Browsing by Author "Yasuda, Ryohei"

It has long been appreciated that the process of learning might invoke a physical change in the brain, establishing a lasting trace of experience. Recent evidence has revealed that this change manifests, at least in part, by the formation of new connections between neurons, as well as the modification of preexisting ones. This so-called structural plasticity of neural circuits – their ability to physically change in response to experience – has remained fixed as a primary point of focus in the field of neuroscience.

A large portion of this effort has been directed towards the study of dendritic spines, small protrusions emanating from neuronal dendrites that constitute the majority of recipient sites of excitatory neuronal connections. The unique, mushroom-like morphology of these tiny structures has earned them considerable attention, with even the earliest observers suggesting that their unique shape affords important functional advantages that would not be possible if synapses were to directly contact dendrites. Importantly, dendritic spines can be formed, eliminated, or structurally modified in response to both neural activity as well as learning, suggesting that their organization reflects the experience of the neural network. As such, elucidating how these structures undergo such rearrangements is of critical importance to understanding both learning and memory.

As dendritic spines are principally composed of the cytoskeletal protein actin, their formation, elimination, and modification requires biochemical signaling networks that can remodel the actin cytoskeleton. As a result, significant effort has been placed into identifying and characterizing such signaling networks and how they are controlled during synaptic activity and learning. Such efforts have highlighted Rho family GTPases – binary signaling proteins central in controlling the dynamics of the actin cytoskeleton – as attractive targets for understanding how the structural modification of spines might be controlled by synaptic activity. While much has been revealed regarding the importance of the Rho GTPases for these processes, the specific spatial and temporal features of their signals that impart such structural changes remains unclear.

The central hypotheses of the following research dissertation are as follows: first, that synaptic activity rapidly initiates Rho GTPase signaling within single dendritic spines, serving as the core mechanism of dendritic spine structural plasticity. Next, that each of the Rho GTPases subsequently expresses a spatially distinct pattern of activation, with some signals remaining highly localized, and some becoming diffuse across a region of the nearby dendrite. The diffusive signals modify the plasticity induction threshold of nearby dendritic spines, and the spatially restricted signals serve to keep the expression of plasticity specific to those spines that receive synaptic input. This combination of differentially spatially regulated signals thus equips the neuronal dendrite with the ability to perform local biochemical computations, potentially establishing an organizational preference for the arrangement of dendritic spines along a dendrite. Finally, the consequences of the differential signal patterns also help to explain several seemingly disparate properties of one of the primary upstream activators of these proteins: brain-derived neurotrophic factor (BDNF).

The first section of this dissertation describes the characterization of the activity patterns of one of the Rho family GTPases, Rac1. Using a novel Förster Resonance Energy Transfer (FRET)- based biosensor in combination with two-photon fluorescence lifetime imaging (2pFLIM) and single-spine stimulation by two-photon glutamate uncaging, the activation profile and kinetics of Rac1 during synaptic stimulation were characterized. These experiments revealed that Rac1 conveys signals to both activated spines as well as nearby, unstimulated spines that are in close proximity to the target spine. Despite the diffusion of this structural signal, however, the structural modification associated with synaptic stimulation remained restricted to the stimulated spine. Thus, Rac1 activation is not sufficient to enlarge spines, but nonetheless likely confers some heretofore-unknown function to nearby synapses.

The next set of experiments set out to detail the upstream molecular mechanisms controlling Rac1 activation. First, it was found that Rac1 activation during sLTP depends on calcium through NMDA receptors and subsequent activation of CaMKII, suggesting that Rac1 activation in this context agrees with substantial evidence linking NMDAR-CaMKII signaling to LTP in the hippocampus. Next, in light of recent evidence linking structural plasticity to another potential upstream signaling complex, BDNF-TrkB, we explored the possibility that BDNF-TrkB signaling functioned in structural plasticity via Rac1 activation. To this end, we first explored the release kinetics of BDNF and the activation kinetics of TrkB using novel biosensors in conjunction with 2p glutamate uncaging. It was found that release of BDNF from single dendritic spines during sLTP induction activates TrkB on that same spine in an autocrine manner, and that this autocrine system was necessary for both sLTP and Rac1 activation. It was also found that BDNF-TrkB signaling controls the activity of another Rho GTPase, Cdc42, suggesting that this autocrine loop conveys both synapse-specific signals (through Cdc42) and heterosynaptic signals (through Rac1).

