Search result for : author:samuel s-h wang

Total 21 result(s) found

Normal cognitive and social development require posterior cerebellar activity.

Cognitive and social capacities require postnatal experience, yet the pathways by which experience guides development are unknown. Here we show that the normal development of motor and nonmotor capacities requires cerebellar activity. Using chemogenetic perturbation of molecular layer interneurons to attenuate cerebellar output in mice, we found that activity of posterior regions in juvenile life modulates adult expression of eyeblink conditioning (paravermal lobule VI, crus I), reversal learning (lobule VI), persistive behavior and novelty-seeking (lobule VII), and social preference (crus I/II). Perturbation in adult life altered only a subset of phenotypes. Both adult and juvenile disruption left gait metrics largely unaffected. Contributions to phenotypes increased with the amount of lobule inactivated. Using an anterograde transsynaptic tracer, we found that posterior cerebellum made strong connections with prelimbic, orbitofrontal, and anterior cingulate cortex. These findings provide anatomical substrates for the clinical observation that cerebellar injury increases the risk of autism.

Aleksandra Badura, Jessica L Verpeut, Julia W Metzger, Talmo D Pereira, Thomas J Pisano, Ben Deverett, Dariya E Bakshinskaya, Samuel S-H Wang

Reliable coding emerges from coactivation of climbing fibers in microbands of cerebellar Purkinje neurons.

The inferior olive projects climbing fiber axons to cerebellar Purkinje neurons, where they trigger calcium-based dendritic spikes. These responses dynamically shape the immediate spike output of Purkinje cells as well as provide an instructive signal to guide long-term plasticity. Climbing fibers typically fire approximately once a second, and the instructive role is distributed over many such firing events. However, transmission of salient information on an immediate basis needs to occur on a shorter timescale during which a Purkinje cell would typically be activated by a climbing fiber only once. Here we show using in vivo calcium imaging in anesthetized mice and rats that sensory events are rapidly and reliably represented by momentary, simultaneous coactivation of microbands of adjacent Purkinje cells. Microbands were sagittally oriented and spanned up to 100 microm mediolaterally, representing hundreds of Purkinje cells distributed over multiple folia. Spontaneous and sensory-evoked microbands followed boundaries that were close or identical to one another and were desynchronized by olivary injection of the gap junction blocker mefloquine, indicating that excitation to the olive is converted to synchronized firing by electrical coupling. One-time activation of microbands could distinguish a sensory response from spontaneous activity with up to 98% accuracy. Given the anatomy of the olivocerebellar system, microband synchrony may shape the output of neurons in the cerebellar nuclei either via powerful inhibition by Purkinje cells or by direct monosynaptic excitation from the inferior olive.

Ilker Ozden, Megan R Sullivan, H Megan Lee, Samuel S-H Wang

Radially expanding transglial calcium waves in the intact cerebellum.

Multicellular glial calcium waves may locally regulate neural activity or brain energetics. Here, we report a diffusion-driven astrocytic signal in the normal, intact brain that spans many astrocytic processes in a confined volume without fully encompassing any one cell. By using 2-photon microscopy in rodent cerebellar cortex labeled with fluorescent indicator dyes or the calcium-sensor protein G-CaMP2, we discovered spontaneous calcium waves that filled approximately ellipsoidal domains of Bergmann glia processes. Waves spread in 3 dimensions at a speed of 4-11 microm/s to a diameter of approximately 50 microm, slowed during expansion, and were reversibly blocked by P2 receptor antagonists. Consistent with the hypothesis that ATP acts as a diffusible trigger of calcium release waves, local ejection of ATP triggered P2 receptor-mediated waves that were refractory to repeated activation. Transglial waves represent a means for purinergic signals to act with local specificity to modulate activity or energetics in local neural circuits.

Tycho M Hoogland, Bernd Kuhn, Werner Göbel, Wenying Huang, Junichi Nakai, Fritjof Helmchen, Jane Flint, Samuel S-H Wang

Identification and clustering of event patterns from in vivo multiphoton optical recordings of neuronal ensembles.

