Development and validation of quantitative optical index of skin blood content.

SIGNIFICANCE: We present an approach to estimate with simple instrumentation the amount of red blood cells in the skin microvasculature, designated as parameter LRBC. Variations of parameter LRBC are shown to reflect local changes in the quantity of skin red blood cells during a venous occlusion challenge. AIM: To validate a simple algebraic model of light transport in skin using the Monte Carlo method and to develop a measure of the red blood cell content in skin microvessels using the Monte Carlo predictions; to guide the development of an instrument to measure experimentally variations of the amount of red blood cells in the skin. APPROACH: Monte Carlo simulations were carried out in a multilayer model of the skin to compute remitted light intensities as a function of distance from the illumination locus for different values of the skin blood content. The simulation results were used to compute parameter LRBC and its variations with local skin blood content. An experimental setup was developed to measure parameter LRBC in human volunteers in whom skin blood content of the forearm increased during temporary interruption of the venous outflow. RESULTS: In the simulations, parameter LRBC was ∼16 µm in baseline conditions, and it increased in near proportion with the blood content of the skin layers. Measuring the diffusely reflected light intensity 0.5 to 1.2 mm away from the illumination locus was optimal to detect appreciable changes of the reflected light intensity as skin blood content was altered. Parameter LRBC measured experimentally on the human forearm was 17 ± 2 µm in baseline conditions it increased at a rate of 4 ± 2 µm / min when venous outflow was temporarily interrupted. CONCLUSION: Parameter LRBC derived experimentally with a two-wavelength diffuse reflectometer can be used to measure local variations of the amount of red blood cells in skin microvessels.

Virtual Flipped Class and Laboratories for Medical Electronics Course.

This paper describes the adaptation of a flipped Biomedical Electronics course with laboratories to remote learning at the start of the Covid-19 pandemic. In class collaborative work on problem sets was replaced by group work (4-5 students) in Zoom breakout sessions. When the groups assembled at random for each class had sufficiently progressed on a problem, a detailed solution was typed on the Multisim circuit simulator desktop (National Instruments) shared on the instructor screen. A laboratory project dealing with the development of an electromyograph (EMG) was redesigned for in-depth exploration of each circuit block composing the EMG circuit that was only feasible with the circuit simulator. The students progressed through the remote section of the course at the same rate as they had in the physical classroom in prior years. Student pairs finished a more complete virtual EMG laboratory project without being hampered by manipulation errors that are typical of novices when assembling hardware circuits. We conclude that a flipped biomedical electronics course can successfully be offered remotely and that virtual electronics laboratories that make use of a circuit simulator can provide a complete and meaningful learning experience.

Image-based ß-adrenergic sweat rate assay captures minimal cystic fibrosis transmembrane conductance regulator function.

BACKGROUND: There is a need to prognosticate the severity of cystic fibrosis (CF) detected by newborn screening (NBS) by early assessment of CF transmembrane conductance regulator (CFTR) protein function. We introduce novel instrumentation and protocol for evaluating CFTR activity as reflected by ß-adrenergically stimulated sweat secretion. METHODS: A pixilated image sensor detects sweat rates. Compounds necessary for maximum sweat gland stimulation are applied by iontophoresis, replacing ID injections. Results are compared to a validated ß-adrenergic assay that measures sweat secretion by evaporation (evaporimetry). RESULTS: Ten healthy controls (HC), 6 heterozygous (carriers), 5 with CFTR-related metabolic syndrome (CRMS)/CF screen-positive, inconclusive diagnosis (CFSPID), and 12 CF individuals completed testing. All individuals with minimal and residual function CFTR mutations had low ratios of ß-adrenergically stimulated sweat rate to cholinergically stimulated sweat rate (ß/chol) as measured by either assay. CONCLUSIONS: ß-Adrenergic assays quantitate CFTR dysfunction in the secretory pathway of sweat glands in CF and CRMS/CFSPID populations. This novel image-sensor and iontophoresis protocol detect CFTR function with minimal and residual function and is a feasible test for young children because it is insensible to movement and it decreases the number of injections. It may also assist to distinguish between CF and CRMS/CFSPID diagnosis.

Measurement of Cardiac Output and Blood Volume During Hemodialysis with Fluorescent Dye Dilution Technique.