The next set of experiments detail one the potential consequences of heterosynaptic Rac1 signaling. The spread of Rac1 activity out of the stimulated spine was found to be necessary for lowering the plasticity threshold at nearby spines, a process known as synaptic crosstalk. This was also true for the Rho family GTPase, RhoA, which shows a similar diffusive activity pattern. Conversely, the activity of Cdc42, a Rho GTPase protein whose activity is highly restricted to stimulated spines, was required only for input-specific plasticity induction. Thus, the spreading of a subset of Rho GTPase signaling into nearby spines modifies the plasticity induction threshold of these spines, increasing the likelihood that synaptic activity at these sites will induce structural plasticity. Importantly, these data suggest that the autocrine BDNF-TrkB loop described above simultaneously exerts control over both homo- and heterosynaptic structural plasticity.

The final set of experiments reveals that the spreading of GTPase activity from stimulated spines helps to overcome the high activation thresholds of these proteins to facilitate nearby plasticity. Both Rac1 and RhoA, the activity of which spread into nearby spines, showed high activation thresholds, making weak stimuli incapable of activating them. Thus, signal spreading from a strongly stimulated spine can lower the plasticity threshold at nearby spines in part by supplementing the activation of high-threshold Rho GTPases at these sites. In contrast, the highly compartmentalized Rho GTPase Cdc42 showed a very low activation threshold, and thus did not require signal spreading to achieve high levels of activity to even a weak stimulus. As a result, synaptic crosstalk elicits cooperativity of nearby synaptic events by first priming a local region of the dendrite with several (but not all) of the factors required for structural plasticity, which then allows even weak inputs to achieve plasticity by means of localized Cdc42 activation.

Taken together, these data reveal a molecular pattern whereby BDNF-dependent structural plasticity can simultaneously maintain input-specificity while also relaying heterosynaptic signals along a local stretch of dendrite via coordination of differential spatial signaling profiles of the Rho GTPase proteins. The combination of this division of spatial signaling patterns and different activation thresholds reveals a unique heterosynaptic coincidence detection mechanism that allows for cooperative expression of structural plasticity when spines are close together, which in turn provides a putative mechanism for how neurons arrange structural modifications during learning.

Multiple lines of evidence reveal that activation of the tropomyosin related kinase B (TrkB) receptor is a critical molecular mechanism underlying status epilepticus (SE) induced epilepsy development. However, the cellular consequences of such signaling remain unknown. To this point, localization of SE-induced TrkB activation to CA1 apical dendritic spines provides an anatomic clue pointing to Schaffer collateral-CA1 synaptic plasticity as one potential cellular consequence of TrkB activation. Here, we combine two-photon glutamate uncaging with two photon fluorescence lifetime imaging microscopy (2pFLIM) of fluorescence resonance energy transfer (FRET)-based sensors to specifically investigate the roles of TrkB and its canonical ligand brain derived neurotrophic factor (BDNF) in dendritic spine structural plasticity (sLTP) of CA1 pyramidal neurons in cultured hippocampal slices of rodents. To begin, we demonstrate a critical role for post-synaptic TrkB and post-synaptic BDNF in sLTP. Building on these findings, we develop a novel FRET-based sensor for TrkB activation that can report both BDNF and non-BDNF activation in a specific and reversible manner. Using this sensor, we monitor the spatiotemporal dynamics of TrkB activity during single-spine sLTP. In response to glutamate uncaging, we report a rapid (onset less than 1 minute) and sustained (lasting at least 20 minutes) activation of TrkB in the stimulated spine that depends on N-methyl-D-aspartate receptor (NMDAR)-Ca2+/Calmodulin dependent kinase II (CaMKII) signaling as well as post-synaptically synthesized BDNF. Consistent with these findings, we also demonstrate rapid, glutamate uncaging-evoked, time-locked release of BDNF from single dendritic spines using BDNF fused to superecliptic pHluorin (SEP). Finally, to elucidate the molecular mechanisms by which TrkB activation leads to sLTP, we examined the dependence of Rho GTPase activity - known mediators of sLTP - on BDNF-TrkB signaling. Through the use of previously described FRET-based sensors, we find that the activities of ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) require BDNF-TrkB signaling. Taken together, these findings reveal a spine-autonomous, autocrine signaling mechanism involving NMDAR-CaMKII dependent BDNF release from stimulated dendritic spines leading to TrkB activation and subsequent activation of the downstream molecules Rac1 and Cdc42 in these same spines that proves critical for sLTP. In conclusion, these results highlight structural plasticity as one cellular consequence of CA1 dendritic spine TrkB activation that may potentially contribute to larger, circuit-level changes underlying SE-induced epilepsy.

