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The Subspine Organization of Actin Fibers Regulates the Structure and Plasticity of Dendritic Spines
Abstract
Synapse function and plasticity depend on the physical structure of dendritic spines as determined by the actin cytoskeleton. We have investigated the organization of filamentous (F-) actin within individual spines on CA1 pyramidal neurons in rat hippocampal slices. Using two-photon photoactivation of green fluorescent protein fused to beta-actin, we found that a dynamic pool of F-actin at the tip of the spine quickly treadmilled to generate an expansive force. The size of a stable F-actin pool at the base of the spine depended on spine volume. Repeated two-photon uncaging of glutamate formed a third pool of F-actin and enlarged the spine. The spine often released this "enlargement pool" into the dendritic shaft, but the pool had to be physically confined by a spine neck for the enlargement to be long-lasting. Ca2+/calmodulin-dependent protein kinase II regulated this confinement. Thus, spines have an elaborate mechanical nature that is regulated by actin fibers.
... Ce mécanisme est nécessaire aux mouvements, comme par exemple à la migration cellulaire, à des processus cellulaires tels que la division cellulaire ou l'endocytose, ou encore aux modifications morphologiques de la cellule en réponse à l'environnement (Blanchoin et al., 2014). Ces modifications de forme concernent entre autres les protrusions cellulaires, dont font partie les épines dendritiques, qui contiennent une concentration très élevée d'actine (Honkura et al., 2008). On considère désormais l'actine comme le principal facteur permettant les variations morphologiques des épines, que ce soit au cours de leur maturation développementale ou en réponse à l'activité. ...
... fois des filaments branchés, des filaments linéaires et quelques faisceaux (ou bundles), de tailles différentes et alignés entre eux de façon parallèle (Korobova & Svitkina, 2010). Dans la tête des épines, la F-actine possède deux pools : un « noyau » de F-actine stable, situé à la base de la tête, et qui constitue les fondations de sa structure, et un pool hautement dynamique, avec un taux de renouvellement élevé, localisé à la périphérie de l'épine, et dont la polymérisation serait responsable de la force de propulsion et des changements morphologiques rapides de l'épine (Honkura et al., 2008). ...
... En effet, grâce à ses interactions avec des protéines exprimées à la membrane de l'épine, telles que les molécules d'adhésion cellulaire ou les récepteurs au glutamate, le pool dynamique de la F-actine traduit les signaux extracellulaires en une modification de sa structure et/ou de sa dynamique, ce qui entraîne des variations de la morphologie de l'épine. Par ailleurs, en activant des épines isolées dans des tranches d'hippocampe par glutamate uncaging, un troisième pool a été identifié : il s'agit d'un pool d'élargissement, présent uniquement dans les épines stimulées et nécessaire à l'augmentation de la taille de l'épine sur le long terme (Honkura et al., 2008) (Figure 22). ...
Les épines dendritiques sont de petites protrusions membranaires qui portent la partie postsynaptique des synapses excitatrices. Les épines sont des structures extrêmement dynamiques : elles subissent des changements de forme et de fonction qui dépendent d’une réorganisation dynamique du cytosquelette d’actine. En effet, la dynamique de l’actine régule la forme de l’épine ainsi que la force de transmission synaptique. L’actine influence cette activité synaptique à travers le contrôle du nombre et de la localisation des récepteurs AMPA (AMPARs), qui assurent la transmission excitatrice rapide. Parmi les protéines qui régulent la dynamique de l’actine, la cofiline, une protéine qui dépolymérise l’actine, favorise le renouvellement dynamique de l’actine. L’inactivation de la cofiline par sa phosphorylation par la LIMK augmente la stabilité de l’actine, ce qui conduit à des altérations de forme d’épine et à un défaut de recrutement des AMPARs pendant la plasticité synaptique. Des anomalies de la dynamique des épines et de leur fonction sont une caractéristique de la plupart des pathologies neuropsychiatriques.La maladie de Huntington (MH) est une pathologie neurodégénérative et génétique caractérisée par la dysfonction et la dégénérescence des neurones du striatum et du cortex adultes. Les symptômes apparaissent à l’âge adulte et incluent des manifestations motrices, cognitives et psychiatriques. La MH est causée par la mutation du gène qui code pour la protéine huntingtine (HTT). Jusqu’à présent, la plupart des études se sont concentrées sur le gain de nouvelles fonctions toxiques de la HTT mutée. Cependant, on considère désormais que les évènements conduisant à la manifestation clinique de la MH sont en partie dus à la modification des fonctions de la HTT normale. La compréhension des fonctions normales de cette protéine est donc cruciale pour élucider les mécanismes cellulaires à l’origine de la MH.Nous montrons que la perte de HTT pendant le développement altère la morphologie des épines dendritiques et l’activité des synapses chez le jeune animal. Plus précisément, la HTT est présente dans le compartiment postsynaptique, et sa déplétion de façon cellule-autonome augmente le nombre d’épines matures et réduit la transmission synaptique excitatrice dépendante des AMPARs. Les AMPARs sont en effet affectés par la perte de HTT, puisque cette dernière réduit leur expression postsynaptique et limite leur recyclage à la suite d’une stimulation synaptique. Le cytosquelette d’actine est par ailleurs plus stable dans les épines déplétées en HTT, et ceci est associé à une hyperactivité de la voie moléculaire RAC1-LIMK-Cofiline. Ces résultats apportent de nouvelles informations quant aux fonctions moléculaires de la HTT dans la régulation de la morphologie des épines et de la physiologie de la synapse, processus qui pourrait être altéré dans le contexte de la MH.
... Decades of intensive analyses have revealed molecules and signaling cascades that regulate the dynamics of actin filaments (F-actins) for formation and structural plasticity of dendritic spines [14,[24][25][26][27][28][29]. Cell adhesion molecules (CAMs) and the extracellular matrix (ECM) proteins are also involved in providing mechanical support for spine structure [2,[30][31][32][33][34][35]. ...
... Actin cytoskeletons in mature spines also undergo dynamic turnover [26,71,133]; F-actins polymerize mainly at the peripheral region, and flow from the periphery toward the center of the spine head [26,36,37,71,84,134]. The activated Rac1 and Cdc42 described above can promote actin polymerization by activating N-WASP and WAVE, respectively, and subsequently activating the Arp2/3 complex [135,136]. ...
... Actin cytoskeletons in mature spines also undergo dynamic turnover [26,71,133]; F-actins polymerize mainly at the peripheral region, and flow from the periphery toward the center of the spine head [26,36,37,71,84,134]. The activated Rac1 and Cdc42 described above can promote actin polymerization by activating N-WASP and WAVE, respectively, and subsequently activating the Arp2/3 complex [135,136]. ...
Dendritic spines are small protrusions arising from dendrites and constitute the major compartment of excitatory post-synapses. They change in number, shape, and size throughout life; these changes are thought to be associated with formation and reorganization of neuronal networks underlying learning and memory. As spines in the brain are surrounded by the microenvironment including neighboring cells and the extracellular matrix, their protrusion requires generation of force to push against these structures. In turn, neighboring cells receive force from protruding spines. Recent studies have identified BAR-domain proteins as being involved in membrane deformation to initiate spine formation. In addition, forces for dendritic filopodium extension and activity-induced spine expansion are generated through cooperation between actin polymerization and clutch coupling. On the other hand, force from expanding spines affects neurotransmitter release from presynaptic terminals. Here, we review recent advances in our understanding of the physical aspects of synapse formation and plasticity, mainly focusing on spine dynamics.
... Finally, the use of photoactivatable GFP-actin fusion (e.g., paGFP-actin) has enabled photon-induced labeling of bulk or single actin molecules so that their fates can be monitored. This labeling approach showed that actin polymerization rates were higher at the spine edge than at the spine base and that actin polymers undergo retrograde flow from the periphery toward the spine base [23]. Live tracking of labeled actin also revealed pools of actin in spines that undergo dramatic expansion in response to stimulation [23]. ...
... This labeling approach showed that actin polymerization rates were higher at the spine edge than at the spine base and that actin polymers undergo retrograde flow from the periphery toward the spine base [23]. Live tracking of labeled actin also revealed pools of actin in spines that undergo dramatic expansion in response to stimulation [23]. The application of actin polymerization inhibitors rapidly arrested spine motility, indicating that actin filament networks are a primary determinant of spine shape and structural plasticity and that actin polymerization is indeed needed for spine expansion [24]. ...
... High-frequency stimulation that causes long-term potentiation (LTP), which is believed to be a proxy for memory formation, promotes spine head enlargement [71,93]. These same stimulations lead to a long-lasting increase in the stable actin pool [23,65]. Not only do spine heads increase in size following these potentiation events, but novel super-resolution imaging approaches revealed that spine necks also increase in diameter [66], an event that may also be driven by the expanding actin network. ...
Neurons transmit and receive information at specialized junctions called synapses. Excitatory synapses form at the junction between a presynaptic axon terminal and a postsynaptic dendritic spine. Supporting the shape and function of these junctions is a complex network of actin filaments and its regulators. Advances in microscopic techniques have enabled studies of the organization of actin at synapses and its dynamic regulation. In addition to highlighting recent advances in the field, we will provide a brief historical perspective of the understanding of synaptic actin at the synapse. We will also highlight key neuronal functions regulated by actin, including organization of proteins in the pre- and post- synaptic compartments and endocytosis of ion channels. We review the evidence that synapses contain distinct actin pools that differ in their localization and dynamic behaviors and discuss key functions for these actin pools. Finally, whole exome sequencing of humans with neurodevelopmental and psychiatric disorders has identified synaptic actin regulators as key disease risk genes. We briefly summarize how genetic variants in these genes impact neurotransmission via their impact on synaptic actin.
... Most synapses between excitatory neurons form on dendritic spines, which are protrusions from dendrites that consist of a large, actin-rich head separated from the dendrite shaft by a thin neck. Actin exists in multiple populations in dendritic spines, with different functions and rates of turnover [1]. Most actin present in spines is dynamic and can change its organisation very rapidly [2]. ...
... Previous studies have suggested there are functionally distinct populations of F-actin in dendritic spines [1,2]. These different populations are involved in maintaining the spine head size [1] and anchoring receptors to the membrane [7], and some are involved in trafficking receptors between membrane compartments [7,25]. ...
... Previous studies have suggested there are functionally distinct populations of F-actin in dendritic spines [1,2]. These different populations are involved in maintaining the spine head size [1] and anchoring receptors to the membrane [7], and some are involved in trafficking receptors between membrane compartments [7,25]. It is possible that Tpm3.1 regulates postsynaptic F-actin populations which are involved in processes distinct from synaptogenesis and clustering of ionotropic glutamate receptors. ...