In vivo multiphoton fluorescence microscopy allows imaging of cellular structures in brain tissue to depths of hundreds of micrometers and, when combined with the use of activity-dependent indicator dyes, opens the possibility of observing intact, functioning neural circuitry. We have developed tools for analyzing in vivo multiphoton data sets to identify responding structures and events in single cells as well as patterns of activity within the neural ensemble. Data were analyzed from populations of cerebellar Purkinje cell dendrites, which generate calcium-based complex action potentials. For image segmentation, active dendrites were identified using a correlation-based method to group covarying pixels. Firing events were extracted from dendritic fluorescence signals with a 95% detection rate and an 8% false-positive rate. Because an event that begins in one movie frame is sometimes not detected until the next frame, detection delays were compensated using a likelihood-based correction procedure. To identify groups of dendrites that tended to fire synchronously, a k-means-based procedure was developed to analyze pairwise correlations across the population. Because repeated runs of k-means often generated dissimilar clusterings, the runs were combined to determine a consensus cluster number and composition. This procedure, termed meta-k-means, gave clusterings as good as individual runs of k-means, was independent of random initial seeding, and allowed the exclusion of outliers. Our methods should be generally useful for analyzing multicellular activity recordings in a variety of brain structures.

Ilker Ozden, H Megan Lee, Megan R Sullivan, Samuel S-H Wang

Order-dependent coincidence detection in cerebellar Purkinje neurons at the inositol trisphosphate receptor.

Associative long-term depression (LTD) at cerebellar parallel fiber-Purkinje cell synapses is sensitive to the temporal order in which the parallel fiber is coactivated with the climbing fiber input, but how order sensitivity is achieved is unknown. Here we show that the cerebellar inositol-1,4,5-trisphosphate (IP3) receptor, whose activation is required for LTD induction, is sensitive in situ to the order of presentation of its coagonists, IP3 and cytoplasmic calcium. By focally photolyzing a novel caged IP3 compound in dendritic spines, we find that pairing IP3 with climbing fiber-mediated calcium entry leads to a large calcium release transient if the climbing fiber is activated up to 100 ms before or up to 500 ms after IP3 uncaging. This asymmetric timing window for coactivation follows the kinetics of calcium removal and IP3 unbinding from the receptor and is not limited by IP3 metabolism. IP3 receptor binding thus acts as an eligibility trace that can drive temporal order-dependent calcium release and LTD induction in Purkinje cells and event order-dependent sensory plasticity in the whole animal.

Dmitry V Sarkisov, Samuel S-H Wang

Synapse specificity of calcium release probed by chemical two-photon uncaging of inositol 1,4,5-trisphosphate.

Biological messengers can be "caged" by adding a single photosensitive group that can be photolyzed by a light flash to achieve spatially and temporally precise biochemical control. Here we report that photolysis of a double-caged form of the second messenger inositol 1,4,5-trisphosphate (IP3) triggers focal calcium release in Purkinje cell somata, dendrites, and spines as measured by two-photon microscopy. In calbindin knock-out Purkinje cells, peak calcium increased with flash energy with higher cooperativity for double-caged IP3 than for conventional single-caged IP3, consistent with a chemical two-photon effect. Spine photolysis of double-caged IP3 led to local calcium release. Uncaging of glycerophosphoryl-myo-inositol 4,5-bisphosphate (gPIP2), a poorly metabolizable IP3 analog, led to less well localized release. Thus, IP3 breakdown is necessary for spine-specificity. IP3- and gPIP2-evoked signals declined from peak with similar, slow time courses, indicating that release lasts hundreds of milliseconds and is terminated not by IP3 degradation but by intrinsic receptor dynamics. Based on measurements of spine-dendrite coupling, IP3-evoked calcium signals are expected to be at least 2.4-fold larger in their spine of origin than in nearby spines, allowing IP3 to act as a synapse-specific second messenger. Unexpectedly, single-caged IP3 led to less release in somata and was ineffective in dendrites and spines. Calcium release using caged gPIP2 was inhibited by the addition of single-caged IP3, suggesting that single-caged IP3 is an antagonist of calcium release. Caging at multiple sites may be an effective general approach to reducing residual receptor interaction.

Dmitry V Sarkisov, Shari E Gelber, Jeffery W Walker, Samuel S-H Wang

In vivo calcium imaging of circuit activity in cerebellar cortex.

