Project description:Dendritic spines are major loci of excitatory inputs and undergo activity-dependent structural changes that contribute to synaptic plasticity and memory formation. Despite the existence of various classification types of spines, how they arise and which molecular components trigger their structural plasticity remain elusive. microRNAs (miRNAs) have emerged as critical regulators of synapse development and plasticity via their control of gene expression. Brain-specific miR-134s likely regulate the morphological maturation of spines, but their subcellular distributions and functional impacts have rarely been assessed. Here, we exploited atomic force microscopy to visualize in situ miR-134s, which indicated that they are mainly distributed at nearby dendritic shafts and necks of spines. The abundance of miR-134s varied between morphologically and functionally distinct spine types, and their amounts were inversely correlated with their postulated maturation stages. Moreover, spines exhibited reduced contents of miR-134s when selectively stimulated with beads containing brain-derived neurotropic factor (BDNF). Taken together, in situ visualizations of miRNAs provided unprecedented insights into the "inverse synaptic-tagging" roles of miR-134s that are selective to inactive/irrelevant synapses and potentially a molecular means for modifying synaptic connectivity via structural alteration.
Project description:Metabotropic GABA(B) receptors play a fundamental role in modulating the excitability of neurons and circuits throughout the brain. These receptors influence synaptic transmission by inhibiting presynaptic release or activating postsynaptic potassium channels. However, their ability to directly influence different types of postsynaptic glutamate receptors remains unresolved. Here we examine GABA(B) receptor modulation in layer 2/3 pyramidal neurons from the mouse prefrontal cortex. We use two-photon laser-scanning microscopy to study synaptic modulation at individual dendritic spines. Using two-photon optical quantal analysis, we first demonstrate robust presynaptic modulation of multivesicular release at single synapses. Using two-photon glutamate uncaging, we then reveal that GABA(B) receptors strongly inhibit NMDA receptor calcium signals. This postsynaptic modulation occurs via the PKA pathway and does not affect synaptic currents mediated by AMPA or NMDA receptors. This form of GABA(B) receptor modulation has widespread implications for the control of calcium-dependent neuronal function.
Project description:Fast calcium transients (<10 ms) remain difficult to analyse in cellular microdomains, yet they can modulate key cellular events such as trafficking, local ATP production by endoplasmic reticulum-mitochondria complex (ER-mitochondria complex), or spontaneous activity in astrocytes. In dendritic spines receiving synaptic inputs, we show here that in the presence of a spine apparatus (SA), which is an extension of the smooth ER, a calcium-induced calcium release (CICR) is triggered at the base of the spine by the fastest calcium ions arriving at a Ryanodyne receptor (RyR). The mechanism relies on the asymmetric distributions of RyRs and sarco/ER calcium-ATPase (SERCA) pumps that we predict using a computational model and further confirm experimentally in culture and slice hippocampal neurons. The present mechanism for which the statistics of the fastest particles arriving at a small target, followed by an amplification, is likely to be generic in molecular transduction across cellular microcompartments, such as thin neuronal processes, astrocytes, endfeets, or protrusions.
Project description:Calcium/calmodulin-dependent protein kinase II (CaMKII) and neuronal store-operated calcium entry (nSOCE) have been implicated in the development of Alzheimer's disease (AD). nSOCE is involved in regulation of dendritic spine shape, particularly in stability of mushroom spines that play role in formation of strong synapses. CaMKII is involved in regulation of induction of long-term potentiation, that is needed for shaping of memory. In the present study, we demonstrated that inhibition of kinase activity of CaMKII by KN-62 decreases nSOCE amplitude in soma of primary hippocampal neurons. We have shown that knockdown of CaMKIIβ leads to the downregulation of nSOCE in dendritic spines. In agreement with previously published data, we have also observed that CaMKIIβ knockdown causes mushroom spine loss in primary hippocampal culture. The effect of CaMKIIβ knockdown on the nSOCE may be associated with a decrease of dendritic spine head size.
Project description:A subanesthetic dose of ketamine causes acute psychotomimetic symptoms and sustained antidepressant effects. In prefrontal cortex, the prevailing disinhibition hypothesis posits that N-methyl-d-aspartate receptor (NMDAR) antagonists such as ketamine act preferentially on GABAergic neurons. However, cortical interneurons are heterogeneous. In particular, somatostatin-expressing (SST) interneurons selectively inhibit dendrites and regulate synaptic inputs, yet their response to systemic NMDAR antagonism is unknown. Here, we report that ketamine acutely suppresses the activity of SST interneurons in the medial prefrontal cortex of the awake mouse. The deficient dendritic inhibition leads to greater synaptically evoked calcium transients in the apical dendritic spines of pyramidal neurons. By manipulating NMDAR signaling via GluN2B knockdown, we show that ketamine's actions on the dendritic inhibitory mechanism has ramifications for frontal cortex-dependent behaviors and cortico-cortical connectivity. Collectively, these results demonstrate dendritic disinhibition and elevated calcium levels in dendritic spines as important local-circuit alterations driven by the administration of subanesthetic ketamine.