Tropomyosin (Tpm) has been regarded as the master regulator of actin dynamics. Tpms regulate the binding of the various proteins involved in restructuring actin. The actin cytoskeleton is the predominant cytoskeletal structure in dendritic spines. Its regulation is critical for spine formation and long-term activity-dependent changes in synaptic strength. The Tpm isoform Tpm3.1 is enriched in dendritic spines, but its role in regulating the synapse structure and function is not known. To determine the role of Tpm3.1, we studied the synapse structure and function of cultured hippocampal neurons from transgenic mice overexpressing Tpm3.1. We recorded hippocampal field excitatory postsynaptic potentials (fEPSPs) from brain slices to examine if Tpm3.1 overexpression alters long-term synaptic plasticity. Tpm3.1-overexpressing cultured neurons did not show a significantly altered dendritic spine morphology or synaptic activity. Similarly, we did not observe altered synaptic transmission or plasticity in brain slices. Furthermore, expression of Tpm3.1 at the postsynaptic compartment does not increase the local F-actin levels. The results suggest that although Tpm3.1 localises to dendritic spines in cultured hippocampal neurons, it does not have any apparent impact on dendritic spine morphology or function. This is contrary to the functional role of Tpm3.1 previously observed at the tip of growing neurites, where it increases the F-actin levels and impacts growth cone dynamics.
... By inducing CALI 10 min after sLTP induction, both the enrichment of CFL-GFP and the increase in spine volume were reversed. sLTP is accompanied by decreased actin turnover within dendritic spines (12). To establish whether the enriched CFL and result-ing cofilactin formation are involved in this process, we tested whether CALI of CFL-SN can restore actin turnover using photoactivatable GFP (PAGFP)-fused actin (12). ...
... sLTP is accompanied by decreased actin turnover within dendritic spines (12). To establish whether the enriched CFL and result-ing cofilactin formation are involved in this process, we tested whether CALI of CFL-SN can restore actin turnover using photoactivatable GFP (PAGFP)-fused actin (12). Photoactivation of PAGFP-actin at the tip of dendritic spines revealed actin turnover within the body of the dendritic spine, which slowed after the induction of sLTP (Fig. 1C). ...
Where and when of memory consolidation
Episodic memory is initially encoded in the hippocampus and later transferred to other brain regions for long-term storage. Synaptic plasticity underlies learning and plays a critical role in memory consolidation. However, it remains largely unknown where and when synaptic plasticity occurs and how it shapes the neuronal representation. Goto et al . developed a new tool for controlling early structural long-term potentiation (sLTP). By selectively manipulating sLTP, the authors showed that the local circuitry in hippocampal area CA1 is required for memory formation shortly after the encoding event. The local circuitry is also important for offline memory consolidation within 24 hours. The anterior cingulate cortex, another brain region directly connected with area CA1, is crucial for memory consolidation during sleep on the second night. —PRS
... Dendritic spine enlargement by synaptic activation is thought to increase synaptic efficacy underlying learning and memory (Honkura et al., 2008;Bosch et al., 2014). F-actin retrograde flow in spines has been monitored by using photoactivatable fluorescent proteins or by combination of super resolution microscope and fluorescent proteins (Honkura et al., 2008;Tatavarty et al., 2009Tatavarty et al., , 2012Frost et al., 2010;Chazeau et al., 2014). ...
... Dendritic spine enlargement by synaptic activation is thought to increase synaptic efficacy underlying learning and memory (Honkura et al., 2008;Bosch et al., 2014). F-actin retrograde flow in spines has been monitored by using photoactivatable fluorescent proteins or by combination of super resolution microscope and fluorescent proteins (Honkura et al., 2008;Tatavarty et al., 2009Tatavarty et al., , 2012Frost et al., 2010;Chazeau et al., 2014). However, photoactivation of fluorescent proteins requires irradiation light with specific wavelength and intensity, which induces photobleaching and causes phototoxicity to cells (Lippincott-Schwartz and Patterson, 2009;Banaz et al., 2019). ...
Dendritic spine enlargement by synaptic activation is thought to increase synaptic efficacy underlying learning and memory. This process requires forces generated by actin polymerization and actin-adhesion coupling (clutch coupling). Here, we describe a protocol to monitor actin filament retrograde flow and actin polymerization within spines using a standard epi-fluorescence microscope. In combination with chemical long-term potentiation, this protocol allows us to quantify clutch coupling efficiency and actin polymerization rate, which are essential variables for generating forces for activity-dependent spine enlargement.
For complete details on the use and execution of this protocol, please refer to Kastian et al. (2021).
... F-actin can form macrostructures of a higher order, such as bundles and meshes, when actin filaments are cross-linked by specific actin-binding proteins, such as fimbrin, α-actinin, filamin, and also CaMKII (Hotulainen and Hoogenraad 2010;Chazeau et al. 2014;Chazeau and Giannone 2016). Although these structures are already present in naïve spines, the induction of LTP creates new F-actin macrostructures specifically at the potentiated spine with novel properties of higher stability, spine confinement, and different binding capacities (Honkura et al. 2008). One of these macrostructures is the one created by cofilin and F-actin. ...
After a synapse undergoes long-term potentiation (LTP), it acquires a newly remodeled molecular and structural organization. This reorganization over time is explained by a hypothetical structure called a synaptic tag, which is specifically formed at potentiated synapses, not at unstimulated ones, and captures newly synthesized proteins to persistently stabilize the potentiated state. However, to date, the molecular identity of the synaptic tag remains unclear. Based on several lines of experimental evidence, we propose that remodeled filamentous (F-) actin and CaMKII together form the synaptic tag by modifying the postsynaptic cytoskeletal structure to capture newly synthesized synaptic proteins. Liquid–liquid phase separation, a biophysical property of biological macromolecules, also plays a key role in this process. F-actin and CaMKII both fulfill the criteria to be the tag: they are specifically enriched at potentiated synapses without requiring new protein synthesis and persist for at least 1 h. Additionally, the intrinsic binding capacity of F-actin and CaMKII is ideal for capturing newly synthesized proteins at the synapse, thereby consolidating the synaptic structure and function and eventually allowing memory persistence.
... Disturbances in actin dynamics, which regulates both the somatodendritic compartment of neurons (Han et al., 2017) and dendritic spines (Honkura et al., 2008;Hotulainen and Hoogenraad, 2010), could account for both the smaller size and lower dendritic spine density of L3PN in schizophrenia; indeed, expression levels of transcripts in actin-regulating pathways are altered in DLPFC L3PNs in schizophrenia (Datta et al., 2017;Datta et al., 2015) and correlated with lower dendritic spine density (Hill et al., 2006;Ide and Lewis, 2010). Thus, our findings that COX4I1 mRNA levels scale similarly with somal size in both UC and schizophrenia subjects supports the interpretation that lower COX4I1 expression is an appropriate consequence of L3PNs receiving less excitatory drive in schizophrenia. ...
Background
Dorsolateral prefrontal cortex (DLPFC) dysfunction in schizophrenia appears to reflect alterations in layer 3 pyramidal neurons (L3PNs), including smaller cell bodies and lower expression of mitochondrial energy production genes. However, prior somal size studies used biased strategies for identifying L3PNs, and somal size and levels of energy production markers have not been assessed in individual L3PNs.
Study design
We combined fluorescent in situ hybridization (FISH) of vesicular glutamate transporter 1 (VGLUT1) mRNA and immunohistochemical-labeling of NeuN to determine if the cytoplasmic distribution of VGLUT1 mRNA permits the unbiased identification and somal size quantification of L3PNs. Dual-label FISH for VGLUT1 mRNA and cytochrome C oxidase subunit 4I1 (COX4I1) mRNA, a marker of energy production, was used to assess somal size and COX4I1 transcript levels in individual DLPFC L3PNs from schizophrenia (12 males; 2 females) and unaffected comparison (13 males; 1 female) subjects.
Study results
Measures of L3PN somal size with NeuN immunohistochemistry or VGLUT1 mRNA provided nearly identical results (ICC = 0.96, p < 0.0001). Mean somal size of VGLUT1-identified L3PNs was 8.7% smaller (p = 0.004) and mean COX4I1 mRNA levels per L3PN were 16.7% lower (p = 0.01) in schizophrenia. These measures were correlated across individual L3PNs in both subject groups (rrm = 0.81–0.86).
Conclusions
This preliminary study presents a novel method for combining unbiased neuronal identification with quantitative assessments of somal size and mRNA levels. We replicated findings of smaller somal size and lower COX4I1 mRNA levels in DLPFC L3PNs in schizophrenia. The normal scaling of COX4I1 mRNA levels with somal size in schizophrenia suggests that lower markers of energy production are secondary to L3PN morphological alterations in the illness.
... Long-term potentiation (LTP), the cellular correlate of learning and memory, induces both spinogenesis and spine enlargement (Matsuzaki et al., 2004;Harvey and Svoboda, 2007;Bourne and Harris, 2011;Watson et al., 2016). LTP triggers branched actin polymerization adjacent to the post-synaptic density, thereby promoting spine growth and the consolidation of the potentiated state (Matsuzaki et al., 2004;Okamoto et al., 2004;Honkura et al., 2008;Bosch et al., 2014). Actin remodeling is therefore a key driver of spine formation and structural plasticity. ...
Structural plasticity, the ability of dendritic spines to change their volume in response to synaptic stimulation, is an essential determinant of synaptic strength and long-term potentiation (LTP), the proposed cellular substrate for learning and memory. Branched actin polymerization is a major force driving spine enlargement and sustains structural plasticity. The WAVE Regulatory Complex (WRC), a pivotal branched actin regulator, controls spine morphology and therefore structural plasticity. However, the molecular mechanisms that govern WRC activation during spine enlargement are largely unknown. Here we identify a critical role for Neogenin and its ligand RGMa (Repulsive Guidance Molecule a) in promoting spine enlargement through the activation of WRC-mediated branched actin remodeling. We demonstrate that Neogenin regulates WRC activity by binding to the highly conserved Cyfip/Abi binding pocket within the WRC. We find that after Neogenin or RGMa depletion, the proportions of filopodia and immature thin spines are dramatically increased, and the number of mature mushroom spines concomitantly decreased. Wildtype Neogenin, but not Neogenin bearing mutations in the Cyfip/Abi binding motif, is able to rescue the spine enlargement defect. Furthermore, Neogenin depletion inhibits actin polymerization in the spine head, an effect that is not restored by the mutant. We conclude that RGMa and Neogenin are critical modulators of WRC-mediated branched actin polymerization promoting spine enlargement. This study also provides mechanistic insight into Neogenin’s emerging role in LTP induction.
... If one considers the nature of the synaptic tag, one strong candidate is the actin cytoskeleton. It is well established that morphological changes observed in dendritic spines, induced by synaptic plasticity, are strongly associated with the actin cytoskeleton dynamics (Honkura et al., 2008;Matsuzaki et al., 2004). The dynamics of actin is modulated by several actin-binding proteins (ABPs) such as CaMKII, Cofilin, and Drebrin (Hayashi et al., 1996;Okamoto et al., 2009Okamoto et al., , 2004. ...
