Bachelor of Science, Technische Universitat Munchen (2011)
Doctor of Philosophy, Johns Hopkins University (2019)
Jun Ding, Postdoctoral Faculty Sponsor
Visualizing synaptic plasticity in vivo by large-scale imaging of endogenous AMPA receptors.
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
AMPA Receptors Exist in Tunable Mobile and Immobile Synaptic Fractions In Vivo.
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
An optimized CRISPR/Cas9 approach for precise genome editing in neurons.
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
Cortical Synaptic AMPA Receptor Plasticity during Motor Learning
2020; 105 (5): 895-+
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
Lamina-specific AMPA receptor dynamics following visual deprivation in vivo
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
An ultrasensitive biosensor for high-resolution kinase activity imaging in awake mice.
Nature chemical biology
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
Single-fluorophore biosensors for sensitive and multiplexed detection of signalling activities
NATURE CELL BIOLOGY
2018; 20 (10): 1215-+
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
Dynamic imaging of AMPA receptor trafficking in vitro and in vivo
CURRENT OPINION IN NEUROBIOLOGY
2017; 45: 51–58
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
Homer1a drives homeostatic scaling-down of excitatory synapses during sleep
2017; 355 (6324): 511-+
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
Synaptic Organization of the Neuronal Circuits of the Claustrum
JOURNAL OF NEUROSCIENCE
2016; 36 (3): 773-784
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