All Publications
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A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic events.
Neuron
2024
Abstract
A remaining challenge for genetically encoded voltage indicators (GEVIs) is the reliable detection of excitatory postsynaptic potentials (EPSPs). Here, we developed ASAP5 as a GEVI with enhanced activation kinetics and responsivity near resting membrane potentials for improved detection of both spiking and subthreshold activity. ASAP5 reported action potentials (APs) in vivo with higher signal-to-noise ratios than previous GEVIs and successfully detected graded and subthreshold responses to sensory stimuli in single two-photon trials. In cultured rat or human neurons, somatic ASAP5 reported synaptic events propagating centripetally and could detect ∼1-mV EPSPs. By imaging spontaneous EPSPs throughout dendrites, we found that EPSP amplitudes decay exponentially during propagation and that amplitude at the initiation site generally increases with distance from the soma. These results extend the applications of voltage imaging to the quantal response domain, including in human neurons, opening up the possibility of high-throughput, high-content characterization of neuronal dysfunction in disease.
View details for DOI 10.1016/j.neuron.2024.08.019
View details for PubMedID 39305894
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Achieving optical transparency in live animals with absorbing molecules.
Science (New York, N.Y.)
2024; 385 (6713): eadm6869
Abstract
Optical imaging plays a central role in biology and medicine but is hindered by light scattering in live tissue. We report the counterintuitive observation that strongly absorbing molecules can achieve optical transparency in live animals. We explored the physics behind this observation and found that when strongly absorbing molecules dissolve in water, they can modify the refractive index of the aqueous medium through the Kramers-Kronig relations to match that of high-index tissue components such as lipids. We have demonstrated that our straightforward approach can reversibly render a live mouse body transparent to allow visualization of a wide range of deep-seated structures and activities. This work suggests that the search for high-performance optical clearing agents should focus on strongly absorbing molecules.
View details for DOI 10.1126/science.adm6869
View details for PubMedID 39236186
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Oligodendrocytes and myelin limit neuronal plasticity in visual cortex.
Nature
2024
Abstract
Developmental myelination is a protracted process in the mammalian brain1. One theory for why oligodendrocytes mature so slowly posits that myelination may stabilize neuronal circuits and temper neuronal plasticity as animals age2-4. We tested this theory in the visual cortex, which has a well-defined critical period for experience-dependent neuronal plasticity5. During adolescence, visual experience modulated the rate of oligodendrocyte maturation in visual cortex. To determine whether oligodendrocyte maturation in turn regulates neuronal plasticity, we genetically blocked oligodendrocyte differentiation and myelination in adolescent mice. In adult mice lacking adolescent oligodendrogenesis, a brief period of monocular deprivation led to a significant decrease in visual cortex responses to the deprived eye, reminiscent of the plasticity normally restricted to adolescence. This enhanced functional plasticity was accompanied by a greater turnover of dendritic spines and coordinated reductions in spine size following deprivation. Furthermore, inhibitory synaptic transmission, which gates experience-dependent plasticity at the circuit level, was diminished in the absence of adolescent oligodendrogenesis. These results establish a critical role for oligodendrocytes in shaping the maturation and stabilization of cortical circuits and support the concept of developmental myelination acting as a functional brake on neuronal plasticity.
View details for DOI 10.1038/s41586-024-07853-8
View details for PubMedID 39169185
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Cortico-basal ganglia plasticity in motor learning.
Neuron
2024
Abstract
One key function of the brain is to control our body's movements, allowing us to interact with the world around us. Yet, many motor behaviors are not innate but require learning through repeated practice. Among the brain's motor regions, the cortico-basal ganglia circuit is particularly crucial for acquiring and executing motor skills, and neuronal activity in these regions is directly linked to movement parameters. Cell-type-specific adaptations of activity patterns and synaptic connectivity support the learning of new motor skills. Functionally, neuronal activity sequences become structured and associated with learned movements. On the synaptic level, specific connections become potentiated during learning through mechanisms such as long-term synaptic plasticity and dendritic spine dynamics, which are thought to mediate functional circuit plasticity. These synaptic and circuit adaptations within the cortico-basal ganglia circuitry are thus critical for motor skill acquisition, and disruptions in this plasticity can contribute to movement disorders.