Synapses change their weights in response to neuronal activity and in turn, neuronal networks alter their response properties and ultimately allow the brain to store information as memories. As for memories, not all events are maintained over time. Maintenance of synaptic plasticity depends on the interplay between functional changes at synapses and the synthesis of plasticity-related proteins that are involved in stabilizing the initial functional changes. Different forms of synaptic plasticity coexist in time and across the neuronal dendritic area. Thus, homosynaptic plasticity refers to activity-dependent synaptic modifications that are input-specific, whereas heterosynaptic plasticity relates to changes in non-activated synapses. Heterosynaptic forms of plasticity, such as synaptic cooperation and competition allow neurons to integrate events that occur separated by relatively large time windows, up to one hour. Here, we show that activation of Cdc42, a Rho GTPase that regulates actin cytoskeleton dynamics, is necessary for the maintenance of long-term potentiation (LTP) in a time-dependent manner. Inhibiting Cdc42 activation does not alter the time-course of LTP induction and its initial expression but blocks its late maintenance. We show that Cdc42 activation is involved in the phosphorylation of cofilin, a protein involved in modulating actin filaments and that weak and strong synaptic activation leads to similar levels on cofilin phosphorylation, despite different levels of LTP expression. We show that Cdc42 activation is required for synapses to interact by cooperation or competition, supporting the hypothesis that modulation of the actin cytoskeleton provides an activity-dependent and time-restricted permissive state of synapses allowing synaptic plasticity to occur. We found that under competition, the sequence in which synapses are activated determines the degree of LTP destabilization, demonstrating that competition is an active destabilization process. Taken together, we show that a dynamic actin cytoskeleton is necessary for the expression of homosynaptic and heterosynaptic forms of plasticity. Determining the temporal and spatial rules that determine whether synapses cooperate or compete will allow us to understand how memories are associated.
Graphical Abstract
Highlights
Cdc42 is required for the maintenance of homosynaptic synaptic plasticity
Weak and strong stimulation modulate actin by cofilin phosphorylation
Cdc42 activation is necessary for heterosynaptic cooperation and competition
Synaptic competition is an active destabilization process
The time-window of synaptic cooperation and competition is activity dependent
... β-actin is highly dynamic in neurons and undergoes coordinated assembly, disassembly, and treadmilling. A highly dynamic fast actin pool can be detected in neurons with a time constant less than a minute, while other species take 17 min or more to recover [25]. We next asked whether Cdk12 affects fast β-actin dynamics. ...
The role of cyclin-dependent kinases (CDKs) that are ubiquitously expressed in the adult nervous system remains unclear. Cdk12 is enriched in terminally differentiated neurons where its conical role in the cell cycle progression is redundant. We find that in adult neurons Cdk12 acts a negative regulator of actin formation, mitochondrial dynamics and neuronal physiology. Cdk12 maintains the size of the axon at sites proximal to the cell body through the transcription of homeostatic enzymes in the 1-carbon by folate pathway which utilize the amino acid homocysteine. Loss of Cdk12 leads to elevated homocysteine and in turn leads to uncontrolled F-actin formation and axonal swelling. Actin remodeling further induces Drp1-dependent fission of mitochondria and the breakdown of axon-soma filtration barrier allowing soma restricted cargos to enter the axon. We demonstrate that Cdk12 is also an essential gene for long-term neuronal survival and loss of this gene causes age-dependent neurodegeneration. Hyperhomocysteinemia, actin changes, and mitochondrial fragmentation are associated with several neurodegenerative conditions such as Alzheimer's disease and we provide a candidate molecular pathway to link together such pathological events. Cell Death Discovery (2023) 9:348 ; https://doi.
... The actin cytoskeleton is found in various regions within a dendritic spine, such as postsynaptic densities (PSD), and spine necks and core regions (Peng et al., 2004;Korobova and Svitkina, 2010;Bar et al., 2016). But actin dynamics vary in each subcellular region (Honkura et al., 2008), possibly regulated by various actin-binding proteins (dos Remedios et al., 2003;Pollard and Cooper, 2009) in region-specific ways. Drebrin and Ca 2+ /calmodulin-dependent protein kinase II (CaMKIIβ) are F-actin-binding proteins in dendritic spines (Ishikawa et al., 1994;Hayashi et al., 1996;Shen et al., 1998;Kim et al., 2015). ...
Dendritic spines are unique postsynaptic structures that emerge from the dendrites of neurons. They undergo activity-dependent morphological changes known as structural plasticity. The changes involve actin cytoskeletal remodeling, which is regulated by actin-binding proteins. CaMKII is a crucial molecule in synaptic plasticity. Notably, CaMKIIβ subtype is known to bind to filamentous-actin and is closely involved in structural plasticity. We have shown that CaMKIIβ binds to drebrin, and is localized in spines as both drebrin-dependent and drebrin-independent pools. However, the nanoscale relationship between drebrin and CaMKIIβ within dendritic spines has not been clarified. In this study, we used stochastic optical reconstruction microscopy (STORM) to examine the detailed localization of these proteins. STORM imaging showed that CaMKIIβ co-localized with drebrin in the core region of spines, and localized in the submembrane region of spines without drebrin. Interestingly, the dissociation of CaMKIIβ and drebrin in the core region was induced by NMDA receptor activation. In drebrin knockdown neurons, CaMKIIβ was decreased in the core region but not in the submembrane region. Together it indicates that the clustering of CaMKIIβ in the spine core region is dependent on drebrin. These findings suggest that drebrin-dependent CaMKIIβ is in a standby state before its activation.
... Indeed, ethanol disrupts synaptic transmission at dMSN synapses [36,37]. Moreover, ethanol exposure increases dMSN dendritic length, presumably increasing the synaptic contacts made onto spines [35,38]. This is a possible mechanism by which ethanol consumption can strengthen glutamatergic input signaling onto dMSNs [34,35]. ...
Endocannabinoids (eCB) and cannabinoid receptor 1 (CB1) play important roles in mediating short- and long-term synaptic plasticity in many brain regions involved in learning and memory, as well as the reinforcing effects of misused substances. Ethanol-induced plasticity and neuroadaptations predominantly occur in striatal direct pathway projecting medium spiny neurons (dMSNs). It is hypothesized that alterations in eCB neuromodulation may be involved. Recent work has implicated a role of eCB 2-arachidonoylglycerol (2-AG) in the rewarding effects of ethanol. However, there is insufficient research to answer which cellular subtype is responsible for mediating the 2-AG eCB signal that might be involved in the rewarding properties of ethanol and the mechanisms by which that occurs. To examine the role of dMSN mediated 2-AG signaling in ethanol related synaptic transmission and behaviors, we used conditional knockout mice in which the 2-AG-synthesizing enzyme diacylglycerol lipase α (DGLα) was deleted in dMSNs, DGLα D1-Cre+ . Using acute brain slice photometry and a genetically encoded fluorescent eCB sensor, GRAB eCB2.0, to assess real-time eCB mediated activity of sensorimotor inputs from primary motor cortices (M1/M2) to the dorsolateral striatum, we showed that DGLα D1-Cre+ mice had blunted evoked eCB-mediated presynaptic eCB signaling compared to littermate controls. Furthermore, ethanol induced eCB inhibition was significantly reduced in DGLα D1-Cre+ deficient mice. Additionally, there was a reduction in the duration of loss of righting reflex (LORR) to a high dose of ethanol in the DGLα D1-Cre+ mice compared to controls. These mice also showed a male-specific decrease in ethanol preference accompanied by an increase in ethanol-induced water consumption in a voluntary drinking paradigm. There were no significant differences observed in sucrose and quinine consumption between the genotypes. These findings reveal a novel role for dMSN mediated 2-AG signaling in modulating ethanol effects on presynaptic function and behavior.
... Actin rests in the polymerized filamentous form in a major proportion and in a soluble depolymerized monomeric form in minor proportion. Total actin is higher in dendritic spines when compared with dendritic shafts (Honkura et al. 2008). During synaptic activity, morphological changes of dendritic spines occurs by actin polymerization and the cross-talks between actin polymerization and/or depolymerization determine the motility, growth and shape of the dendritic spines during their maturation (Chidambaram et al. 2019;Walker and Herskowitz 2021). ...
Depression and Alzheimer´s disease (AD) are two disorders highly prevalent worldwide. Depression affects more than 300 million people worldwide while AD affects 60% to 80% of the 55 million cases of dementia. Both diseases are affected by aging with high prevalence in elderly and share not only the main brain affected areas but also several physiopathological mechanisms. Depression disease is already ascribed as a risk factor to the development of AD. Despite the wide diversity of pharmacological treatments currently available in clinical practice for depression management, they remain associated to a slow recovery process and to treatment-resistant depression. On the other hand, AD treatment is essentially based in symptomatology relieve. Thus, the need for new multi-target treatments arises. Herein, we discuss the current state-of-art regarding the contribution of the endocannabinoid system (ECS) in synaptic transmission processes, synapses plasticity and neurogenesis and consequently the use of exogenous cannabinoids in the treatment of depression and on delaying the progression of AD. Besides the well-known imbalance of neurotransmitter levels, including serotonin, noradrenaline, dopamine and glutamate, recent scientific evidence highlights aberrant spine density, neuroinflammation, dysregulation of neurotrophic factor levels and formation of amyloid beta (Aβ) peptides, as the main physiopathological mechanisms compromised in depression and AD. The contribution of the ECS in these mechanisms is herein specified as well as the pleiotropic effects of phytocannabinoids. At the end, it became evident that Cannabinol, Cannabidiol, Cannabigerol, Cannabidivarin and Cannabichromene may act in novel therapeutic targets, presenting high potential in the pharmacotherapy of both diseases.
... The study suggested that some tiny structures, such as spine necks, tended to be swollen by conditional chemical fixation (Tamada et al. 2020). Because spine shape critically depends on the arrangement of actin, which is easily influenced by exposure to aldehydes (Honkura et al. 2008), this might contribute to the differences between cryo-and chemical-fixed tissue. The value of FIB/SEM required in the future will depend on the extent to which it reveals the native ultrastructure and whether these cells are representative of the physiological state (Hoffman et al. 2020). ...
Morphological analysis of organelles is one of the important clues for understanding the cellular conditions and mechanisms occurring in cells. In particular, nanoscale information within crowded intracellular organelles of tissues provide more direct implications when compared to analyses of cells in culture or isolation. However, there are some difficulties in detecting individual shape using light microscopy, including super-resolution microscopy. Transmission electron microscopy (TEM), wherein the ultrastructure can be imaged at the membrane level, cannot determine the whole structure, and analyze it quantitatively. Volume EM, such as focused ion beam/scanning electron microscopy (FIB/SEM), can be a powerful tool to explore the details of three-dimensional ultrastructures even within a certain volume, and to measure several parameters from them. In this review, the advantages of FIB/SEM analysis in organelle studies are highlighted along with the introduction of mitochondrial analysis in injured motor neurons. This would aid in understanding the morphological details of mitochondria, especially those distributed in the cell bodies as well as in the axon initial segment (AIS) in mouse tissues. These regions have not been explored thus far due to the difficulties encountered in accessing their images by conditional microscopies. Some mechanisms of nerve regeneration have also been discussed with reference to the obtained findings. Finally, future perspectives on FIB/SEM are introduced. The combination of biochemical and genetic understanding of organelle structures and a nanoscale understanding of their three-dimensional distribution and morphology will help to match achievements in genomics and structural biology.
... Although straight filaments are also present in the spine head, they are more common in the neck part, or in immature filopodia (Korobova and Svitkina, 2010). Actin remodeling is tightly regulated in connection with synaptic plasticity and is therefore important in learning and memory as well as in neuronal network functions (Schubert and Dotti, 2007;Honkura et al., 2008;Rudy, 2015). This is in accordance with the accumulating evidence that abnormal actin regulation in dendritic ...
Actin cytoskeleton predominantly regulates the formation and maintenance of synapses by controlling dendritic spine morphology and motility. To visualize actin dynamics, actin molecules can be labelled by genetically fusing fluorescent proteins to actin monomers, actin-binding proteins or single-chain anti-actin antibodies. In the present study, we compared the dendritic effect of EGFP-actin, LifeAct-TagGFP2 (LifeAct-GFP) and Actin-Chromobody-TagGFP2 (AC-GFP) in mouse cultured hippocampal neurons using unbiased quantitative methods.