In vivo two-photon calcium imaging provides the opportunity to monitor activity in multiple components of neural circuitry at once. Here we report the use of bulk-loading of fluorescent calcium indicators to record from axons, dendrites, and neuronal cell bodies in cerebellar cortex in vivo. In cerebellar folium crus IIa of anesthetized rats, we imaged the labeled molecular layer and identified all major cellular structures: Purkinje cells, interneurons, parallel fibers, and Bergmann glia. Using extracellular stimuli we evoked calcium transients corresponding to parallel fiber beam activity. This beam activity triggered prolonged calcium transients in interneurons, consistent with in vitro evidence for synaptic activation of N-methyl-d-aspartate receptors via glutamate spillover. We also observed spontaneous calcium transients in Purkinje cell dendrites that were identified as climbing-fiber-evoked calcium spikes by their size, time course, and sensitivity to AMPA receptor antagonist. Two-photon calcium imaging of bulk-loaded cerebellar cortex is thus well suited to optically monitor synaptic processing in the intact cerebellum.

Megan R Sullivan, Axel Nimmerjahn, Dmitry V Sarkisov, Fritjof Helmchen, Samuel S-H Wang

Speed limits in the cerebellum: constraints from myelinated and unmyelinated parallel fibers.

Cerebellar parallel fibers are among the thinnest known vertebrate axons and represent an extreme anatomical adaptation. Until now a systematic examination of their properties across species has not been carried out. We used transmission electron microscopy and light microscopy to compare parallel fibers in mammals of different brain sizes. From mouse to macaque, the average unmyelinated parallel fiber diameter was 0.2-0.3 microm, consistent with the idea that they are evolutionarily selected for compactness. Average unmyelinated parallel fiber diameter scaled up slightly with brain size, and across species the estimated total conduction time is 5-10 ms. However, these conduction times can vary by milliseconds, and unmyelinated PFs consume large amounts of energy per action potential. These functional disadvantages are overcome in myelinated parallel fibers, which we found in the deep regions nearest the Purkinje cell layer in marmoset, cat and macaque. These axons were 0.4-1.1 microm wide, have expected conduction times of 0.5-1.0 ms, and may convey fast feedforward inhibition via basket cells to Purkinje cells.

Krysta D Wyatt, Patima Tanapat, Samuel S-H Wang

Sensory-driven enhancement of calcium signals in individual Purkinje cell dendrites of awake mice.

Climbing fibers (CFs) are thought to contribute to cerebellar plasticity and learning by triggering a large influx of dendritic calcium in the postsynaptic Purkinje cell (PC) to signal the occurrence of an unexpected sensory event. However, CFs fire about once per second whether or not an event occurs, raising the question of how sensory-driven signals might be distinguished from a background of ongoing spontaneous activity. Here, we report that in PC dendrites of awake mice, CF-triggered calcium signals are enhanced when the trigger is a sensory event. In addition, we show that a large fraction of the total enhancement in each PC dendrite can be accounted for by an additional boost of calcium provided by sensory activation of a non-CF input. We suggest that sensory stimulation may modulate dendritic voltage and calcium concentration in PCs to increase the strength of plasticity signals during cerebellar learning.

Farzaneh Najafi, Andrea Giovannucci, Samuel S-H Wang, Javier F Medina

Fast calcium sensor proteins for monitoring neural activity.

A major goal of the BRAIN Initiative is the development of technologies to monitor neuronal network activity during active information processing. Toward this goal, genetically encoded calcium indicator proteins have become widely used for reporting activity in preparations ranging from invertebrates to awake mammals. However, slow response times, the narrow sensitivity range of Ca and in some cases, poor signal-to-noise ratio still limit their usefulness. Here, we review recent improvements in the field of neural activity-sensitive probe design with a focus on the GCaMP family of calcium indicator proteins. In this context, we present our newly developed Fast-GCaMPs, which have up to 4-fold accelerated off-responses compared with the next-fastest GCaMP, GCaMP6f. Fast-GCaMPs were designed by destabilizing the association of the hydrophobic pocket of calcium-bound calmodulin with the RS20 binding domain, an intramolecular interaction that protects the green fluorescent protein chromophore. Fast-GCaMP6f-RS06 and Fast-GCaMP6f-RS09 have rapid off-responses in stopped-flow fluorimetry, in neocortical brain slices, and in the intact cerebellum . Fast-GCaMP6f variants should be useful for tracking action potentials closely spaced in time, and for following neural activity in fast-changing compartments, such as axons and dendrites. Finally, we discuss strategies that may allow tracking of a wider range of neuronal firing rates and improve spike detection.