Repetitive Ca2+ transients in dendritic spines induce various forms of synaptic plasticity by transmitting information encoded in their frequency and amplitude. CaMKII plays a critical role in decoding these Ca2+ signals to initiate long-lasting synaptic plasticity. However, the properties of CaMKII that mediate Ca2+ decoding in spines remain elusive. Here, I measured CaMKII activity in spines using fast-framing two-photon fluorescence lifetime imaging. Following each repetitive Ca2+ elevations, CaMKII activity increased in a stepwise manner. This signal integration, at the time scale of seconds, critically depended on Thr286 phosphorylation. In the absence of Thr286 phosphorylation, only by increasing the frequency of repetitive Ca2+ elevations could high peak CaMKII activity or plasticity be induced. In addition, I measured the association between CaMKII and Ca2+/CaM during spine plasticity induction. Unlike CaMKII activity, association of Ca2+/CaM to CaMKII plateaued at the first Ca2+ elevation event. This result indicated that integration of Ca2+ signals was initiated by the binding of Ca2+/CaM and amplified by the subsequent increases in Thr286-phosphorylated form of CaMKII. Together, these findings demonstrate that CaMKII functions as a leaky integrator of repetitive Ca2+ signals during the induction of synaptic plasticity, and that Thr286 phosphorylation is critical for defining the frequencies of such integration.

The late phase of long-term synaptic potentiation (LTP) at glutamatergic synapses, which is thought to underlie the long lasting memory (at least hours), requires gene transcription in the nucleus. However, it remains elusive how signaling initiated at synapses during induction of LTP is transmitted into the nucleus to commence transcription. Using a combination of two-photon glutamate uncaging and a genetically encoded FRET sensor, I found that induction of synapse-specific LTP at only a few (3-7) dendritic spines leads to pronounced activation of extracellular signal-regulated kinase (ERK) in the nucleus and downstream phosphorylation of transcription factors, cAMP-response element-binding protein (CREB) and E26-like protein-1 (Elk-1). The underlying molecular mechanism of this nuclear ERK activation was investigated: it seems to involve activation of NMDA receptors, metabotrophic glutamate receptors, and the classical Ras pathway. I also found that the spatial pattern of synaptic stimulation matters: spatially dispersed stimulation over multiple dendritic branches activated nuclear ERK much more efficiently than clustered stimulation within a single dendritic branch. In sum, these results suggest that biochemical signals could be transmitted from individual spines to the nucleus following LTP induction and that such synapse-to-nucleus signaling requires integration across multiple dendritic branches.

Membrane trafficking in dendritic spines is critical for regulating the number of channels and spine structure during synaptic plasticity. Here I report two small Rab GTPases, Rab4 and Rab10, oppositely regulate AMPA receptors (AMPARs) trafficking and structural plasticity of dendritic spines. Combining two-photon glutamate uncaging with two-photon fluorescence lifetime imaging microscopy (2pFLIM), I found that Rab4 is transiently activated whereas Rab10 is persistently inactivated in the stimulated spines during structural long-term potentiation (sLTP). Inhibition of Rab4 signaling has no effect on GluA1 endocytosis but inhibits activity-dependent GluA1 exocytosis. Conversely, disruption of Rab10 signaling inhibits GluA1 endocytosis while enhancing activity-dependent GluA1 exocytosis. In summary, these results uncover a new mechanism to establish the specificity and directionality of AMPARs trafficking and sLTP via distinct regulations of Rab4 and Rab10 signaling.