The actin-binding probes LifeAct-GFP and AC-GFP showed similar affinity to F-actin, but in contrast to EGFP-actin, they did not reveal subtle changes in actin remodelling between mushroom shaped spines and filopodia. All tested actin probes colocalized with phalloidin similarly, however, the enrichment of LifeAct-GFP in dendritic spines was remarkably lower compared to the other constructs.
LifeAct-GFP expression was tolerated at a higher expression level compared to EGFP-actin and AC-GFP with only subtle differences identified in dendritic spine morphology and protrusion density. While EGFP-actin and LifeAct-GFP expression did not alter dendritic arborization, AC-GFP expressing neurons displayed a reduced dendritic tree. Thus, although all tested actin probes may be suitable for actin imaging studies, certain limitations should be considered before performing experiments with a particular actin labelling probe in primary neurons.
[Media: see text] [Media: see text]
... whereas remodelling of actin networks within dendritic spines has also been shown to be crucial for activity-dependent structural changes (Okamoto et al. 2004;Honkura et al. 2008;Frost et al. 2010 50 | P a g e and receptor number on the surface have all been reported to be responsible for synaptic potentiation (Isaac, Nicoll, and Malenka 1995;Roche et al. 1996;Benke et al. 1998;S.-H. Shi et al. 1999). ...
Synapses are the main site of communication between neurons in the central nervous system. These specialised cell-cell contacts are initiated by cell adhesion molecules at the pre- and post-synapse, that interact with one another to form trans-synaptic complexes, and recruit molecules regulating synapse maturation, specificity and function.Leucine Rich Repeat Transmembrane Protein 2 (LRRTM2) is a synaptic adhesion molecule that binds to pre-synaptic neurexin and is exclusively localised and enriched at excitatory synapses where it exhibits low membrane dynamics. Interestingly, LRRTM2 is involved in synaptic transmission and plasticity and regulates the surface levels of AMPARs, the main glutamatergic receptors responsible for fast neurotransmission in the brain.In my PhD, I investigated the molecular mechanisms underlying LRRTM2 stabilisation and trafficking at excitatory synapses, as well as the interplay between LRRTM2 and AMPARs.We demonstrated that the C-terminal domain of LRRTM2 controls its compartmentalisation in dendrites as well as its enrichment and compaction at synapses. Surprisingly, LRRTM2 synaptic confinement was found to be independent of its PDZ-like binding domain and was instead regulated by a recently identified YxxC intracellular sequence. We further confirmed that this sequence was critical for LRRTM2 trafficking and exocytosis and observed the existence of intracellular LRRTM2-containing vesicles inside spines. Regarding the interplay between LRRTM2 and AMPARs, we showed for the first time, that the recently identified neurexin-binding site in LRRTM2 (E348) is responsible for membrane stabilisation of synaptic AMPARs.These results demonstrate that the intracellular region of LRRTM2 controls its synaptic clustering, membrane dynamics, and confinement, while extracellular binding interfaces are involved in stabilising AMPARs at the plasma membrane.
... Inspection of hippocampal neurons in primary cultures expressing synaptopodin tagged with monomeric red fluorescent protein (mRFP-synaptopodin) and the cytosolic marker EGFP (enhanced green fluorescent protein) confirmed the presence of synaptopodin in 33 ± 8% of spines, where it strongly colocalized with a pool of F-actin as detected by phalloidin ( Fig. 1 E and F and SI Appendix, Fig. S1F ) (12). Consistent with EM observations, mRFP-synaptopodin was concentrated close to the neck of the spine (the high-magnification fields in Fig. 1E), where a stable pool of F-actin is reported to reside, and was absent from the head where branched F-actin predominates (Fig. 1F ) (26). In addition, in a subset of cultured hippocampal neurons, endogenous synaptopodin localized to the axonal initial segment where the cisternal organelle is observed by EM (SI Appendix, Fig. S1G) (11,27). ...
The spine apparatus is a specialized compartment of the neuronal smooth endoplasmic reticulum (ER) located in a subset of dendritic spines. It consists of stacks of ER cisterns that are interconnected by an unknown dense matrix and are continuous with each other and with the ER of the dendritic shaft. While this organelle was first observed over 60 y ago, its molecular organization remains a mystery. Here, we performed in vivo proximity proteomics to gain some insight into its molecular components. To do so, we used the only known spine apparatus-specific protein, synaptopodin, to target a biotinylating enzyme to this organelle. We validated the specific localization in dendritic spines of a small subset of proteins identified by this approach, and we further showed their colocalization with synaptopodin when expressed in nonneuronal cells. One such protein is Pdlim7, an actin binding protein not previously identified in spines. Pdlim7, which we found to interact with synaptopodin through multiple domains, also colocalizes with synaptopodin on the cisternal organelle, a peculiar stack of ER cisterns resembling the spine apparatus and found at axon initial segments of a subset of neurons. Moreover, Pdlim7 has an expression pattern similar to that of synaptopodin in the brain, highlighting a functional partnership between the two proteins. The components of the spine apparatus identified in this work will help elucidate mechanisms in the biogenesis and maintenance of this enigmatic structure with implications for the function of dendritic spines in physiology and disease.
... It is well known that synaptic activity, in the form of long-term potentiation (LTP), increases calcium influx in dendritic spines through AMPA and NMDA receptors (Fortin et al., 2010;Lu et al., 2001). LTPinduced calcium influx activates several downstream pathways resulting in increased actin polymerization in individual spines that are potentiated (Bosch et al., 2014;Fukazawa et al., 2003;Honkura et al., 2008). A parsimonious explanation of how actin filaments (f-actin) could influence MT invasion of spines is that f-actin could serve as a "gate" or "ramp" in the axon shaft and could physically deflect MTs into specific spines that are potentiated ( Figure 3). ...
Microtubules (MT) are elongated, tubular, cytoskeletal structures formed from polymerization of tubulin dimers. They undergo continuous cycles of polymerization and depolymerization, primarily at their plus ends, termed dynamic instability. Although this is an intrinsic property of MTs, there are a myriad of MT-associated proteins that function in regulating MT dynamic instability and other dynamic processes that shape the MT array. Additionally, MTs assemble into long, semi-rigid structures which act as substrates for long-range, motor-driven transport of many different types of cargoes throughout the cell. Both MT dynamics and motor-based transport play important roles in the function of every known type of cell. Within the last fifteen years many groups have shown that MT dynamics and transport play ever-increasing roles in the neuronal function of mature neurons. Not only are neurons highly polarized cells, but they also connect with one another through synapses to form complex networks. Here we will focus on exciting studies that have illuminated how MTs function both pre-synaptically in axonal boutons and post-synaptically in dendritic spines. It is becoming clear that MT dynamics and transport both serve important functions in synaptic plasticity. Thus, it is not surprising that disruption of MTs, either through hyperstabilization or destabilization, has profound consequences for learning and memory. Together, the studies described here suggest that MT dynamics and transport play key roles in synaptic function and when disrupted result in compromised learning and memory.
... It has long been speculated that memory storage in the brain implicates altering the strength of large assemblies of interconnected neurons (Nadel et al., 1975), where synaptic plasticity may constitute the physical correlates of memory storage (Kandel and Squire, 2000). However, the "intrinsic electrical activity" in neurons (Llinás, 1988) that includes both passive and active membrane characteristics, does not include the neuronal cytoskeleton, which is essential to experience-related plasticity, and changes associated with neural stimulation (Honkura et al., 2008). ...
Dendritic spines (DS) are tiny protrusions implicated in excitatory postsynaptic responses in the CNS. To achieve their function, DS concentrate a high density of ion channels and dynamic actin networks in a tiny specialized compartment. However, to date there is no direct information on DS ionic conductances. Here, we used several experimental techniques to obtain direct electrical information from DS of the adult mouse hippocampus. First, we optimized a method to isolate DS from the dissected hippocampus. Second, we used the lipid bilayer membrane (BLM) reconstitution and patch clamping techniques and obtained heretofore unavailable electrical phenotypes on ion channels present in the DS membrane. Third, we also patch clamped DS directly in cultured adult mouse hippocampal neurons, to validate the electrical information observed with the isolated preparation. Electron microscopy and immunochemistry of PDS-95 and NMDA receptors and intrinsic actin networks confirmed the enrichment of the isolated DS preparation, showing open and closed DS, and multi-headed DS. The preparation was used to identify single channel activities and “whole-DS” electrical conductance. We identified NMDA and Ca ²⁺ -dependent intrinsic electrical activity in isolated DS and in situ DS of cultured adult mouse hippocampal neurons. In situ recordings in the presence of local NMDA, showed that individual DS intrinsic electrical activity often back-propagated to the dendrite from which it sprouted. The DS electrical oscillations were modulated by changes in actin cytoskeleton dynamics by addition of the F-actin disrupter agent, cytochalasin D, and exogenous actin-binding proteins. The data indicate that DS are elaborate excitable electrical devices, whose activity is a functional interplay between ion channels and the underlying actin networks. The data argue in favor of the active contribution of individual DS to the electrical activity of neurons at the level of both the membrane conductance and cytoskeletal signaling.
... Although straight filaments are also present in the spine head, they are more common in the neck part, or in immature filopodia (Korobova and Svitkina, 2010). Actin remodelling is tightly regulated in connection with synaptic plasticity and is therefore important in learning and memory as well as in neuronal network functions (Schubert and Dotti, 2007;Honkura et al., 2008;Rudy, 2015). This is in accordance with the accumulating evidence that abnormal actin regulation in dendritic spines plays an important role in different neurological diseases and thus research of actin regulation in dendritic spines is crucial in understanding these pathologies (Joensuu, Lanoue and Hotulainen, 2018;Pelucchi, Stringhi and Marcello, 2020). ...
Actin cytoskeleton predominantly regulates the formation and maintenance of synapses by controlling dendritic spine morphology and motility. To visualize actin dynamics, actin molecules can be labelled by genetically fusing fluorescent proteins to actin monomers or using fluorescently tagged actin-binding proteins or single-chain anti-actin antibodies. However, the effects of these labels on the morphology of neurons have not been quantitatively compared yet. In the present study, we analysed Actin-Chromobody-GFP, LifeAct-GFP and EGFP-actin with respect to their effects on actin-related features in mouse cultured hippocampal neurons.
The actin-binding probes LifeAct and Actin-Chromobody showed similar affinity to F-actin, and along with EGFP-actin, were enriched in dendritic protrusions. In contrast to EGFP-actin, neither of these constructs was able to detect subtle changes in actin remodelling between mature mushroom shaped spine and less developed filopodia. None of the compared probes altered filopodial motility compared to control EGFP expression, however, within 24 hours expression, minor changes in dendritic spine morphology and density were visible. Furthermore, while EGFP-actin and LifeAct-GFP expression did not alter dendritic arborization, AC-GFP expressing neurons displayed a reduced dendritic arborization. We therefore conclude that careful consideration of cellular consequences is required before performing experiments with a particular actin labelling probe in primary neurons.
