45 minutes in an environmental chamber. One trial employed continuous inner forearm cooling (COOL) with 5°C water passing through aluminum heat exchangers, while the other had no cooling (CONTROL). Heat was removed from the body at an average rate of 30.3±6.6W during the COOL trial resulting in an attenuation of TGI rise (CONTROL: 2.46±0.70, COOL: 2.03±0.63°C·h-1; p=0.002). The change in heart rate from the 10th minute to the end of exercise, as an indicator of cardiovascular drift, was reduced (CONTROL: 20±7, COOL: 17±6beats·min-1; p=0.050) and end-exercise thermal comfort was improved in the COOL trial with a trend for reduced rating of perceived exertion (p=0.055). Findings suggest that continuous cooling of the inner forearms can attenuate the rise of TGI and help mitigate the risk of heat injury during exercise in hot and humid conditions.

Bayesian estimation of vocal function measures using laryngeal high-speed videoendoscopy and glottal airflow estimates: An in vivo case study.

This study introduces the in vivo application of a Bayesian framework to estimate subglottal pressure, laryngeal muscle activation, and vocal fold contact pressure from calibrated transnasal high-speed videoendoscopy and oral airflow data. A subject-specific, lumped-element vocal fold model is estimated using an extended Kalman filter and two observation models involving glottal area and glottal airflow. Model-based inferences using data from a vocally healthy male individual are compared with empirical estimates of subglottal pressure and reference values for muscle activation and contact pressure in the literature, thus providing baseline error metrics for future clinical investigations.

The dopamine D2 receptor can directly recruit and activate GRK2 without G protein activation.

The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) that is critical for many central nervous system functions. The D2R carries out these functions by signaling through two transducers: G proteins and ß-arrestins (ßarrs). Selectively engaging either the G protein or ßarr pathway may be a way to improve drugs targeting GPCRs. The current model of GPCR signal transduction posits a chain of events where G protein activation ultimately leads to ßarr recruitment. GPCR kinases (GRKs), which are regulated by G proteins and whose kinase action facilitates ßarr recruitment, bridge these pathways. Therefore, ßarr recruitment appears to be intimately tied to G protein activation via GRKs. Here we sought to understand how GRK2 action at the D2R would be disrupted when G protein activation is eliminated and the effect of this on ßarr recruitment. We used two recently developed biased D2R mutants that can preferentially interact either with G proteins or ßarrs as well as a ßarr-biased D2R ligand, UNC9994. With these functionally selective tools, we investigated the mechanism whereby the ßarr-preferring D2R achieves ßarr pathway activation in the complete absence of G protein activation. We describe how direct, G protein-independent recruitment of GRK2 drives interactions at the ßarr-preferring D2R and also contributes to ßarr recruitment at the WT D2R. Additionally, we found an additive interaction between the ßarr-preferring D2R mutant and UNC9994. These results reveal that the D2R can directly recruit GRK2 without G protein activation and that this mechanism may have relevance to achieving ßarr-biased signaling.

Comparison of pulse contour, aortic Doppler ultrasound and bioelectrical impedance estimates of stroke volume during rapid changes in blood pressure.

NEW FINDINGS: What is the central question of this study? Pulse contour analysis of the finger arterial pressure by Windkessel modelling is commonly used to estimate stroke volume continuously. But is it valid during dynamic changes in blood pressure? What is the main finding and its importance? Second-by-second analysis revealed that pulse contour analysis underestimated stroke volume by up to 25% after standing from a squat, and 16% after standing thigh-cuff release, when compared with aortic Doppler ultrasound estimates. These results reveal that pulse contour analysis of stroke volume should be interpreted with caution during rapid changes in physiological state. ABSTRACT: Dynamic measurements of stroke volume (SV) and cardiac output provide an index of central haemodynamics during transitional states, such as postural changes and onset of exercise. The most widely used method to assess dynamic fluctuations in SV is the Modelflow method, which uses the arterial blood pressure waveform along with age- and sex-specific aortic properties to compute beat-to-beat estimates of aortic flow. Modelflow has been validated against more direct methods in steady-state conditions, but not during dynamic changes in physiological state, such as active orthostatic stress testing. In the present study, we compared the dynamic SV responses from Modelflow (SVMF ), aortic Doppler ultrasound (SVU/S ) and bioelectrical impedance analysis (SVBIA ) during two different orthostatic stress tests, a squat-to-stand (S-S) transition and a standing bilateral thigh-cuff release (TCR), in 15 adults (six females). Second-by-second analysis revealed that when compared with estimates of SV by aortic Doppler ultrasound, Modelflow underestimated SV by up to 25% from 3 to 11 s after standing from the squat position and by up to 16% from 3 to 7 s after TCR (P < 0.05). The SVMF and SVBIA were similar during the first minute of the S-S transition, but were different 3 s after TCR and at intermittent time points between 34 and 44 s (P < 0.05). These findings indicate that the physiological conditions elicited by orthostatic stress testing violate some of the inherent assumptions of Modelflow and challenge models used to interpret bioelectrical impedance responses, resulting in an underestimation in SV during rapid changes in physiological state.