View details for DOI 10.1016/j.neuron.2024.06.014
View details for PubMedID 39002543
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Stimulus-dependent synaptic plasticity underlies neuronal circuitry refinement in the mouse primary visual cortex.
Cell reports
2024; 43 (4): 113966
Abstract
Perceptual learning improves our ability to interpret sensory stimuli present in our environment through experience. Despite its importance, the underlying mechanisms that enable perceptual learning in our sensory cortices are still not fully understood. In this study, we used in vivo two-photon imaging to investigate the functional and structural changes induced by visual stimulation in the mouse primary visual cortex (V1). Our results demonstrate that repeated stimulation leads to a refinement of V1 circuitry by decreasing the number of responsive neurons while potentiating their response. At the synaptic level, we observe a reduction in the number of dendritic spines and an overall increase in spine AMPA receptor levels in the same subset of neurons. In addition, visual stimulation induces synaptic potentiation in neighboring spines within individual dendrites. These findings provide insights into the mechanisms of synaptic plasticity underlying information processing in the neocortex.
View details for DOI 10.1016/j.celrep.2024.113966
View details for PubMedID 38507408
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Adolescent oligodendrogenesis and myelination restrict experience-dependent neuronal plasticity in adult visual cortex.
bioRxiv : the preprint server for biology
2023
Abstract
Developmental myelination is a protracted process in the mammalian brain. One theory for why oligodendrocytes mature so slowly posits that myelination may stabilize neuronal circuits and temper neuronal plasticity as animals age. We tested this hypothesis in the visual cortex, which has a well-defined critical period for experience-dependent neuronal plasticity.To prevent myelin progression, we conditionally deleted Myrf, a transcription factor necessary for oligodendrocyte maturation, from oligodendrocyte precursor cells (Myrf cKO) in adolescent mice. To induce experience-dependent plasticity, adult control and Myrf cKO mice were monocularly deprived by eyelid suture. Functional and structural neuronal plasticity in the visual cortex were assessed in vivo by intrinsic signal optical imaging and longitudinal two photon imaging of dendritic spines, respectively.During adolescence, visual experience modulated the rate of oligodendrocyte maturation in visual cortex. Myrf deletion from oligodendrocyte precursors during adolescence led to inhibition of oligodendrocyte maturation and myelination that persisted into adulthood. Following monocular deprivation, visual cortex activity in response to visual stimulation of the deprived eye remained stable in adult control mice, as expected for post-critical period animals. By contrast, visual cortex responses to the deprived eye decreased significantly following monocular deprivation in adult Myrf cKO mice, reminiscent of the plasticity observed in adolescent mice. Furthermore, visual cortex neurons in adult Myrf cKO mice had fewer dendritic spines and a higher level of spine turnover. Finally, monocular deprivation induced spatially coordinated spine size decreases in adult Myrf cKO, but not control, mice.These results demonstrate a critical role for oligodendrocytes in shaping the maturation and stabilization of cortical circuits and support the concept of myelin acting as a brake on neuronal plasticity during development.
View details for DOI 10.1101/2023.09.29.560231
View details for PubMedID 37808666
View details for PubMedCentralID PMC10557765
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A positively tuned voltage indicator for extended electrical recordings in the brain.