... Bleb-forming conditions can be further exacerbated by a decrease in RhoA activity or lack of RhoA activation/modulation, consistent with the observation made for cells expressing Lphn3 and T783M but also denoted by A247S receptor variant's inaptitude to follow suit as well as D615N and R465S. Interestingly, neuronal properties emanating from RhoA modulation deficiency correlate with cognition impairments linked to the essential contribution of actin cytoskeleton assembly/disassembly dynamics in synaptic plasticity paradigms [51,59,60]. ...
Latrophilin-3 (Lphn3; also known as ADGRL3) is a member of the adhesion G Protein Coupled Receptor subfamily, which participates in the stabilization and maintenance of neuronal networks by mediating intercellular adhesion through heterophilic interactions with transmembrane ligands. Polymorphisms modifying the Lphn3 gene are associated with attention-deficit/hyperactivity disorder (ADHD) in children and its persistence into adulthood. How these genetic alterations affect receptor function remains unknown. Here, we conducted the functional validation of distinct ADHD-related Lphn3 variants bearing mutations in the receptor’s adhesion motif-containing extracellular region. We found that all variants tested disrupted the ability of Lphn3 to stabilize intercellular adhesion in a manner that was distinct between ligands classes, but which did not depend on ligand-receptor interaction parameters, thus pointing to altered intrinsic receptor signaling properties. Using G protein signaling biosensors, we determined that Lphn3 couples to Gαi1, Gαi2, Gαs, Gαq, and Gα13. However, all ADHD-related receptor variants consistently lacked intrinsic as well as ligand-dependent Gα13 coupling efficiency while maintaining unaltered coupling to Gαi, Gαs, and Gαq. Consistent with these alterations, actin remodeling functions as well as actin-relevant RhoA signaling normally displayed by the constitutively active Lphn3 receptor were impeded by select receptor variants, thus supporting additional signaling defects. Taken together, our data point to Gα13 selective signaling impairments as representing a disease-relevant pathogenicity pathway that can be inherited through Lphn3 gene polymorphisms. This study highlights the intricate interplay between Lphn3 GPCR functions and the actin cytoskeleton in modulating neurodevelopmental cues related to ADHD etiology.
... Assuming that actin PTMs and binding proteins are maintained in a similar way to that of MTs, the extracted system can be used to study regions such as dendritic spines and growth cones. In dendritic spines, when neurons are stimulated, AF density increases and nearby MTs are signaled to invade (Gray et al., 1982;Gu et al., 2008;Honkura et al., 2008;X. Hu et al., 2011;Merriam et al., 2013;Westrum et al., 1980). ...
The organization of structurally polarized microtubules into networks is critical for efficient cargo transport mediated by the molecular motors dynein and kinesin. The motility properties of molecular motors are best understood in simplified reconstituted systems using single microtubule filaments, as well as in cells with radial microtubule arrangements and axonal compartments with uniformly oriented microtubule arrays. However, it is not understood how active transport occurs in environments with more complicated cytoskeletal geometries, such as the mixed polarity microtubule arrays found in the dendrites of neurons. Here we focus on the plus-end directed kinesin-4 KIF21B motor that is associated with retrograde biased cargo movement in dendrites, despite the mixed polarity microtubule organization. How KIF21B achieves this net directional bias, as well as whether KIF21B is primarily responsible for retrograde directed motility is not known. To understand this, we examined KIF21B motility on mixed polarity microtubule arrays within in vitro systems of increasing complexity and in live neurons. In reconstituted systems with recombinant KIF21B and engineered dynamic antiparallel microtubule bundles or extracted mixed polarity dendritic microtubule arrays, the nucleotide-independent microtubule binding regions of KIF21B were shown to modulate microtubule dynamics and promote directional track switching. For analysis of KIF21B motility, existing methods to automate motor tracking were not ideal, and we developed a segmentation tool called Cega, to detect purified fluorescently labeled kinesin motors moving within a system with high background noise. Interestingly, KIF21B motors did not display the net directional bias along stabilized extracted dendritic microtubule arrays, as seen by KIF21B in live cells. This in combination with the dramatic stabilization of microtubule dynamics by KIF21B suggested that directional bias required microtubule remodeling by KIF21B motors, and thus would only be observed along native dynamic microtubule arrays. Unsurprisingly, KIF21B optogenetic recruitment to dendritic cargo induced net retrograde movement, and both native microtubule dynamics and the secondary microtubule binding regions of KIF21B were required to achieve this directional bias. These results suggest a mechanism where teams of cargo bound KIF21B motors coordinate nucleotide-sensitive and insensitive microtubule binding sites to regulate microtubule stability and promote track switching and ultimately achieve net retrograde movement along the mixed polarity microtubule arrays of dendrites.
... How the presence of Synpo leads to spine stabilization is unknown but a potential mechanism 560 may involve modification of actin cytoskeleton dynamics. Congruent with its association with the 561 SA, Synpo is present at the base of the head and in the neck of mushroom spines, regions in 562 23 which F-actin is less dynamic (Honkura et al, 2008) and concentrated between the lamellae of 563 the SA (Capani et al, 2001). By enhancing α-actinin-dependent F-actin bundling (Asanuma et ...
Dendritic spines, actin-rich protrusions forming the postsynaptic sites of excitatory synapses, undergo activity-dependent molecular and structural remodeling. Activation of Group 1 metabotropic glutamate receptors (mGluR1 and mGluR5) by synaptic or pharmacological stimulation, induces LTD, but whether this is accompanied with spine elimination remains unresolved. A subset of telencephalic mushroom spines contains the spine apparatus (SA), an enigmatic organelle composed of stacks of smooth endoplasmic reticulum, whose formation depends on the expression of the actin-bundling protein Synaptopodin. Allocation of Synaptopodin to spines appears governed by cell-intrinsic mechanisms as the relative frequency of spines harboring Synaptopodin is conserved in vivo and in vitro. Here we show that expression of Synaptopodin/SA in spines is required for induction of mGluR-LTD at Schaffer collateral-CA1 synapses of male mice. Post-mGluR-LTD, mushroom spines lacking Synaptopodin/SA are selectively lost, whereas spines harboring it are preserved. This process, dependent on activation of mGluR1 but not mGluR5, is conserved in mature mouse neurons and rat neurons of both sexes. Mechanistically, we find that mGluR1 supports physical retention of Synaptopodin within excitatory spine synapses during LTD while triggering lysosome-dependent degradation of the protein residing in dendritic shafts. Together, these results reveal a cellular mechanism, dependent on mGluR1, which enables selective preservation of stronger spines containing Synaptopodin/SA while eliminating weaker ones and potentially countering spurious strengthening by de novo recruitment of Synaptopodin. Overall, our results identify spines with Synaptopodin/SA as the locus of mGluR-LTD and underscore the importance of the molecular microanatomy of spines in synaptic plasticity.
... Inspection of hippocampal neurons in primary cultures expressing mRFP-synaptopodin and the cytosolic marker EGFP confirmed presence of synaptopodin in spines (Deller et al., 2000) where it strongly colocalized with a pool of F-actin as detected by phalloidin ( Figures 1E-F). Consistent with EM observations, mRFP-synaptopodin was concentrated close to the neck of the spine ( Figure 1E, see high magnification fields) where the stable pool of actin is reported to reside, and was absent from the head where branched F-actin predominates ( Figure 1F) (Honkura et al., 2008). In addition, in a subset of cultured hippocampal neurons endogenous synaptopodin localized to axonal initial segments where the cisternal organelle is observed by EM ( Figure S1E) Peters et al., 1968). ...
The spine apparatus is a specialization of the neuronal ER in dendritic spines consisting of stacks of interconnected cisterns separated by a dense matrix. Synaptopodin, a specific actin binding protein of the spine apparatus, is essential for its formation, but the underlying mechanisms remain unknown. We show that synaptopodin, when expressed in fibroblasts, forms actin-rich structures with connections to the ER, and that an ER-tethered synaptopodin assembles into liquid condensates. We also identified protein neighbors of synaptopodin in spines by in vivo proximity biotinylation. We validated a small subset of such proteins and showed that they co-assemble with synaptopodin in living cells. One of them is Pdlim7, an actin binding protein not previously identified in spines, and we show its precise colocalization with synaptopodin. We suggest that the matrix of the spine apparatus has the property of a liquid protein condensate generated by a multiplicity of low affinity interactions.
Graphical abstract
... Although Rac3 knockout hippocampal neurons do not display any obvious alterations, the double Rac1-Rac3 knockout results in a much more severe phenotype, especially characterized by a significant deficiency in the formation of dendritic spines as compared to the single Rac1 knockout [75,78]. In line with this observation, the specificity of Rac3 function of synaptic plasticity is highlighted by the effect of Rac3 re-expression in Rac1-Rac3 double knockout, which results in a significantly more relevant increase in spine size as compared to Rac1 re-expression [79,80]. Eventually, Rac3 was found to specifically interact with β-1 spectrin, a regulator of actin cytoskeleton organization, further substantiating the distinctive role of this protein in comparison to other Rac subfamily members in the modulation of the morphology and the functional dynamic aspects of dendritic spines [81]. ...
Rho family guanosine triphosphatases (GTPases) regulate cellular signaling and cytoskeletal dynamics, playing a pivotal role in cell adhesion, migration, and cell cycle progression. The Rac subfamily of Rho GTPases consists of three highly homologous proteins, Rac 1–3. The proper function of Rac1 and Rac3, and their correct interaction with guanine nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs) are crucial for neural development. Pathogenic variants affecting these delicate biological processes are implicated in different medical conditions in humans, primarily neurodevelopmental disorders (NDDs). In addition to a direct deleterious effect produced by genetic variants in the RAC genes, a dysregulated GTPase activity resulting from an abnormal function of GEFs and GAPs has been involved in the pathogenesis of distinctive emerging conditions. In this study, we reviewed the current pertinent literature on Rac-related disorders with a primary neurological involvement, providing an overview of the current knowledge on the pathophysiological mechanisms involved in the neuro-RACopathies.
... However newly formed spines (thin and elongated) acquire post synaptic density (PSD) leading to enlargement of the head to become classical mushroom-shaped spines. Increase in the spine volume is associated with the accumulation of additional α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and reorganization of actin cytoskeleton [27]. Cytoskeleton determines the shape of the spines, and actin is the major cytoskeletal protein in dendritic spines which polymerizes to filamentous actin (F-actin) through complex interactions with actin binding proteins (ABPs). ...
Dendritic spines are small, thin, hair-like protrusions found on the dendritic processes of neurons. They serve as independent compartments providing large amplitudes of Ca2+ signals to achieve synaptic plasticity, provide sites for newer synapses, facilitate learning and memory. One of the common and severe complication of neurodegenerative disease is cognitive impairment, which is said to be closely associated with spine pathologies viz., decreased in spine density, spine length, spine volume, spine size etc. Many treatments targeting neurological diseases have shown to improve the spine structure and distribution. However, concise data on the various modulators of dendritic spines are imperative and a need of the hour. Hence, in this review we made an attempt to consolidate the effects of various pharmacological (cholinergic, glutamatergic, GABAergic, serotonergic, adrenergic, and dopaminergic agents) and non-pharmacological modulators (dietary interventions, enriched environment, yoga and meditation) on dendritic spines structure and functions. These data suggest that both the pharmacological and non-pharmacological modulators produced significant improvement in dendritic spine structure and functions and in turn reversing the pathologies underlying neurodegeneration. Intriguingly, the non-pharmacological approaches have shown to improve intellectual performances both in preclinical and clinical platforms, but still more technology-based evidence needs to be studied. Thus, we conclude that a combination of pharmacological and non-pharmacological intervention may restore cognitive performance synergistically via improving dendritic spine number and functions in various neurological disorders.