Synaptic plasticity is the leading candidate for the cellular/molecular basis of learning and memory. One of the key molecules involved in synaptic plasticity is Calcium/calmodulin-dependent Kinase II (CaMKII). Synaptic plasticity can be expressed at a single dendritic spine independent of its neighboring dendritic spines. Here, we investigated how long the activity of CaMKII lasts during synaptic plasticity of single dendritic spines. We found that CaMKII activity lasted ~2 minutes during synaptic plasticity and was restricted to the dendritic spines undergoing synaptic plasticity while nearby dendritic spines did not show any change in the level of CaMKII activity. Our experimental data argue against the persistent activation of CaMKII in dendritic spines undergoing synaptic plasticity and suggest that the activity of CaMKII is a spine-specific biochemical signal necessary for synapse-specificity of synaptic plasticity. We provide a biophysical explanation of how spine-specific CaMKII activation can be achieved during synaptic plasticity. We also found that CaMKII is activated by highly localized calcium influx in the proximity of Voltage-dependent Calcium Channels (VDCCs) and a different set of VDCCs and their respective Ca2+ nanodomains are responsible for the differential activation of CaMKII between dendritic spines and dendritic shafts.

Multifunctional calcium/calmodulin dependent protein kinases (CaMKs) are key regulators of spine structural plasticity and long-term potentiation (LTP) in neurons. CaMKs have promiscuous and overlapping substrate recognition motifs, and are distinguished in their regulatory role based on differences in the spatiotemporal dynamics of activity. While the function and activity of CaMKII in synaptic plasticity has been extensively studied, that of CaMKI, another major class of CaMK required for LTP, still remain elusive.

Here, we develop a Förster’s Resonance Energy Transfer (FRET) based sensor to measure the spatiotemporal activity dynamics of CaMK1. We monitored CaMKI activity using 2-photon fluorescence lifetime imaging, while inducing LTP in single dendritic spines of rat (Rattus Norvegicus, strain Sprague Dawley) hippocampal CA1 pyramidal neurons using 2-photon glutamate uncaging. Using RNA-interference and pharmacological means, we also characterize the role of CaMKI during spine structural plasticity.

We found that CaMKI was rapidly and transiently activated with a rise time of ~0.3 s and decay time of ~1 s in response to each uncaging pulse. Activity of CaMKI spread out of the spine. Phosphorylation of CaMKI by CaMKK was required for this spreading and for the initial phase of structural LTP. Combined with previous data showing that CaMKII is restricted to the stimulated spine and required for long-term maintenance of structural LTP, these results suggest that CaMK diversity allows the same incoming signal – calcium – to independently regulate distinct phases of LTP by activating different CaMKs with distinct spatiotemporal dynamics.

Learning and memory is one of the critical components of the human experience. In one model of memory, hippocampal LTP, it is believed that the trafficking of AMPA receptors to the synapse is a fundamental process, yet the spatiotemporal kinetics of the process remain under dispute. In this work, we imaged the trafficking of AMPA receptors by combining two-photon glutamate uncaging on single spines with a fluorescent reporter for surface AMPA receptors. We found that AMPA receptors are trafficked to the spine at the same time as the spine size is increasing. Using a bleaching protocol, we found that the receptors that reach the spine come from a combination of the surface and endosomal pools. Imaging exocytosis in real time, we found that the exocytosis rate increases briefly (~1 min.), both in the spine and neighbouring dendrite. Finally, we performed pharmacological and genetic manipulations of signaling pathways, and found that the Ras-ERK signaling pathway is necessary for AMPAR exocytosis.

In a set of related experiments, we also investigated the capacity of single spines to undergo potentiation multiple times. By stimulating spines twice using glutamate uncaging, we found that there is a refractory period for synaptic plasticity in spines during which they cannot further be potentiated. We furthermore found that inducing plasticity in a given spine inhibits plasticity at nearby spines.