Nature methods
2023; 20 (7): 1104-1113
Abstract
Genetically encoded voltage indicators (GEVIs) enable optical recording of electrical signals in the brain, providing subthreshold sensitivity and temporal resolution not possible with calcium indicators. However, one- and two-photon voltage imaging over prolonged periods with the same GEVI has not yet been demonstrated. Here, we report engineering of ASAP family GEVIs to enhance photostability by inversion of the fluorescence-voltage relationship. Two of the resulting GEVIs, ASAP4b and ASAP4e, respond to 100-mV depolarizations with ≥180% fluorescence increases, compared with the 50% fluorescence decrease of the parental ASAP3. With standard microscopy equipment, ASAP4e enables single-trial detection of spikes in mice over the course of minutes. Unlike GEVIs previously used for one-photon voltage recordings, ASAP4b and ASAP4e also perform well under two-photon illumination. By imaging voltage and calcium simultaneously, we show that ASAP4b and ASAP4e can identify place cells and detect voltage spikes with better temporal resolution than commonly used calcium indicators. Thus, ASAP4b and ASAP4e extend the capabilities of voltage imaging to standard one- and two-photon microscopes while improving the duration of voltage recordings.
View details for DOI 10.1038/s41592-023-01913-z
View details for PubMedID 37429962
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Motor learning selectively strengthens cortical and striatal synapses of motor engram neurons.
Neuron
2022
Abstract
Learning and consolidation of new motor skills require plasticity in the motor cortex and striatum, two key motor regions of the brain. However, how neurons undergo synaptic changes and become recruited during motor learning to form a memory engram remains unknown. Here, we train mice on a motor learning task and use a genetic approach to identify and manipulate behavior-relevant neurons selectively in the primary motor cortex (M1). We find that the degree of M1 engram neuron reactivation correlates with motor performance. We further demonstrate that learning-induced dendritic spine reorganization specifically occurs in these M1 engram neurons. In addition, we find that motor learning leads to an increase in the strength of M1 engram neuron outputs onto striatal spiny projection neurons (SPNs) and that these synapses form clusters along SPN dendrites. These results identify a highly specific synaptic plasticity during the formation of long-lasting motor memory traces in the corticostriatal circuit.
View details for DOI 10.1016/j.neuron.2022.06.006
View details for PubMedID 35809573
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Visualizing synaptic plasticity in vivo by large-scale imaging of endogenous AMPA receptors.
eLife
2021; 10
Abstract
Elucidating how synaptic molecules such as AMPA receptors mediate neuronal communication and tracking their dynamic expression during behavior is crucial to understand cognition and disease, but current technological barriers preclude large-scale exploration of molecular dynamics in vivo. We have developed a suite of innovative methodologies that break through these barriers: a new knockin mouse line with fluorescently tagged endogenous AMPA receptors, two-photon imaging of hundreds of thousands of labeled synapses in behaving mice, and computer-vision-based automatic synapse detection. Using these tools, we can longitudinally track how the strength of populations of synapses changes during behavior. We used this approach to generate an unprecedentedly detailed spatiotemporal map of synapses undergoing changes in strength following sensory experience. More generally, these tools can be used as an optical probe capable of measuring functional synapse strength across entire brain areas during any behavioral paradigm, describing complex system-wide changes with molecular precision.
View details for DOI 10.7554/eLife.66809
View details for PubMedID 34658338
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AMPA Receptors Exist in Tunable Mobile and Immobile Synaptic Fractions In Vivo.
eNeuro
2021
Abstract
AMPA receptor (AMPAR) mobility within synapses has been extensively studied in vitro However, whether similar mobility properties apply to AMPARs in vivo has yet to be determined. Here, we use two-photon-fluorescence recovery after photobleaching (FRAP) to study AMPAR mobility within individual dendritic spines in live animals using an overexpression vector. We demonstrate the existence of mobile and immobile fractions of AMPARs across multiple cortical regions and layers. Additionally, we found that AMPAR mobility can be altered in vivo in response to administration of corticosterone, a condition that mimics exposure to stress.Significance StatementOur work provides novel insight to receptor mobility within intact brains of live mice using live two-photon microscopy through cranial windows. In vivo assessment of protein mobility within mammalian neuronal synapses have thus far been limited. Here, within this system, we are able to confirm that there are both mobile and immobile AMPA receptor (AMPAR) fractions in vivo and that these fractions are similar across different cortical regions and layers. Additionally, we reveal that the proportion of mobile to immobile receptor fraction may be altered by administration of corticosterone, a condition that mimics stress response, suggesting AMPAR mobility is acutely modulated in vivo.