... Actin structures regulate stabilization of dendritic spines critical to spine establishment and maturation, spine plasticity, and synapse functions (Svitkina, 2018;Kanjhan et al., 2016) [ Fig. 1]. The actin enriched dendritic spines comprise both dynamic and stable actin structures shown using the photoactivatable form of green fluorescent protein (GFP) fused to actin (Honkura et al., 2008;Halpain, 2000). When the photoactivatable form of GFP-actin is activated in dendritic spines, fluorescence decays from the activated molecules are noticed in two phases with time constants. ...
Neural networks with precise connection are compulsory for learning and memory. Various cellular events occur during the genesis of dendritic spines to their maturation, synapse formation, stabilization of the synapse, and proper signal transmission. The cortical actin cytoskeleton and its multiple regulatory proteins are playing crucial for the above cellular events. The different types of ionotropic glutamate receptors (iGluRs) present on the postsynaptic density (PSD) are also essential for learning and memory. Interaction of the iGluRs in association of their auxiliary proteins with actin cytoskeleton regulated by actin-binding proteins (ABPs) are required for precise long-term potentiation (LTP) and long-term depression (LTD). There has been a quest to understand the mechanistic detail of synapse function involving these receptors with dynamic actin cytoskeleton. A major, emerging area of investigation is the relationship between ABPs and iGluRs in synapse development. In this review we have summarized the current understanding of iGluRs functioning with respect to the actin cytoskeleton, scaffolding proteins, and their regulators. The AMPA, NMDA, Delta and Kainate receptors needs the stable underlying actin cytoskeleton to anchor through synaptic proteins for precise synapse formation. The different types of ABPs present in neurons play a critical role in dynamizing/stabilizing the actin cytoskeleton needed for iGluRs function.
... In addition, they can filter the electrical component of synaptic signals and amplify spine head depolarization [20][21][22] (but see [23][24][25]). Both spine heads and spine necks are remodeled depending on neuronal activity [9,26,27] and in pathology [28,29]. While the relationship between spine morphology and function is widely acknowledged, and although dendritic spines are known to participate in different neural circuits depending on their location in the dendritic tree [30], the extent of synaptic ultrastructural diversity along individual identified dendrites has not been quantified, and the consequences of this variability on signal compartmentalization and dendritic integration remain to be investigated. ...
Pyramidal neurons (PNs) are covered by thousands of dendritic spines receiving excitatory synaptic inputs. The ultrastructure of dendritic spines shapes signal compartmentalization, but ultrastructural diversity is rarely taken into account in computational models of synaptic integration. Here, we developed a 3D correlative light–electron microscopy (3D-CLEM) approach allowing the analysis of specific populations of synapses in genetically defined neuronal types in intact brain circuits. We used it to reconstruct segments of basal dendrites of layer 2/3 PNs of adult mouse somatosensory cortex and quantify spine ultrastructural diversity. We found that 10% of spines were dually innervated and 38% of inhibitory synapses localized to spines. Using our morphometric data to constrain a model of synaptic signal compartmentalization, we assessed the impact of spinous versus dendritic shaft inhibition. Our results indicate that spinous inhibition is locally more efficient than shaft inhibition and that it can decouple voltage and calcium signaling, potentially impacting synaptic plasticity.
The problem of frequency coding is closely related to the studies of inhibitory transmission as a factor of neural network plasticity. The rewiew presents basic mechanisms of inhibitory control of spatio-temporal pattern of neural activity during signal processing. Current views are analyzed in respect of dynamic synapses, their instability and variation within the ongoing activity. The results presented here demonstrate that short-term plasticity operates with the combined contribution of excitatory and inhibitory synapses. The role of GABAergic potentials in modulation of intracellular messenger’s activity is discussed, including those implicated in postsynaptic modifications of excitatory and inhibitory transmission. The main topics concerning the molecular mechanisms centered on the lateral diffusion of GABAA receptors. The data of many reports argue for coordinating role of actin cytoskeleton. It is proposed that postsynaptic mechanisms underlying GABAA plasticity may be activated in result of fast adaptation of actin cytoskeleton and associated proteins to disbalance between excitation and inhibition.
Synaptic plasticity is important for learning and memory formation; it describes the strengthening or weakening of connections between synapses. The postsynaptic part of excitatory synapses resides in dendritic spines, which are small protrusions on the dendrites. One of the key features of synaptic plasticity is its correlation with the size of these spines. A long-lasting synaptic strength increase [long-term potentiation (LTP)] is only possible through the reconfiguration of the actin spine cytoskeleton. Here, we develop an experimentally informed three-dimensional computational model in a moving boundary framework to investigate this reconfiguration. Our model describes the reactions between actin and actin-binding proteins leading to the cytoskeleton remodeling and their effect on the spine membrane shape to examine the spine enlargement upon LTP. Moreover, we find that the incorporation of perisynaptic elements enhances spine enlargement upon LTP, exhibiting the importance of accounting for these elements when studying structural LTP. Our model shows adaptation to repeated stimuli resulting from the interactions between spine proteins and mechanical forces.
JOURNAL/nrgr/04.03/01300535-990000000-00151/inline-graphic1/v/2023-12-22T072556Z/r/image-tiff
Morphological alterations in dendritic spines have been linked to changes in functional communication between neurons that affect learning and memory. Kinesin-4 KIF21A helps organize the microtubule-actin network at the cell cortex by interacting with KANK1; however, whether KIF21A modulates dendritic structure and function in neurons remains unknown. In this study, we found that KIF21A was distributed in a subset of dendritic spines, and that these KIF21A-positive spines were larger and more structurally plastic than KIF21A-negative spines. Furthermore, the interaction between KIF21A and KANK1 was found to be critical for dendritic spine morphogenesis and synaptic plasticity. Knockdown of either KIF21A or KANK1 inhibited dendritic spine morphogenesis and dendritic branching, and these deficits were fully rescued by coexpressing full-length KIF21A or KANK1, but not by proteins with mutations disrupting direct binding between KIF21A and KANK1 or binding between KANK1 and talin1. Knocking down KIF21A in the hippocampus of rats inhibited the amplitudes of long-term potentiation induced by high-frequency stimulation and negatively impacted the animals’ cognitive abilities. Taken together, our findings demonstrate the function of KIF21A in modulating spine morphology and provide insight into its role in synaptic function.
Many experiments suggest that long-term information associated with neuronal memory resides collectively in dendritic spines. However, spines can have a limited size due to metabolic and neuroanatomical constraints, which should effectively limit the amount of encoded information in excitatory synapses. This study investigates how much information can be stored in the population of sizes of dendritic spines, and whether it is optimal in any sense. It is shown here, using empirical data for several mammalian brains across different regions and physiological conditions, that dendritic spines nearly maximize entropy contained in their volumes and surface areas for a given mean size in cortical and hippocampal regions. Although both short- and heavy-tailed fitting distributions approach $$90-100\%$$ 90 - 100 % of maximal entropy in the majority of cases, the best maximization is obtained primarily for short-tailed gamma distribution. We find that most empirical ratios of standard deviation to mean for spine volumes and areas are in the range $$1.0\pm 0.3$$ 1.0 ± 0.3 , which is close to the theoretical optimal ratios coming from entropy maximization for gamma and lognormal distributions. On average, the highest entropy is contained in spine length ( $$4-5$$ 4 - 5 bits per spine), and the lowest in spine volume and area ( $$2-3$$ 2 - 3 bits), although the latter two are closer to optimality. In contrast, we find that entropy density (entropy per spine size) is always suboptimal. Our results suggest that spine sizes are almost as random as possible given the constraint on their size, and moreover the general principle of entropy maximization is applicable and potentially useful to information and memory storing in the population of cortical and hippocampal excitatory synapses, and to predicting their morphological properties.
Myristoylated, alanine-rich C-kinase substrate (MARCKS) is an F-actin and phospholipid binding protein implicated in numerous cellular activities, including the regulation of morphology in neuronal dendrites and dendritic spines. MARCKS contains a lysine-rich effector domain that mediates its binding to plasma membrane phosphatidylinositol-4,5-biphosphate (PI(4,5)P 2 ) in a manner controlled by PKC and calcium/calmodulin. In neurons, manipulations of MARCKS concentration and membrane targeting strongly affect the numbers, shapes, and F-actin properties of dendritic spines, but the mechanisms remain unclear. Here, we tested the hypothesis that the effects of MARCKS on dendritic spine morphology are due to its capacity to regulate the availability of plasma membrane PI(4,5)P 2 . We observed that the concentration of free PI(4,5)P 2 on the dendritic plasma membrane was inversely proportional to the concentration of MARCKS. Endogenous PI(4,5)P 2 levels were increased or decreased, respectively, by acutely overexpressing either phosphatidylinositol-4-phosphate 5-kinase (PIP5K) or inositol polyphosphate 5-phosphatase (5ptase). PIP5K, like MARCKS depletion, induced severe spine shrinkage; 5ptase, like constitutively membrane-bound MARCKS, induced aberrant spine elongation. These phenotypes involved changes in actin properties driven by the F-actin severing protein cofilin. Collectively, these findings support a model in which neuronal activity regulates actin-dependent spine morphology through antagonistic interactions of MARCKS and PI(4,5)P 2 .
Synaptic overproduction and elimination is a regular developmental event in the mammalian brain. In the cerebral cortex, synaptic overproduction is almost exclusively correlated with glutamatergic synapses located on dendritic spines. Therefore, analysis of changes in spine density on different parts of the dendritic tree in identified classes of principal neurons could provide insight into developmental reorganization of specific microcircuits.
The activity-dependent stabilization and selective elimination of the initially overproduced synapses is a major mechanism for generating diversity of neural connections beyond their genetic determination. The largest number of overproduced synapses was found in the monkey and human cerebral cortex. The highest (exceeding adult values by two- to threefold) and most protracted overproduction (up to third decade of life) was described for associative layer IIIC pyramidal neurons in the human dorsolateral prefrontal cortex.
Therefore, the highest proportion and extraordinarily extended phase of synaptic spine overproduction is a hallmark of neural circuitry in human higher-order associative areas. This indicates that microcircuits processing the most complex human cognitive functions have the highest level of developmental plasticity. This finding is the backbone for understanding the effect of environmental impact on the development of the most complex, human-specific cognitive and emotional capacities, and on the late onset of human-specific neuropsychiatric disorders, such as autism and schizophrenia.
Recent research extends our understanding of brain processes beyond just action potentials and chemical transmissions within neural circuits, emphasizing the mechanical forces generated by excitatory synapses on dendritic spines to modulate presynaptic function. From in vivo and in vitro studies, we outline five central principles of synaptic mechanics in brain function: P1: Stability – Underpinning the integral relationship between the structure and function of the spine synapses. P2: Extrinsic dynamics – Highlighting synapse-selective structural plasticity which plays a crucial role in Hebbian associative learning, distinct from pathway-selective long-term potentiation (LTP) and depression (LTD). P3: Neuromodulation – Analyzing the role of G-protein-coupled receptors, particularly dopamine receptors, in time-sensitive modulation of associative learning frameworks such as Pavlovian classical conditioning and Thorndike’s reinforcement learning (RL). P4: Instability – Addressing the intrinsic dynamics crucial to memory management during continual learning, spotlighting their role in “spine dysgenesis” associated with mental disorders. P5: Mechanics – Exploring how synaptic mechanics influence both sides of synapses to establish structural traces of short- and long-term memory, thereby aiding the integration of mental functions. We also delve into the historical background and foresee impending challenges.