Aleksandra Badura, Xiaonan Richard Sun, Andrea Giovannucci, Laura A Lynch, Samuel S-H Wang

Synthesis and biological evaluation of bis-CNB-GABA, a photoactivatable neurotransmitter with low receptor interference and chemical two-photon uncaging properties.

Photoactivatable "caged" neurotransmitters allow optical control of neural tissue with high spatial and temporal precision. However, the development of caged versions of the chief vertebrate inhibitory neurotransmitter, γ-amino butyric acid (GABA), has been limited by the propensity of caged GABAs to interact with GABA receptors. We describe herein the synthesis and application of a practically useful doubly caged GABA analog, termed bis-α-carboxy-2-nitrobenzyl-GABA (bis-CNB-GABA). Uncaging of bis-CNB-GABA evokes inward GABAergic currents in cerebellar molecular layer interneurons with rise times of 2 ms, comparable to flash duration. Response amplitudes depend on the square of flash intensity, as expected for a chemical two-photon uncaging effect. Importantly, prior to uncaging, bis-CNB-GABA is inactive at the GABAA receptor, evoking no changes in holding current in voltage-clamped neurons and showing an IC50 of at least 2.5 mM as measured using spontaneous GABAergic synaptic currents. Bis-CNB-GABA is stable in solution, with an estimated half-life of 98 days in the light. We expect that bis-CNB-GABA will prove to be an effective tool for high-resolution chemical control of brain circuits.

Diana D Shi, Federico F Trigo, Martin F Semmelhack, Samuel S-H Wang

Coding of stimulus strength via analog calcium signals in Purkinje cell dendrites of awake mice.

The climbing fiber input to Purkinje cells acts as a teaching signal by triggering a massive influx of dendritic calcium that marks the occurrence of instructive stimuli during cerebellar learning. Here, we challenge the view that these calcium spikes are all-or-none and only signal whether the instructive stimulus has occurred, without providing parametric information about its features. We imaged ensembles of Purkinje cell dendrites in awake mice and measured their calcium responses to periocular airpuffs that serve as instructive stimuli during cerebellar-dependent eyeblink conditioning. Information about airpuff duration and pressure was encoded probabilistically across repeated trials, and in two additional signals in single trials: the synchrony of calcium spikes in the Purkinje cell population, and the amplitude of the calcium spikes, which was modulated by a non-climbing fiber pathway. These results indicate that calcium-based teaching signals in Purkinje cells contain analog information that encodes the strength of instructive stimuli trial-by-trial.

Farzaneh Najafi, Andrea Giovannucci, Samuel S-H Wang, Javier F Medina

The cerebellum, sensitive periods, and autism.

Cerebellar research has focused principally on adult motor function. However, the cerebellum also maintains abundant connections with nonmotor brain regions throughout postnatal life. Here we review evidence that the cerebellum may guide the maturation of remote nonmotor neural circuitry and influence cognitive development, with a focus on its relationship with autism. Specific cerebellar zones influence neocortical substrates for social interaction, and we propose that sensitive-period disruption of such internal brain communication can account for autism's key features.

Samuel S-H Wang, Alexander D Kloth, Aleksandra Badura

Calcium-based dendritic excitability and its regulation in the deep cerebellar nuclei.

The deep cerebellar nuclei (DCN) convey the final output of the cerebellum and are a major site of activity-dependent plasticity. Here, using patch-clamp recording and two-photon calcium imaging in rat brain slices, we demonstrate that DCN dendrites exhibit three hallmarks of active amplification of electrical signals. First, they produce calcium transients with rise times of tens of milliseconds, comparable in amplitude and duration to calcium spikes in other neurons. Second, calcium signal amplitudes are undiminished along the length of dendrites to the farthest distances from the soma. Third, they can generate calcium signals even in the presence of tetrodotoxin, a sodium channel blocker that abolishes somatic action potential initiation. DCN calcium transients do require the action of T-type calcium channels, a common voltage-gated conductance in excitable dendrites. Dendritic calcium influx was evoked by release from hyperpolarization, peaked within tens of milliseconds, and was observed in both transient- and weak-rebound-firing neurons. In a survey across the DCN, transient-burst rebound firing, which was accompanied by the most rapid calcium flux, was more common in lateral nucleus than in interpositus nucleus and was not seen in medial nucleus. Rebound firing and calcium transients were not present in animals shipped 1-3 days before recording, a condition associated with elevated maternal and pup corticosterone and reduced pup body weight. Rebounds could be restored by the protein kinase C activator phorbol 12-myristate-13-acetate. Thus local calcium-based dendritic excitability supports a stage of presomatic amplification that is under regulation by stress and neuromodulatory influence.