View details for DOI 10.1523/ENEURO.0015-21.2021
View details for PubMedID 33906969
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An optimized CRISPR/Cas9 approach for precise genome editing in neurons.
eLife
2021; 10
Abstract
The efficient knock-in of large DNA fragments to label endogenous proteins remains especially challenging in non-dividing cells such as neurons. We developed Targeted Knock-In with Two (TKIT) guides as a novel CRISPR/Cas9 based approach for efficient, and precise, genomic knock-in. Through targeting non-coding regions TKIT is resistant to INDEL mutations. We demonstrate TKIT labeling of endogenous synaptic proteins with various tags, with efficiencies up to 42% in mouse primary cultured neurons. Utilizing in utero electroporation or viral injections in mice TKIT can label AMPAR subunits with Super Ecliptic pHluorin, enabling visualization of endogenous AMPARs in vivo using two-photon microscopy. We further use TKIT to assess the mobility of endogenous AMPARs using fluorescence recovery after photobleaching. Finally, we show that TKIT can be used to tag AMPARs in rat neurons, demonstrating precise genome editing in another model organism and highlighting the broad potential of TKIT as a method to visualize endogenous proteins.
View details for DOI 10.7554/eLife.65202
View details for PubMedID 33689678
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From Neurons to Cognition: Technologies for Precise Recording of Neural Activity Underlying Behavior.
BME frontiers
2020; 2020: 7190517
Abstract
Understanding how brain activity encodes information and controls behavior is a long-standing question in neuroscience. This complex problem requires converging efforts from neuroscience and engineering, including technological solutions to perform high-precision and large-scale recordings of neuronal activity in vivo as well as unbiased methods to reliably measure and quantify behavior. Thanks to advances in genetics, molecular biology, engineering, and neuroscience, in recent decades, a variety of optical imaging and electrophysiological approaches for recording neuronal activity in awake animals have been developed and widely applied in the field. Moreover, sophisticated computer vision and machine learning algorithms have been developed to analyze animal behavior. In this review, we provide an overview of the current state of technology for neuronal recordings with a focus on optical and electrophysiological methods in rodents. In addition, we discuss areas that future technological development will need to cover in order to further our understanding of the neural activity underlying behavior.
View details for DOI 10.34133/2020/7190517
View details for PubMedID 37849967
View details for PubMedCentralID PMC10521756
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Cortical Synaptic AMPA Receptor Plasticity during Motor Learning
NEURON
2020; 105 (5): 895-+
Abstract
Modulation of synaptic strength through trafficking of AMPA receptors (AMPARs) is a fundamental mechanism underlying synaptic plasticity, learning, and memory. However, the dynamics of AMPAR trafficking in vivo and its correlation with learning have not been resolved. Here, we used in vivo two-photon microscopy to visualize surface AMPARs in mouse cortex during the acquisition of a forelimb reaching task. Daily training leads to an increase in AMPAR levels at a subset of spatially clustered dendritic spines in the motor cortex. Surprisingly, we also observed increases in spine AMPAR levels in the visual cortex. There, synaptic potentiation depends on the availability of visual input during motor training, and optogenetic inhibition of visual cortex activity impairs task performance. These results indicate that motor learning induces widespread cortical synaptic potentiation by increasing the net trafficking of AMPARs into spines, including in non-motor brain regions.