Intradialytic hypotensive events (IDH) accompanied by deleterious decreases of the cardiac output complicate up to 25% of hemodialysis treatments. Monitoring options available to track hemodynamic changes during hemodialysis have been found ineffective to anticipate the occurrence of IDH. We have assembled opto-electronic instrumentation that uses the fluorescence of a small bolus of indocyanine green dye injected in the hemodialysis circuit to estimate cardiac output and blood volume based on indicator dilution principles in patients receiving hemodialysis. The instrument and technique were tested in 24 adult end-stage renal failure subjects during 64 hemodialysis sessions. A single calibration factor could be used across subjects and across time. Intra-subject variability of the measurements over time was <10%. Stroke volume index (SVI) (mean ± SEM = 34 ± 1 vs. 39 ± 2 mL m-2) and central blood volume (CBV) index (783 ± 36 vs. 881 ± 33 mL m-2) were lower at the beginning of the sessions in which IDH eventually occurred. Cardiac index, SVI, and CBV index decreased with hemodialysis in all treatment sessions but the decrease was more intense in the IDH sessions. We conclude that hemodynamic monitoring can be implemented in patients receiving hemodialysis with minimal disruption of the treatment and could help understand intradialytic hypotension.

Novel technique of a multifunctional electrosurgical system for minimally invasive surgery.

Bipolar electrosurgery in the minimally invasive endoscopic surgery theater has been traditionally limited to the use of standard bipolar forceps, which are minimally versatile, have a limited range of motion, and are associated with visualization and handling constraints. The authors designed a novel surgical device system in which commonly used surgical instruments (suction, microscissors, micrograspers, and dissectors) co-function as individually insulated and modular electrodes for bipolar electrosurgery. In this feasibility study, the successful use of these prototypes in endonasal endoscopic transsphenoidal surgery was demonstrated on 2 human cadavers, and in an in vivo arterial coagulation model on 2 live rats. This prototype system provided improved bipolar instrument mobility, minimized the requirement to exchange surgical instruments when performing electrosurgery, and allowed for new maneuvers that optimized surgical workflow, such as the ability to suction blood and smoke while cauterizing. This multifunctional bipolar cautery system may improve surgical efficiency and workflow and facilitate surgical microdissection and electrocautery during minimally invasive, endoscopic, robotic or traditional open surgery.

Exercise alters resting-state functional connectivity of motor circuits in parkinsonian rats.

Few studies have examined changes in functional connectivity after long-term aerobic exercise. We examined the effects of 4 weeks of forced running wheel exercise on the resting-state functional connectivity (rsFC) of motor circuits of rats subjected to bilateral 6-hydroxydopamine lesion of the dorsal striatum. Our results showed substantial similarity between lesion-induced changes in rsFC in the rats and alterations in rsFC reported in Parkinson's disease subjects, including disconnection of the dorsolateral striatum. Exercise in lesioned rats resulted in: (1) normalization of many of the lesion-induced alterations in rsFC, including reintegration of the dorsolateral striatum into the motor network; (2) emergence of the ventrolateral striatum as a new broadly connected network hub; and (3) increased rsFC among the motor cortex, motor thalamus, basal ganglia, and cerebellum. Our results showed for the first time that long-term exercise training partially reversed lesion-induced alterations in rsFC of the motor circuits, and in addition enhanced functional connectivity in specific motor pathways in the parkinsonian rats, which could underlie recovery in motor functions observed in these animals.

Exercise training reinstates cortico-cortical sensorimotor functional connectivity following striatal lesioning: development and application of a subregional-level analytic toolbox for perfusion autoradiographs of the rat brain.