Accumulating evidence from a wide range of studies, including behavioral, cellular, molecular and computational findings, support a key role of dendrites in the encoding and recall of new memories. Dendrites can integrate synaptic inputs in non-linear ways, provide the substrate for local protein synthesis and facilitate the orchestration of signaling pathways that regulate local synaptic plasticity. These capabilities allow them to act as a second layer of computation within the neuron and serve as the fundamental unit of plasticity. As such, dendrites are integral parts of the memory engram, namely the physical representation of memories in the brain and are increasingly studied during learning tasks. Here, we review experimental and computational studies that support a novel, dendritic view of the memory engram that is centered on non-linear dendritic branches as elementary memory units. We highlight the potential implications of dendritic engrams for the learning and memory field and discuss future research directions.
Alzheimer's disease (AD) is a neurodegenerative disease that manifests its pathology through synaptic damage, mitochondrial abnormalities, microRNA deregulation, hormonal imbalance, increased astrocytes & microglia, accumulation of amyloid β (Aβ) and phosphorylated Tau in the brains of AD patients. Despite extensive research, the effective treatment of AD is still unknown. Tau hyperphosphorylation and mitochondrial abnormalities are involved in the loss of synapses, defective axonal transport and cognitive decline in patients with AD. Mitochondrial dysfunction is evidenced by enhanced mitochondrial fragmentation, impaired mitochondrial dynamics, mitochondrial biogenesis and defective mitophagy in AD. Hence, targeting mitochondrial proteins might be a promising therapeutic strategy in treating AD. Recently, dynamin-related protein 1 (Drp1), a mitochondrial fission protein, has gained attention due to its interactions with Aβ and hyperphosphorylated Tau, altering mitochondrial morphology, dynamics, and bioenergetics. These interactions affect ATP production in mitochondria. A reduction in Drp1 GTPase activity protects against neurodegeneration in AD models. This article provides a comprehensive overview of Drp1's involvement in oxidative damage, apoptosis, mitophagy, and axonal transport of mitochondria. We also highlighted the interaction of Drp1 with Aβ and Tau, which may contribute to AD progression. In conclusion, targeting Drp1 could be a potential therapeutic approach for preventing AD pathology.
Neuropathic pain is a common, debilitating chronic pain condition caused by damage or a disease affecting the somatosensory nervous system. Understanding the pathophysiological mechanisms underlying neuropathic pain is critical for developing new therapeutic strategies to treat chronic pain effectively. Tiam1 is a Rac1 guanine nucleotide exchange factor (GEF) that promotes dendritic and synaptic growth during hippocampal development by inducing actin cytoskeletal remodeling. Here, using multiple neuropathic pain animal models, we show that Tiam1 coordinates synaptic structural and functional plasticity in the spinal dorsal horn via actin cytoskeleton reorganization and synaptic NMDAR stabilization and that these actions are essential for the initiation, transition, and maintenance of neuropathic pain. Furthermore, an antisense oligonucleotides (ASO) targeting spinal Tiam1 persistently alleviate neuropathic pain sensitivity. Our findings suggest that Tiam1-coordinated synaptic functional and structural plasticity underlies the pathophysiology of neuropathic pain and that intervention of Tiam1-mediated maladaptive synaptic plasticity has long-lasting consequences in neuropathic pain management.
Do dendritic spines, which comprise the postsynaptic component of most excitatory synapses, exist only for their structural dynamics, receptor trafficking, and chemical and electrical compartmentation? The answer is no. Simultaneous investigation of both spine and presynaptic terminals has recently revealed a novel feature of spine synapses. Spine enlargement pushes the presynaptic terminals with muscle-like force and augments the evoked glutamate release for up to 20 min. We now summarize the evidence that such mechanical transmission shares critical features in common with short-term potentiation (STP) and may represent the cellular basis of short-term and working memory. Thus, spine synapses produce the force of learning to leave structural traces for both short and long-term memories.
Background: Synaptic plasticity requires constant adaptation of functional and structural features at individual synaptic connections. Rapid re-modulation of the synaptic actin cytoskeleton provides the scaffold orchestrating both morphological and functional modifications. A major regulator of actin polymerization not only in neurons but also in various other cell types is the actin-binding protein profilin. While profilin is known to mediate the ADP to ATP exchange at actin monomers through its direct interaction with G-actin, it additionally is able to influence actin dynamics by binding to membrane-bound phospholipids as phosphatidylinositol (4,5)-bisphosphate (PIP2) as well as several other proteins containing poly-L-proline motifs including actin modulators like Ena/VASP, WAVE/WASP or formins. Notably, these interactions are proposed to be mediated by a fine-tuned regulation of post-translational phosphorylation of profilin. However, while phosphorylation sites of the ubiquitously expressed isoform profilin1 have been described and analyzed previously, there is still only little known about the phosphorylation of the profilin2a isoform predominantly expressed in neurons.
Methods: Here, utilizing a knock-down/knock-in approach, we replaced endogenously expressed profilin2a by (de)phospho-mutants of S137 known to alter actin-, PIP2 and PLP-binding properties of profilin2a and analyzed their effect on general actin dynamics as well as activity-dependent structural plasticity.
Results and Discussion: Our findings suggest that a precisely timed regulation of profilin2a phosphorylation at S137 is needed to mediate actin dynamics and structural plasticity bidirectionally during long-term potentiation and long-term depression, respectively.
Dendritic spines host glutamatergic excitatory synapses and compartmentalize biochemical signalling underlying synaptic plasticity. The narrow spine neck that connects the spine head with its parent dendrite is the crucial structural element of this compartmentalization. Both neck morphology and its molecular composition differentially regulate exchange of molecular signals between the spine and rest of the neuron. Although these spine neck properties themselves show activity-dependent plasticity, it remains unclear what functional role spine neck plasticity plays in synaptic plasticity expression. To address this, we built a data-constrained biophysical computational model of AMPA receptor (AMPAR) trafficking and intracellular signalling involving Ca ²⁺ calmodulin-dependent kinase II (CaMKII) and the phosphatase calcineurin in hippocampal CA1 neurons, which provides new mechanistic insights into spatiotemporal AMPAR dynamics during long-term potentiation (LTP). Using the model, we tested how plasticity of neck morphology and of neck septin7 barrier, which specifically restricts membrane protein diffusion, affect LTP. We found that spine neck properties control LTP by regulating the balance between AMPAR and calcineurin escape from the spine. Neck plasticity that increases spine-dendrite coupling reduces LTP by allowing more AMPA receptors to diffuse away from the synapse. Surprisingly, neck plasticity that decreases spine-dendrite coupling can also reduce LTP by trapping calcineurin, which dephosphorylates AMPARs. Further simulations showed that the precise timescale of neck plasticity, relative to AMPAR and enzyme diffusion and phosphorylation dynamics, critically regulates LTP. These results suggest a new mechanistic and experimentally-testable theory for how spine neck plasticity regulates synaptic plasticity.
Dendritic spines are submicron, subcellular compartments whose shape is defined by actin filaments and associated proteins. Accurately mapping the cytoskeleton is a challenge, given the small size of its components. It remains unclear whether the actin-associated structures analyzed in dendritic spines of neurons in vitro apply to dendritic spines of intact, mature neurons in situ. Here, we combined advanced preparative methods with multitilt serial section electron microscopy (EM) tomography and computational analysis to reveal the full three-dimensional (3D) internal architecture of spines in the intact brains of male mice at nanometer resolution. We compared hippocampal (CA1) pyramidal cells and cerebellar Purkinje cells in terms of the length distribution and connectivity of filaments, their branching-angles and absolute orientations, and the elementary loops formed by the network. Despite differences in shape and size across spines and between spine heads and necks, the internal organization was remarkably similar in both neuron types and largely homogeneous throughout the spine volume. In the tortuous mesh of highly branched and interconnected filaments, branches exhibited no preferred orientation except in the immediate vicinity of the cell membrane. We found that new filaments preferentially split off from the convex side of a bending filament, consistent with the behavior of Arp2/3-mediated branching of actin under mechanical deformation. Based on the quantitative analysis, the spine cytoskeleton is likely subject to considerable mechanical force in situ.
Effective treatments that prevent or reduce drug relapse vulnerability should be developed to relieve the high burden of drug addiction on society. This will only be possible by enhancing the understanding of the molecular mechanisms underlying the neurobiology of addiction. Recent experimental data have shown that dendritic spines, small protrusions from the dendrites that receive excitatory input, of spiny neurons in the nucleus accumbens exhibit morphological changes during drug exposure and withdrawal. Moreover, these changes relate to the characteristic drug-seeking behavior of addiction. However, due to the complexity of the dendritic spines, we do not yet fully understand the processes underlying their structural changes in response to different inputs. We propose that biophysical models can enhance the current understanding of these processes by incorporating different, and sometimes, discrepant experimental data to identify the shared underlying mechanisms and generate experimentally testable hypotheses. This review aims to give an up-to-date report on biophysical models of dendritic spines, focusing on those models that describe their shape changes, which are well-known to relate to learning and memory. Moreover, it examines how these models can enhance our understanding of the effect of the drugs and the synaptic changes during withdrawal, as well as during neurodegenerative disease progression such as Alzheimer’s disease.
In dendrites and synapses in the neuronal circuit, temporal and spatial trains of Ca²⁺ transients are triggered as a consequence of 4-dimenstional patterns of synaptic transmission, local dendritic spikes and action potential firing. Among downstream Ca²⁺ effectors, Ca²⁺/calmodulin-dependent kinase II (CaMKII) and Ca²⁺/calmodulin-dependent phosphatase calcineurin (CaN) interactively and competitively regulate essential neuronal functions, such as bidirectional synaptic plasticity, gene expression, learning and memory. New developments in optical imaging and local optical manipulation revealed distinctive spatiotemporal features of bidirectional dendritic spine structural plasticity that are co-regulated by CaMKII and CaN. We created a novel set of genetically-encoded fluorescent probes to specifically investigate key activation processes of CaMKII and CaN in living neurons. Multiplex FRET imaging approaches revealed distinct spatiotemporal properties of CaMKII and CaN co-activation in dendrites and synapses that likely underlie the biochemical machinery to decode information represented in the patterned neuronal input to decide properties of spine structural plasticity. We also created new orthogonal color variants of linearly performing Ca²⁺ indicators XCaMPs that can be easily multiplexed with a number of other fluorescent probes. These advances facilitate future investigation on how biochemical decoding is achieved in neurons in the living brain, and will shed new light on complex brain information dynamics at the crossroad of neurochemistry, pathophysiology and neuro-inspired engineering.