Project description:Scaffolding proteins interact with membrane receptors to control signaling pathways and cellular functions. However, the dynamics and specific roles of interactions between different components of scaffold complexes are poorly understood because of the dearth of methods available to monitor binding interactions. Using a unique combination of single-cell bioluminescence resonance energy transfer imaging in living neurons and electrophysiological recordings, in this paper, we depict the role of glutamate receptor scaffold complex remodeling in space and time to control synaptic transmission. Despite a broad colocalization of the proteins in neurons, we show that spine-confined assembly/disassembly of this scaffold complex, physiologically triggered by sustained activation of synaptic NMDA (N-methyl-d-aspartate) receptors, induces physical association between ionotropic (NMDA) and metabotropic (mGlu5a) synaptic glutamate receptors. This physical interaction results in an mGlu5a receptor-mediated inhibition of NMDA currents, providing an activity-dependent negative feedback loop on NMDA receptor activity. Such protein scaffold remodeling represents a form of homeostatic control of synaptic excitability.
Project description:During sensory deprivation, the barrel cortex undergoes expansion of a functional column representing spared inputs (spared column), into the neighboring deprived columns (representing deprived inputs) which are in turn shrunk. As a result, the neurons in a deprived column simultaneously increase and decrease their responses to spared and deprived inputs, respectively. Previous studies revealed that dendritic spines are remodeled during this barrel map plasticity. Because cofilin1, a predominant regulator of actin filament turnover, governs both the expansion and shrinkage of the dendritic spine structure in vitro, it hypothetically regulates both responses in barrel map plasticity. However, this hypothesis remains untested. Using lentiviral vectors, we knocked down cofilin1 locally within layer 2/3 neurons in a deprived column. Cofilin1-knocked-down neurons were optogenetically labeled using channelrhodopsin-2, and electrophysiological recordings were targeted to these knocked-down neurons. We showed that cofilin1 knockdown impaired response increases to spared inputs but preserved response decreases to deprived inputs, indicating that cofilin1 dependency is dissociated in these two types of barrel map plasticity. To explore the structural basis of this dissociation, we then analyzed spine densities on deprived column dendritic branches, which were supposed to receive dense horizontal transcolumnar projections from the spared column. We found that spine number increased in a cofilin1-dependent manner selectively in the distal part of the supragranular layer, where most of the transcolumnar projections existed. Our findings suggest that cofilin1-mediated actin dynamics regulate functional map plasticity in an input-specific manner through the dendritic spine remodeling that occurs in the horizontal transcolumnar circuits. These new mechanistic insights into transcolumnar plasticity in adult rats may have a general significance for understanding reorganization of neocortical circuits that have more sophisticated columnar organization than the rodent neocortex, such as the primate neocortex.
Project description:Dendritic spines are the primary postsynaptic sites of excitatory neurotransmission in the brain. They exhibit a remarkable morphological variety, ranging from thin protrusions, to stubby shapes, to bulbous mushroom shapes. The remodeling of spines is thought to regulate the strength of the synaptic connection, which depends vitally on the number and the spatial distribution of AMPA-type glutamate receptors (AMPARs). We present numerical and analytical analyses demonstrating that this shape strongly affects AMPAR diffusion. We report a pronounced suppression of the receptor exit rate out of spines with decreasing neck radius. Thus, mushroomlike spines become highly effective at retaining receptors in the spine head. Moreover, we show that the postsynaptic density further enhances receptor trapping, particularly in mushroomlike spines local exocytosis in the spine head, in contrast to release at the base, provides rapid and specific regulatory control of AMPAR concentration at synapses.
Project description:BACKGROUND:The Na+,K+-ATPase plays an important role for ion homeostasis in virtually all mammalian cells, including neurons. Despite this, there is as yet little known about the isoform specific distribution in neurons. RESULTS:With help of superresolving stimulated emission depletion microscopy the spatial distribution of Na+,K+-ATPase in dendritic spines of cultured striatum neurons have been dissected. The found compartmentalized distribution provides a strong evidence for the confinement of neuronal Na+,K+-ATPase (?3 isoform) in the postsynaptic region of the spine. CONCLUSIONS:A compartmentalized distribution may have implications for the generation of local sodium gradients within the spine and for the structural and functional interaction between the sodium pump and other synaptic proteins. Superresolution microscopy has thus opened up a new perspective to elucidate the nature of the physiological function, regulation and signaling role of Na+,K+-ATPase from its topological distribution in dendritic spines.
Project description:The entry of calcium into dendritic spines can trigger a sequence of biochemical reactions that begins with the activation of calmodulin (CaM) and ends with long-term changes to synaptic strengths. The degree of activation of CaM can depend on highly local elevations in the concentration of calcium and the duration of transient increases in calcium concentration. Accurate measurement of these local changes in calcium is difficult because the spaces are so small and the numbers of molecules are so low. We have therefore developed a Monte Carlo model of intracellular calcium dynamics within the spine that included calcium binding proteins, calcium transporters and ion channels activated by voltage and glutamate binding. The model reproduced optical recordings using calcium indicator dyes and showed that without the dye the free intracellular calcium concentration transient was much higher than predicted from the fluorescent signal. Excitatory postsynaptic potentials induced large, long-lasting calcium gradients across the postsynaptic density, which activated CaM. When glutamate was released at the synapse 10 ms before an action potential occurred, simulating activity patterns that strengthen hippocampal synapses, the calcium gradient and activation of CaM in the postsynaptic density were much greater than when the order was reversed, a condition that decreases synaptic strengths, suggesting a possible mechanism underlying the induction of long-term changes in synaptic strength. The spatial and temporal mechanisms for selectivity in CaM activation demonstrated here could be used in other signaling pathways.