Current rodent connectome projects are revealing brain structural connectivity with unprecedented resolution and completeness. How subregional structural connectivity relates to subregional functional interactions is an emerging research topic. We describe a method for standardized, mesoscopic-level data sampling from autoradiographic coronal sections of the rat brain, and for correlation-based analysis and intuitive display of cortico-cortical functional connectivity (FC) on a flattened cortical map. A graphic user interface "Cx-2D" allows for the display of significant correlations of individual regions-of-interest, as well as graph theoretical metrics across the cortex. Cx-2D was tested on an autoradiographic data set of cerebral blood flow (CBF) of rats that had undergone bilateral striatal lesions, followed by 4 weeks of aerobic exercise training or no exercise. Effects of lesioning and exercise on cortico-cortical FC were examined during a locomotor challenge in this rat model of Parkinsonism. Subregional FC analysis revealed a rich functional reorganization of the brain in response to lesioning and exercise that was not apparent in a standard analysis focused on CBF of isolated brain regions. Lesioned rats showed diminished degree centrality of lateral primary motor cortex, as well as neighboring somatosensory cortex-changes that were substantially reversed in lesioned rats following exercise training. Seed analysis revealed that exercise increased positive correlations in motor and somatosensory cortex, with little effect in non-sensorimotor regions such as visual, auditory, and piriform cortex. The current analysis revealed that exercise partially reinstated sensorimotor FC lost following dopaminergic deafferentation. Cx-2D allows for standardized data sampling from images of brain slices, as well as analysis and display of cortico-cortical FC in the rat cerebral cortex with potential applications in a variety of autoradiographic and histologic studies.

Functional brain activation during retrieval of visceral pain-conditioned passive avoidance in the rat.

This study assessed functional brain activation in rats during expectation of visceral pain. Male rats were trained in step-down passive avoidance (PA) for 2 days. Upon stepping down from a platform, conditioned animals received noxious colorectal distension delivered through a colorectal balloon, whereas the balloon in control rats remained uninflated. On day 3, PA behavior was assessed while [(14)C]-iodoantipyrine was infused intravenously, followed by immediate euthanasia. Regional cerebral blood flow-related tissue radioactivity (rCBF) was analyzed by statistical parametric mapping using 3-dimensional brains reconstructed from autoradiographic brain slice images. Associated with retrieved PA behavior, conditioned rats compared with control subjects showed increases in rCBF in sensory (anterior insula, somatosensory cortex), limbic/paralimbic regions (anterior cingulate, prelimbic cortex, amygdala), all regions previously reported to show activation during acute visceral pain. Increases in rCBF were also noted in the dorsal hippocampus, nucleus accumbens, and caudate putamen, regions associated with retrieval of PA. Organization of the underlying brain network was further delineated by functional connectivity analysis. This revealed in conditioned rats a strongly and positively connected corticostriatal cluster (cingulate, prelimbic cortex, caudate putamen). The amygdala and cerebellar hemispheres formed another positively connected cluster, which was negatively connected with the corticostriatal cluster, suggesting corticolimbic modulation. Prelimbic cortex, nucleus accumbens, and anterior insula emerged in conditioned animals as hubs. Our results show that during retrieval of PA, brain areas implicated in PA expression as well as those implicated in acute visceral pain processing were recruited, in line with findings from human brain imaging studies on pain expectation.

Antenatal maternal stress alters functional brain responses in adult offspring during conditioned fear.

Antenatal maternal stress has been shown in rodent models and in humans to result in altered behavioral and neuroendocrine responses, yet little is known about its effects on functional brain activation. Pregnant female rats received a daily foot-shock stress or sham-stress two days after testing plug-positive and continuing for the duration of their pregnancy. Adult male offspring (age 14 weeks) with and without prior maternal stress (MS) were exposed to an auditory fear conditioning (CF) paradigm. Cerebral blood flow (CBF) was assessed during recall of the tone cue in the nonsedated, nontethered animal using the ((14))C-iodoantipyrine method, in which the tracer was administered intravenously by remote activation of an implantable minipump. Regional CBF distribution was examined by autoradiography and analyzed by statistical parametric mapping in the three-dimensionally reconstructed brains. Presence of fear memory was confirmed by behavioral immobility ("freezing"). Corticosterone plasma levels during the CF paradigm were measured by ELISA in a separate group of rats. Antenatal MS exposure altered functional brain responses to the fear conditioned cue in adult offspring. Rats with prior MS exposure compared to those without demonstrated heightened fear responsivity, exaggerated and prolonged corticosterone release, increased functional cerebral activation of limbic/paralimbic regions (amygdala, ventral hippocampus, insula, ventral striatum, and nucleus accumbens), the locus coeruleus, and white matter, and deactivation of medial prefrontal cortical regions. Dysregulation of corticolimbic circuits may represent risk factors in the future development of anxiety disorders and associated alterations in emotional regulation.

Powering an implantable minipump with a multi-layered printed circuit coil for drug infusion applications in rodents.