Haemodynamic and cerebrovascular effects of intermittent lower-leg compression as countermeasure to orthostatic stress.

NEW FINDINGS: What is the central question of this study? Does smartly timed intermittent compression of the lower legs alter cerebral blood velocity and oxygenation during acute orthostatic challenges? What is the main finding and its importance? Intermittent compression timed to the local diastolic phase increased the blood flux through the legs and heart after two different orthostatic stress tests. Cerebral blood velocity improved during the first minute of recovery, and indices of cerebral tissue oxygenation remained elevated for 2 min. These results provide promise for the use of lower-leg active compression as a therapeutic tool for individuals vulnerable to initial orthostatic hypotension and orthostatic stress. ABSTRACT: Intermittent compression of the lower legs provides the possibility of improving orthostatic tolerance by actively promoting venous return and improving central haemodynamics. We tested the hypothesis that intermittent compression of 65 mmHg timed to occur only within the local diastolic phase of each cardiac cycle would attenuate the decrease in blood pressure and improve cerebral haemodynamics during the first minute of recovery from two different orthostatic stress tests. Fourteen subjects (seven female) performed four squat-to-stand transitions and four repeats of standing bilateral thigh-cuff occlusion and release (TCR), with intermittent compression of the lower legs applied in half of the trials. Blood flow in the superficial femoral artery, mean arterial pressure, Doppler ultrasound cardiac output, total peripheral resistance, middle cerebral artery blood velocity (MCAv) and cerebral tissue saturation index (TSI%) were monitored. With both orthostatic stress tests, there was a significant compression × time interaction for superficial femoral artery flow (P < 0.001). The hypotensive state was attenuated with intermittent compression despite decreased total peripheral resistance (squat-to-stand, compression × time interaction, P < 0.001; TCR, compression × time interaction, P = 0.002) as a consequence of elevated cardiac output in both tests (P < 0.001). Intermittent compression also increased MCAv (P = 0.001) and TSI% (P < 0.001) during the squat-to-stand transition and during TCR (MCAv and TSI%, compression × time interaction, P < 0.001). Intermittent compression of the lower legs during quiet standing after an active orthostatic challenge augmented local, central and cerebral haemodynamics, providing potential as a therapeutic tool for individuals vulnerable to orthostatic stress.

The effect of high-speed videoendoscopy configuration on reduced-order model parameter estimates by Bayesian inference.

Bayesian inference has been previously demonstrated as a viable inverse analysis tool for estimating subject-specific reduced-order model parameters and uncertainties. However, previous studies have relied upon simulated glottal area waveforms with superimposed random noise as the measurement. In practice, high-speed videoendoscopy is used to measure glottal area, which introduces practical imaging effects not captured in simulated data, such as viewing angle, frame rate, and camera resolution. Herein, high-speed videos of the vocal folds were approximated by recording the trajectories of physical vocal fold models controlled by a symmetric body-cover model. Twenty videos were recorded, varying subglottal pressure, cricothyroid activation, and viewing angle, with frame rate and video resolution varied by digital video manipulation. Bayesian inference was used to estimate subglottal pressure and cricothyroid activation from glottal area waveforms extracted from the videos. The resulting estimates show off-axis viewing of 10° can lead to a 10% bias in the estimated subglottal pressure. A viewing model is introduced such that viewing angle can be included as an estimated parameter, which alleviates estimate bias. Frame rate and pixel resolution were found to primarily affect uncertainty of parameter estimates up to a limit where spatial and temporal resolutions were too poor to resolve the glottal area. Since many high-speed cameras have the ability to sacrifice spatial for temporal resolution, the findings herein suggest that Bayesian inference studies employing high-speed video should increase temporal resolutions at the expense of spatial resolution for reduced estimate uncertainties.

Elucidation of G-protein and ß-arrestin functional selectivity at the dopamine D2 receptor.

The neuromodulator dopamine signals through the dopamine D2 receptor (D2R) to modulate central nervous system functions through diverse signal transduction pathways. D2R is a prominent target for drug treatments in disorders where dopamine function is aberrant, such as schizophrenia. D2R signals through distinct G-protein and ß-arrestin pathways, and drugs that are functionally selective for these pathways could have improved therapeutic potential. How D2R signals through the two pathways is still not well defined, and efforts to elucidate these pathways have been hampered by the lack of adequate tools for assessing the contribution of each pathway independently. To address this, Evolutionary Trace was used to produce D2R mutants with strongly biased signal transduction for either the G-protein or ß-arrestin interactions. These mutants were used to resolve the role of G proteins and ß-arrestins in D2R signaling assays. The results show that D2R interactions with the two downstream effectors are dissociable and that G-protein signaling accounts for D2R canonical MAP kinase signaling cascade activation, whereas ß-arrestin only activates elements of this cascade under certain conditions. Nevertheless, when expressed in mice in GABAergic medium spiny neurons of the striatum, the ß-arrestin-biased D2R caused a significant potentiation of amphetamine-induced locomotion, whereas the G protein-biased D2R had minimal effects. The mutant receptors generated here provide a molecular tool set that should enable a better definition of the individual roles of G-protein and ß-arrestin signaling pathways in D2R pharmacology, neurobiology, and associated pathologies.