View details for DOI 10.1016/j.neuron.2019.12.005
View details for Web of Science ID 000518860700015
View details for PubMedID 31901303
View details for PubMedCentralID PMC7060107
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Lamina-specific AMPA receptor dynamics following visual deprivation in vivo
ELIFE
2020; 9
Abstract
Regulation of AMPA receptor (AMPAR) expression is central to synaptic plasticity and brain function, but how these changes occur in vivo remains elusive. Here, we developed a method to longitudinally monitor the expression of synaptic AMPARs across multiple cortical layers in awake mice using two-photon imaging. We observed that baseline AMPAR expression in individual spines is highly dynamic with more dynamics in primary visual cortex (V1) layer 2/3 (L2/3) neurons than V1 L5 neurons. Visual deprivation through binocular enucleation induces a synapse-specific and depth-dependent change of synaptic AMPARs in V1 L2/3 neurons, wherein deep synapses are potentiated more than superficial synapses. The increase is specific to L2/3 neurons and absent on apical dendrites of L5 neurons, and is dependent on expression of the AMPAR-binding protein GRIP1. Our study demonstrates that specific neuronal connections, across cortical layers and even within individual neurons, respond uniquely to changes in sensory experience.
View details for DOI 10.7554/eLife.52420
View details for Web of Science ID 000518786700001
View details for PubMedID 32125273
View details for PubMedCentralID PMC7053996
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An ultrasensitive biosensor for high-resolution kinase activity imaging in awake mice.
Nature chemical biology
2020
Abstract
Protein kinases control nearly every facet of cellular function. These key signaling nodes integrate diverse pathway inputs to regulate complex physiological processes, and aberrant kinase signaling is linked to numerous pathologies. While fluorescent protein-based biosensors have revolutionized the study of kinase signaling by allowing direct, spatiotemporally precise kinase activity measurements in living cells, powerful new molecular tools capable of robustly tracking kinase activity dynamics across diverse experimental contexts are needed to fully dissect the role of kinase signaling in physiology and disease. Here, we report the development of an ultrasensitive, second-generation excitation-ratiometric protein kinase A (PKA) activity reporter (ExRai-AKAR2), obtained via high-throughput linker library screening, that enables sensitive and rapid monitoring of live-cell PKA activity across multiple fluorescence detection modalities, including plate reading, cell sorting and one- or two-photon imaging. Notably, in vivo visual cortex imaging in awake mice reveals highly dynamic neuronal PKA activity rapidly recruited by forced locomotion.
View details for DOI 10.1038/s41589-020-00660-y
View details for PubMedID 32989297
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Single-fluorophore biosensors for sensitive and multiplexed detection of signalling activities
NATURE CELL BIOLOGY
2018; 20 (10): 1215-+
Abstract
Unravelling the dynamic molecular interplay behind complex physiological processes such as neuronal plasticity requires the ability to both detect minute changes in biochemical states in response to physiological signals and track multiple signalling activities simultaneously. Fluorescent protein-based biosensors have enabled the real-time monitoring of dynamic signalling processes within the native context of living cells, yet most commonly used biosensors exhibit poor sensitivity (for example, due to low dynamic range) and are limited to imaging signalling activities in isolation. Here, we address this challenge by developing a suite of excitation ratiometric kinase activity biosensors that offer the highest reported dynamic range and enable the detection of subtle changes in signalling activity that could not be reliably detected previously, as well as a suite of single-fluorophore biosensors that enable the simultaneous tracking of as many as six distinct signalling activities in single living cells.
View details for DOI 10.1038/s41556-018-0200-6
View details for Web of Science ID 000445656400016
View details for PubMedID 30250062
View details for PubMedCentralID PMC6258557
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Dynamic imaging of AMPA receptor trafficking in vitro and in vivo
CURRENT OPINION IN NEUROBIOLOGY
2017; 45: 51–58
Abstract
Modulation of synaptic strength through trafficking of AMPA receptors is a fundamental mechanism underlying synaptic plasticity and has been shown to be an important process in higher brain functions such as learning and memory. Many studies have used live time-lapse imaging of fluorescently tagged AMPA receptors to directly monitor their membrane trafficking in the basal state as well as during synaptic plasticity. While most of these studies are performed in vitro using neuronal cell cultures, in the past years technological advances have enabled the imaging of synaptic proteins in vivo in intact organisms. This has allowed for visualization of synaptic plasticity on a molecular level in living and behaving animals. Here, we discuss key studies and approaches using dynamic imaging to visualize AMPA receptor trafficking in vitro as well as imaging synaptic proteins, including AMPA receptors, in vivo.