We report the use of a multi-layer printed coil circuit for powering (36-94 mW) an implantable microbolus infusion pump (MIP) that can be activated remotely for use in drug infusion in nontethered, freely moving small animals. This implantable device provides a unique experimental tool with applications in the fields of animal behavior, pharmacology, physiology, and functional brain imaging. Two different designs are described: a battery-less pump usable when the animal is inside a home-cage surrounded by a primary inductive coil and a pump powered by a rechargeable battery that can be used for studies outside the home-cage. The use of printed coils for powering of small devices by inductive power transfer presents significant advantages over similar approaches using hand-wound coils in terms of ease of manufacturing and uniformity of design. The high efficiency of a class-E oscillator allowed powering of the minipumps without the need for close physical contact of the primary and secondary coils, as is currently the case for most devices powered by inductive power transfer.

A low power, on demand electrothermal valve for wireless drug delivery applications.

We present a low power, on demand Parylene MEMS electrothermal valve. A novel Omega-shaped thermal resistive element requires low power (approximately mW) and enables rapid valve opening (approximately ms). Using both finite element analysis and valve opening experiments, a robust resistive element design for improved valve opening performance in water was obtained. In addition, a thermistor, as an inrush current limiter, was added into the valve circuit to provide variable current ramping. Wireless activation of the valve using RF inductive power transfer was demonstrated.

Estimation of indocyanine green concentration in blood from fluorescence emission: application to hemodynamic assessment during hemodialysis.

There is considerable interest in assessing cardiovascular function noninvasively in patients receiving hemodialysis. A possible approach is to measure the blood concentration of bolus-injected indocyanine green dye and to apply the dye-dilution method for estimating cardiac output and blood volume. Blood ICG concentration can be derived from a measurement of the ICG fluorescence through the dialysis tubing if a simple and unique calibration relationship can be established between transmural fluorescence intensity and blood ICG concentration. We investigated this relationship using Monte Carlo simulations of light transport in blood with varying hematocrit and ICG concentrations and performed empiric measurements of optical absorption and ICG fluorescence emission to confirm our findings. The ICG fluorescence intensity measured at the blood surface, as well as the light intensity remitted by the blood, varied as hematocrit changes modified the absorption and scattering characteristics of the blood. Calibration relationships were developed between fluorescence intensity and ICG concentration that accounted for hematocrit changes. Combining the backreflected fluorescence and the reflected light measured near the point of illumination provided optimal signal intensity, linearity, and robustness to hematocrit changes. These results provide a basis for developing a noninvasive approach to derive optically circulating blood ICG concentration in hemodialysis circuits.

Sex differences in functional brain activation during noxious visceral stimulation in rats.

Studies in healthy human subjects and patients with irritable bowel syndrome suggest sex differences in cerebral nociceptive processing. Here we examine sex differences in functional brain activation in the rat during colorectal distention (CRD), a preclinical model of acute visceral pain. [(14)C]-iodoantipyrine was injected intravenously in awake, non-restrained female rats during 60- or 0-mmHg CRD while electromyographic abdominal activity (EMG) and pain behavior were recorded. Regional cerebral blood flow-related tissue radioactivity was analyzed by statistical parametric mapping from autoradiographic images of three-dimensionally reconstructed brains. Sex differences were addressed by comparing the current data with our previously published data collected from male rats. While sex differences in EMG and pain scores were modest, significant differences were noted in functional brain activation. Females showed widespread changes in limbic (amygdala, hypothalamus) and paralimbic structures (ventral striatum, nucleus accumbens, raphe), while males demonstrated broad cortical changes. Sex differences were apparent in the homeostatic afferent network (parabrachial nucleus, thalamus, insular and dorsal anterior cingulate cortices), in an emotional-arousal network (amygdala, locus coeruleus complex), and in cortical areas modulating these networks (prefrontal cortex). Greater activation of the ventromedial prefrontal cortex and broader limbic/paralimbic changes in females suggest greater engagement of affective mechanisms during visceral pain. Greater cortical activation in males is consistent with the concept of greater cortical inhibitory effects on limbic structures in males, which may relate to differences in attentional and cognitive attribution to visceral stimuli. These findings show remarkable similarities to reported sex differences in brain responses to visceral stimuli in humans.

A Parylene MEMS Electrothermal Valve.