Eve R Schneider, Eugene F Civillico, Samuel S-H Wang

An amplified promoter system for targeted expression of calcium indicator proteins in the cerebellar cortex.

Recording of identified neuronal network activity using genetically encoded calcium indicators (GECIs) requires labeling that is cell type-specific and bright enough for the detection of functional signals. However, specificity and strong expression are often not achievable using the same promoter. Here we present a combinatorial approach for targeted expression and single-cell-level quantification in which a weak promoter is used to drive trans-amplification under a strong general promoter. We demonstrated this approach using recombinant adeno-associated viruses (rAAVs) to deliver the sequence of the GECI D3cpv in the mouse cerebellar cortex. Direct expression under the human synapsin promoter (hSYN) led to high levels of expression (50-100 μM) in five interneuron types of the cerebellar cortex but not in Purkinje cells (PCs) (≤10 μM), yielding sufficient contrast to allow functional signals to be recorded from somata and processes in awake animals using two-photon microscopy. When the hSYN promoter was used to drive expression of the tetracycline transactivator (tTA), a second rAAV containing the bidirectional TET promoter (P(tet)bi) could drive strong D3cpv expression in PCs (10-300 μM), enough to allow reliable complex spike detection in the dendritic arbor. An amplified approach should be of use in monitoring neural processing in selected cell types and boosting expression of optogenetic probes. Additionally, we overcome cell toxicity associated with rAAV injection and/or local GECI overexpression by combining the virus injection with systemic pre-injection of hyperosmotic D-mannitol, and by this double the time window for functional imaging.

Bernd Kuhn, Ilker Ozden, Yulia Lampi, Mazahir T Hasan, Samuel S-H Wang

Widespread state-dependent shifts in cerebellar activity in locomoting mice.

Excitatory drive enters the cerebellum via mossy fibers, which activate granule cells, and climbing fibers, which activate Purkinje cell dendrites. Until now, the coordinated regulation of these pathways has gone unmonitored in spatially resolved neuronal ensembles, especially in awake animals. We imaged cerebellar activity using functional two-photon microscopy and extracellular recording in awake mice locomoting on an air-cushioned spherical treadmill. We recorded from putative granule cells, molecular layer interneurons, and Purkinje cell dendrites in zone A of lobule IV/V, representing sensation and movement from trunk and limbs. Locomotion was associated with widespread increased activity in granule cells and interneurons, consistent with an increase in mossy fiber drive. At the same time, dendrites of different Purkinje cells showed increased co-activation, reflecting increased synchrony of climbing fiber activity. In resting animals, aversive stimuli triggered increased activity in granule cells and interneurons, as well as increased Purkinje cell co-activation that was strongest for neighboring dendrites and decreased smoothly as a function of mediolateral distance. In contrast with anesthetized recordings, no 1-10 Hz oscillations in climbing fiber activity were evident. Once locomotion began, responses to external stimuli in all three cell types were strongly suppressed. Thus climbing and mossy fiber representations can shift together within a fraction of a second, reflecting in turn either movement-associated activity or external stimuli.

Ilker Ozden, Daniel A Dombeck, Tycho M Hoogland, David W Tank, Samuel S-H Wang

Injection of recombinant adenovirus for delivery of genetically encoded calcium indicators into astrocytes of the cerebellar cortex.

The cerebellar cortex contains two astrocyte types: the Bergmann glia of the molecular layer and the velate protoplasmic astrocytes of the granule cell layer. In vivo, these cell types generate both subcellular calcium transients and trans-glial calcium waves. This protocol outlines a method for in vivo calcium imaging in cerebellar astrocytes, using the injection of a replication-incompetent recombinant adenovirus for gene transfer of the fluorescent calcium indicator protein (FCIP) G-CaMP2. The adenovirus contains a cytomegalovirus (CMV) immediate-early (IE) promoter which confines expression of G-CaMP2 to astrocytes. Expression is sufficiently high to allow calcium signals to be recorded in Bergmann glial processes as well as the processes and somata of velate protoplasmic astrocytes. To obtain structural information, G-CaMP2 fused with the brighter chromophore DsRed allows three-dimensional (3D) reconstruction of cells. G-CaMP2 expression lasts for at least 3 wk, enabling long-term functional imaging in both anesthetized and awake animals.