View details for DOI 10.1016/j.conb.2017.03.008
View details for Web of Science ID 000408073900009
View details for PubMedID 28411409
View details for PubMedCentralID PMC5554718
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Homer1a drives homeostatic scaling-down of excitatory synapses during sleep
SCIENCE
2017; 355 (6324): 511-+
Abstract
Sleep is an essential process that supports learning and memory by acting on synapses through poorly understood molecular mechanisms. Using biochemistry, proteomics, and imaging in mice, we find that during sleep, synapses undergo widespread alterations in composition and signaling, including weakening of synapses through removal and dephosphorylation of synaptic AMPA-type glutamate receptors. These changes are driven by the immediate early gene Homer1a and signaling from group I metabotropic glutamate receptors mGluR1/5. Homer1a serves as a molecular integrator of arousal and sleep need via the wake- and sleep-promoting neuromodulators, noradrenaline and adenosine, respectively. Our data suggest that homeostatic scaling-down, a global form of synaptic plasticity, is active during sleep to remodel synapses and participates in the consolidation of contextual memory.
View details for DOI 10.1126/science.aai8355
View details for Web of Science ID 000393183100042
View details for PubMedID 28154077
View details for PubMedCentralID PMC5382711
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Synaptic Organization of the Neuronal Circuits of the Claustrum
JOURNAL OF NEUROSCIENCE
2016; 36 (3): 773-784
Abstract
The claustrum, a poorly understood subcortical structure located between the cortex and the striatum, forms widespread connections with almost all cortical areas, but the cellular organization of claustral circuits remains largely unknown. Based primarily on anatomical data, it has been proposed that the claustrum integrates activity across sensory modalities. However, the extent to which the synaptic organization of claustral circuits supports this integration is unclear. Here, we used paired whole-cell recordings and optogenetic approaches in mouse brain slices to determine the cellular organization of the claustrum. We found that unitary synaptic connections among claustrocortical (ClaC) neurons were rare. In contrast, parvalbumin-positive (PV) inhibitory interneurons were highly interconnected with both chemical and electrical synapses. In addition, ClaC neurons and PV interneurons formed frequent synaptic connections. As suggested by anatomical data, we found that corticoclaustral afferents formed monosynaptic connections onto both ClaC neurons and PV interneurons. However, the responses to cortical input were comparatively stronger in PV interneurons. Consistent with this overall circuit organization, activation of corticoclaustral afferents generated monosynaptic excitatory responses as well as disynaptic inhibitory responses in ClaC neurons. These data indicate that recurrent excitatory circuits within the claustrum alone are unlikely to integrate across multiple sensory modalities. Rather, this cellular organization is typical of circuits sensitive to correlated inputs. Although single ClaC neurons may integrate corticoclaustral input from different cortical regions, these results are consistent with more recent proposals implicating the claustrum in detecting sensory novelty or in amplifying correlated cortical inputs to coordinate the activity of functionally related cortical regions. Significance statement: The function of the claustrum, a brain nucleus found in mammals, remains poorly understood. It has been proposed, based primarily on anatomical data, that claustral circuits play an integrative role and contribute to multimodal sensory integration. Here we show that the principal neurons of the claustrum, claustrocortical (ClaC) projection neurons, rarely form synaptic connections with one another and are unlikely to contribute to broad integration within the claustrum. We show that, although single ClaC neurons may integrate corticoclaustral inputs carrying information for different sensory modalities, the synaptic organization of ClaC neurons, local parvalbumin-positive interneurons within the claustrum, and cortical afferents is also consistent with recent proposals that the claustrum plays a role in detecting salient stimuli or amplifying correlated cortical inputs.
View details for DOI 10.1523/JNEUROSCI.3643-15.2016
View details for Web of Science ID 000368355100013
View details for PubMedCentralID PMC4719014