The first microelectromechanical-system normally closed electrothermal valve constructed using Parylene C is described, which enables both low power (in milliwatts) and rapid operation (in milliseconds). This low-power valve is well suited for applications in wirelessly controlled implantable drug-delivery systems. The simple design was analyzed using both theory and modeling and then characterized in benchtop experiments. Operation in air (constant current) and water (current ramping) was demonstrated. Valve-opening powers of 22 mW in air and 33 mW in water were obtained. Following integration of the valve with catheters, our valve was applied in a wirelessly operated microbolus infusion pump, and the in vivo functionality for the appropriateness of use of this pump for future brain mapping applications in small animals was demonstrated.

Regional brain activation in conscious, nonrestrained rats in response to noxious visceral stimulation.

Preclinical drug development for visceral pain has largely relied on quantifying pseudoaffective responses to colorectal distension (CRD) in restrained rodents. However, the predictive value of changes in simple reflex responses in rodents for the complex human pain experience is not known. Male rats were implanted with venous cannulas and with telemetry transmitters for abdominal electromyographic (EMG) recordings. [(14)C]-iodoantipyrine was injected during noxious CRD (60 mmHg) in the awake, nonrestrained animal. Regional cerebral blood flow (rCBF)-related tissue radioactivity was quantified by autoradiography and analyzed in the three-dimensionally reconstructed brain by statistical parametric mapping. 60-mmHg CRD, compared with controls (0 mmHg) evoked significant increases in EMG activity (267+/-24% vs. 103+/-8%), as well as in behavioral pain score (77+/-6% vs. 3+/-3%). CRD elicited significant increases in rCBF as expected in sensory (insula, somatosensory cortex), and limbic and paralimbic regions (including anterior cingulate cortex and amygdala). Significant decreases in rCBF were seen in the thalamus, parabrachial nucleus, periaqueductal gray, hypothalamus and pons. Correlations of rCBF with EMG and with behavioral pain score were noted in the cingulate, insula, lateral amygdala, dorsal striatum, somatosensory and motor regions. Our findings support the validity of measurements of cerebral perfusion during CRD in the freely moving rat as a model of functional brain changes in human visceral pain. However, not all regions demonstrating significant group differences correlated with EMG or behavioral measures. This suggests that functional brain imaging captures more extensive responses of the central nervous system to noxious visceral distension than those identified by traditional measures.

Fluorescence dilution technique for measurement of cardiac output and circulating blood volume in healthy human subjects.

BACKGROUND: The authors previously validated in an animal model a new indicator dilution technique for measuring cardiac output and circulating blood volume by recording transcutaneously the fluorescence of circulating indocyanine green with an optical probe placed on the skin surface. The current study compared fluorescence dilution recordings recorded from several locations on the human face in terms of signal intensity and stability and estimated the subjects' cardiac output and circulating blood volume from the recordings. METHODS: Fluorescence dilution traces were recorded transcutaneously in six healthy human volunteers after rapid intravenous injection of 1 mg indocyanine green. Three placements of the optical probe were tested: the nose ala, the ear lobe, and the temple area. In three subjects, the recordings were calibrated in terms of circulating indocyanine green concentration to estimate cardiac output and circulating blood volume. RESULTS: Fluorescence dilution traces had the same duration for the three locations, but the recordings obtained from the nose ala and the ear lobe were twice as intense as those from the temple. The fluorescence intensity at each site was linearly related to the local laser Doppler perfusion index. The coefficient of variation for the area under the first pass curve (inversely proportional to cardiac output) was approximately 6% for triplicate measurements at the same location. Cardiac output and circulating blood volume derived from the fluorescence recordings were in the normal range. CONCLUSIONS: The study demonstrates that intense and reproducible fluorescence dilution signals can be measured transcutaneously in healthy humans and could potentially be used to measure cardiac output and circulating blood volume minimally invasively.

Transcutaneous RF-powered implantable minipump driven by a class-E transmitter.

We describe the design and testing of an inductive coupling system used to power an implantable minipump for applications in ambulating rats. A 2 MHz class-E oscillator driver powered a coil transmitter wound around a 33-cm-diameter rat cage. A receiver coil, a filtered rectifier, and a voltage-sensitive switch powered the implant. The implant DC current at the center of the primary coil (5.1 V) exceeded the level required to activate the solenoid valve in the pump. The variations of the implant current in the volume of the primary coil reflected the variations of the estimated coupling coefficient between the two coils. The pump could be activated in-vivo, while accommodating the vertical and horizontal movements of the animal. Advantages of this design include a weight reduction for the implant, an operation independent from a finite power source, and a remote activation/deactivation.