Bernd Kuhn, Tycho M Hoogland, Samuel S-H Wang

Preferential loading of bergmann glia with synthetic acetoxymethyl calcium dyes.

The cerebellar cortex contains two astrocyte types: the Bergmann glia of the molecular layer and the velate protoplasmic astrocytes of the granule cell layer. In vivo, these cell types generate both subcellular calcium transients and trans-glial calcium waves. This protocol outlines a method for in vivo calcium imaging in cerebellar astrocytes of mice which have undergone a cerebellar craniotomy. Multicell bolus loading (MCBL) is performed using the synthetic calcium indicators Fluo-5F AM and Fluo-4 AM. In the cerebellum, a degree of cell-type specificity can be achieved by varying the depth of injection. This protocol describes a loading procedure following craniotomy which allows preferential labeling of Bergmann glia.

Tycho M Hoogland, Bernd Kuhn, Samuel S-H Wang

Cerebellar craniotomy for in vivo calcium imaging of astrocytes.

The cerebellar cortex contains two astrocyte types: the Bergmann glia of the molecular layer and the velate protoplasmic astrocytes of the granule cell layer. In vivo, these cell types generate both subcellular calcium transients and trans-glial calcium waves. It is possible to perform in vivo calcium imaging in cerebellar astrocytes. One method involves injection of a replication-incompetent recombinant adenovirus for gene transfer of a fluorescent calcium indicator protein. A second method uses multicell bolus loading (MCBL) in the molecular layer of the cerebellum with synthetic calcium indicators. This protocol presents a cerebellar craniotomy procedure which can be used to prepare a virus-injected animal for in vivo imaging. It can also be used to prepare an animal for MCBL.

Bernd Kuhn, Tycho M Hoogland, Samuel S-H Wang

Cerebellar involvement in an evidence-accumulation decision-making task.

To make successful evidence-based decisions, the brain must rapidly and accurately transform sensory inputs into specific goal-directed behaviors. Most experimental work on this subject has focused on forebrain mechanisms. Using a novel evidence-accumulation task for mice, we performed recording and perturbation studies of crus I of the lateral posterior cerebellum, which communicates bidirectionally with numerous forebrain regions. Cerebellar inactivation led to a reduction in the fraction of correct trials. Using two-photon fluorescence imaging of calcium, we found that Purkinje cell somatic activity contained choice/evidence-related information. Decision errors were represented by dendritic calcium spikes, which in other contexts are known to drive cerebellar plasticity. We propose that cerebellar circuitry may contribute to computations that support accurate performance in this perceptual decision-making task.

Ben Deverett, Sue Ann Koay, Marlies Oostland, Samuel S-H Wang

Cerebellar granule cells acquire a widespread predictive feedback signal during motor learning.

Cerebellar granule cells, which constitute half the brain's neurons, supply Purkinje cells with contextual information necessary for motor learning, but how they encode this information is unknown. Here we show, using two-photon microscopy to track neural activity over multiple days of cerebellum-dependent eyeblink conditioning in mice, that granule cell populations acquire a dense representation of the anticipatory eyelid movement. Initially, granule cells responded to neutral visual and somatosensory stimuli as well as periorbital airpuffs used for training. As learning progressed, two-thirds of monitored granule cells acquired a conditional response whose timing matched or preceded the learned eyelid movements. Granule cell activity covaried trial by trial to form a redundant code. Many granule cells were also active during movements of nearby body structures. Thus, a predictive signal about the upcoming movement is widely available at the input stage of the cerebellar cortex, as required by forward models of cerebellar control.

Andrea Giovannucci, Aleksandra Badura, Ben Deverett, Farzaneh Najafi, Talmo D Pereira, Zhenyu Gao, Ilker Ozden, Alexander D Kloth, Eftychios Pnevmatikakis, Liam Paninski, Chris I De Zeeuw, Javier F Medina, Samuel S-H Wang