In vivo quantitative assessment of catheter patency in rats.

Formation of fibrin sleeves around catheter tips is a central factor in catheter failure during chronic implantation, and such tissue growth can occur despite administration of anticoagulants. We developed a novel method for monitoring catheter patency. This method recognizes the progressive nature of catheter occlusion, and tracks this process over time through measurement of changes in catheter resistance to a standardized 1 mL bolus infusion from a pressurized reservoir. Two indirect measures of catheter patency were used: (a) reservoir residual pressure and (b) reservoir discharge time. This method was applied to the study of catheter patency in rats comparing the effect of catheter material (silastic, polyurethane, Microrenathanetrade mark), lock solution (heparin, heparin/dexamethasone) and two different cannulation sites (superior vena cava via the external jugular vein, inferior vena cava via the femoral vein). Our findings reveal that application of flexible smaller-size silastic catheters and a dexamethasone lock solution resulted in prolonged catheter patency. Patency could be maintained over nine weeks with the femoral vein catheters, compared with five weeks with the external jugular vein catheters. The current method for measuring catheter patency provides a useful index for the assessment of tissue growth around the catheter tip. The method also provides an objective and quantitative way of comparing changes in catheter patency for different surgical methods and catheter types. Our method improves on the conventional method of assessing catheter occlusion by judging the ability to aspirate from the catheter.

Transcutaneous fluorescence dilution cardiac output and circulating blood volume during hemorrhagic hypovolemia.

BACKGROUND: Cardiac output and circulating blood volume are important parameters for assessing cardiac function in the intensive care setting and during major surgeries. The authors tested in an animal model of hemorrhagic hypovolemia the feasibility of measuring these parameters simultaneously by transcutaneous fluorescence monitoring of an intravenous bolus injection of indocyanine green. METHODS: Fluorescence dilution cardiac output was measured in seven anesthetized rabbits and compared to thermodilution cardiac output. The optical probe used to excite the indocyanine green fluorescence was in contact with the skin above the ear artery. Local heating enhanced blood perfusion of the measurement site. Cardiac output was measured during baseline conditions, during hemorrhagic hypovolemia, and after partial restoration of the blood volume with reinfused blood. Estimates of the circulating blood volume were simultaneously obtained from the analysis of the fluorescence dilution traces. RESULTS: Cardiac output measured by fluorescence dilution (thermodilution) averaged 455 +/- 16 (450 +/- 13) ml/min in baseline conditions and 323 +/- 15 (330 +/- 13) ml/min during hypovolemia. Fluorescence dilution cardiac output was linearly related to thermodilution cardiac output (slope = 1.13 +/- 0.05, ordinate = -50 +/- 19 ml/min, R = 0.92). Interanimal differences explained most of the variance between cardiac output estimates obtained with the two techniques. Circulating blood volume decreased from 204 +/- 5 ml in baseline conditions to 174 +/- 8 ml after bleeding and reflected blood volume changes in this acute bleeding-reinfusion model. CONCLUSIONS: The study extends the applicability of the fluorescence dilution technique for cardiac output measurement to hypovolemic conditions and demonstrates its ability to produce accurate estimates of the circulating blood volume in experimental animals.

Mapping brain function in freely moving subjects.

Expression of many fundamental mammalian behaviors such as, for example, aggression, mating, foraging or social behaviors, depend on locomotor activity. A central dilemma in the functional neuroimaging of these behaviors has been the fact that conventional neuroimaging techniques generally rely on immobilization of the subject, which extinguishes all but the simplest activity. Ideally, imaging could occur in freely moving subjects, while presenting minimal interference with the subject's natural behavior. Here we provide an overview of several approaches that have been undertaken in the past to achieve this aim in both tethered and freely moving animals, as well as in nonrestrained human subjects. Applications of specific radiotracers to single photon emission computed tomography and positron emission tomography are discussed in which brain activation is imaged after completion of the behavioral task and capture of the tracer. Potential applications to clinical neuropsychiatry are discussed, as well as challenges inherent to constraint-free functional neuroimaging. Future applications of these methods promise to increase our understanding of the neural circuits underlying mammalian behavior in health and disease.