Effective treatments that prevent or reduce drug relapse vulnerability should be developed to relieve the high burden of drug addiction to society. This will only be possible by enhancing the understanding of the molecular mechanisms underlying the neurobiology of addiction. Recent experimental data have shown that dendritic spines, small protrusions from the dendrites that receive input from excitatory neurons, from spiny neurons in the nucleus accumbens exhibit morphological changes during drug exposure and withdrawal. Moreover, these changes relate to the characteristic drug-seeking behavior of addiction. However, due to the complexity of the dendritic spines, we do not yet fully understand the processes underlying their structural changes in response to different inputs. We propose that biophysical models can enhance the current understanding of these processes by incorporating different, and sometimes, discrepant experimental data to identify the shared underlying mechanisms and generate experimentally testable hypotheses. This review aims to give an up-to-date report on biophysical models of dendritic spines, focusing on those models that describe their shape changes, which are well-known to relate to learning and memory. Moreover, it examines how these models can enhance our understanding of the effect of the drugs and the synaptic changes during disease progression.
Synaptic transmission involves cell-to-cell communication at the synaptic junction between two neurons, and chemical and electrical forms of this process have been extensively studied. In the brain, excitatory glutamatergic synapses are often made on dendritic spines that enlarge during learning1,2,3,4,5. As dendritic spines and the presynaptic terminals are tightly connected with the synaptic cleft6, the enlargement may have mechanical effects on presynaptic functions7. Here we show that fine and transient pushing of the presynaptic boutons with a glass pipette markedly promotes both the evoked release of glutamate and the assembly of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins8,9,10,11,12—as measured by Förster resonance transfer (FRET) and fluorescence lifetime imaging—in rat slice culture preparations13. Both of these effects persisted for more than 20 minutes. The increased presynaptic FRET was independent of cytosolic calcium (Ca2+), but dependent on the assembly of SNARE proteins and actin polymerization in the boutons. Notably, a low hypertonic solution of sucrose (20 mM) had facilitatory effects on both the FRET and the evoked release without inducing spontaneous release, in striking contrast with a high hypertonic sucrose solution (300 mM), which induced exocytosis by itself14. Finally, spine enlargement induced by two-photon glutamate uncaging enhanced the evoked release and the FRET only when the spines pushed the boutons by their elongation. Thus, we have identified a mechanosensory and transduction mechanism15 in the presynaptic boutons, in which the evoked release of glutamate is enhanced for more than 20 min.
Recent studies have shown high levels of calcium in activated dendritic spines, where the smooth endoplasmic reticulum (SER) is likely to be important for regulating calcium. Here, the dimensions and organization of the SER in hippocampal spines and dendrites were measured through serial electron microscopy and three-dimensional analysis. SER of some form was found in 58% of the immature spines and in 48% of the adult spines. Less than 50% of the small spines at either age contained SER, suggesting that other mechanisms, such as cytoplasmic buffers, regulate ion fluxes within their small volumes. In contrast, >80% of the large mushroom spines of the adult had a spine apparatus, an organelle containing stacks of SER and dense-staining plates. Reconstructed SER occupied 0.001–0.022 μm ³ , which was only 2–3.5% of the total spine volume; however, the convoluted SER membranes had surface areas of 0.12–2.19 μm ² , which were 12 to 40% of the spine surface area. Coated vesicles and multivesicular bodies occurred in some spines, suggesting local endocytotic activity. Smooth vesicles and tubules of SER were found in continuity with the spine plasma membrane and margins of the postsynaptic density (PSD), respectively, suggesting a role for the SER in the addition and recycling of spine membranes and synapses. The amount of SER in the parent dendrites was proportional to the number of spines and synapses originating along their lengths. These measurements support the hypothesis that the SER regulates the ionic and structural milieu of some, but not all, hippocampal dendritic spines.
We used actin-perturbing agents and detergent extraction of primary hippocampal cultures to test directly the role of the actin cytoskeleton in localizing GABAA receptors, AMPA- and NMDA-type glutamate receptors, and potential anchoring proteins at postsynaptic sites. Excitatory postsynaptic sites on dendritic spines contained a high concentration of F-actin that was resistant to cytochalasin D but could be depolymerized using the novel compound latrunculin A. Depolymerization of F-actin led to a 40% decrease in both the number of synaptic NMDA receptor (NMDAR1) clusters and the number of AMPA receptor (GluR1)-labeled spines. The nonsynaptic NMDA receptors appeared to remain clustered and to coalesce in cell bodies. alpha-Actinin-2, which binds both actin and NMDA receptors, dissociated from the receptor clusters, but PSD-95 remained associated with both the synaptic and nonsynaptic receptor clusters, consistent with a proposed cross-linking function. AMPA receptors behaved differently; on GABAergic neurons, the clusters redistributed to nonsynaptic sites, whereas on pyramidal neurons, many of the clusters appeared to disperse. Furthermore, in control neurons, AMPA receptors were detergent extractable from pyramidal cell spines, whereas AMPA receptors on GABAergic neurons and NMDA receptors were unextractable. GABAA receptors were not dependent on F-actin for the maintenance or synaptic localization of clusters. These results indicate fundamental differences in the mechanisms of receptor anchoring at postsynaptic sites, both regarding the anchoring of a single receptor (the AMPA receptor) in pyramidal cells versus GABAergic interneurons and regarding the anchoring of different receptors (AMPA vs NMDA receptors) at a single class of postsynaptic sites on pyramidal cell dendritic spines.
Serial electron microscopy and 3-D reconstructions of dendritic spines from hippocampal area CA 1 dendrites were obtained to evaluate 2 questions about relationships between spine geometry and synaptic efficacy. First, under what biophysical conditions are the spine necks likely to reduce the magnitude of charge transferred from the synapses on the spine heads to the recipient dendrite? Simulation software provided by Charles Wilson (1984) was used to determine that if synaptic conductance is 1 nS or less, only 1% of the hippocampal spine necks are sufficiently thin and long to reduce charge transfer by more than 10%. If synaptic conductance approaches 5 nS, however, 33% of the hippocampal spine necks are sufficiently thin and long to reduce charge transfer by more than 10%. Second, is spine geometry associated with other anatomical indicators of synaptic efficacy, including the area of the postsynaptic density and the number of vesicles in the presynaptic axon? Reconstructed spines were graphically edited into head and neck compartments, and their dimensions were measured, the areas of the postsynaptic densities (PSD) were measured, and all of the vesicles in the presynaptic axonal varicosities were counted. The dimensions of the spine head were well correlated with the area of PSD and the number of vesicles in the presynaptic axonal varicosity. Spine neck diameter and length were not correlated with PSD area, head volume, or the number of vesicles. These results suggest that the dimensions of the spine head, but not the spine neck, reflect differences in synaptic efficacy. We suggest that the constricted necks of hippocampal dendritic spines might reduce diffusion of activated molecules to neighboring synapses, thereby attributing specificity to activated or potentiated synapses.
Antibodies against actin were used to corroborate the presence of actin as a major component protein of isolated brain postsynaptic densities. The same antibodies also were used as an immunohistochemical stain to study the distribution of actin in sections of intact brain tissue. This showed two major sites where actin is concentrated: smooth muscle cells around blood vessels and postsynaptic sites. In the postsynaptic area the highest concentration of actin occurs in postsynaptic densities and there also is intense staining in the surrounding cytoplasm, especially within dendritic spines. Antiactin staining was much weaker in other parts of neurons and in glial cells. The high concentration of actin in dendritic spines may be related to shape changes that these structures have been found to undergo in response to prolonged afferent stimulation.
Neurons contain distinct compartments including dendrites, dendritic spines, axons and synaptic terminals. The molecular mechanisms that generate and distinguish these compartments, although largely unknown, may involve the small GTPases Rac and Cdc42, which appear to regulate actin polymerization. Having shown that perturbations of Rac1 activity block the growth of axons but not dendrites of Drosophila neurons, we investigated whether this also applies to mammals by examining transgenic mice expressing constitutively active human Rac1 in Purkinje cells. We found that these mice were ataxic and had a reduction of Purkinje-cell axon terminals in the deep cerebellar nuclei, whereas the dendritic trees grew to normal height and branched extensively. Unexpectedly, the dendritic spines of Purkinje cells in developing and mature cerebella were much reduced in size but increased in number. These 'mini' spines often form supernumerary synapses. These differential effects of perturbing Rac1 activity indicate that there may be distinct mechanisms for the elaboration of axons, dendrites and dendritic spines.
To determine the role of dendritic filopodia in the genesis of excitatory synaptic contacts and dendritic spines in hippocampal area CA1, serial section electron microscopy and three-dimensional analysis of 16 volumes of neuropil from nine male rat pups, aged postnatal day 1 (P1) through P12, were performed. The analysis revealed that numerous dendritic filopodia formed asymmetric synaptic contacts with axons and with filopodia extending from axons, especially during the first postnatal week. At P1, 22 +/- 5.5% of synapses occurred on dendritic filopodia, with 19 +/- 5.9% on filopodia at P4, 20 +/- 8.0% at P6, decreasing to 7.2 +/- 4.7% at P12 (p < 0.02). Synapses were found at the base and along the entire length of filopodia, with many filopodia exhibiting multiple synaptic contacts. In all, 162 completely traceable dendritic filopodia received 255 asymmetric synaptic contacts. These synapses were found at all parts of filopodia with equal frequency, usually occurring on fusiform swellings of the diameter. Most synaptic contacts (53 +/- 11%) occurred directly on dendritic shafts during the first postnatal week. A smaller but still substantial portion (32 +/- 12%) of synapses were on shafts at P12 (p < 0.036). There was a highly significant (p < 0.0002) increase in the proportion of dendritic spine synapses with age, rising from just 4.9 +/- 4.3% at P1 to 37 +/- 14% at P12. The concurrence of primarily shaft and filopodial synapses in the first postnatal week suggests that filopodia recruit shaft synapses that later give rise to spines through a process of outgrowth.
The role of actin filaments in synaptic function has been studied in the CA1 region of the rat hippocampal slice. Bath application (2 hr) of the actin polymerization inhibitor latrunculin B did not substantially affect the shape of dendrites or spines. However, this and other drugs that affect actin did affect synaptic function. Bath-applied latrunculin B reduced the synaptic response. Several lines of evidence indicate that a component of this effect is presynaptic. To specifically test for a postsynaptic role for actin, latrunculin B or phalloidin, an actin filament stabilizer, was perfused into the postsynaptic neuron. The magnitude of long-term potentiation (LTP) was decreased at times when baseline transmission was not yet affected. Longer applications produced a decrease in baseline AMPA receptor (AMPAR)-mediated transmission. The magnitude of the NMDA receptor-mediated transmission was unaffected, indicating a specific effect on the AMPAR. These results suggest that postsynaptic actin filaments are involved in a dynamic process required to maintain AMPAR-mediated transmission and to enhance it during LTP.
Postembedding immunogold labeling was used to determine the relationship between AMPA and NMDA receptor density and size of Schaffer collateral-commissural (SCC) synapses of the adult rat. All SCC synapses expressed NMDA receptors. AMPA and NMDA receptors were colocalized in at least 75% of SCC synapses; the ratio of AMPA to NMDA receptors was a linear function of postsynaptic density (PSD) diameter, with AMPA receptor number dropping to zero at a PSD diameter of approximately 180 nm. These findings indicate that 'silent' SCC synapses are smaller than the majority of SCC synapses at which AMPA and NMDA receptors are colocalized. Thus synapse size may determine important properties of SCC synapses.
The hypothesis that dynamic actin filaments participate in specific aspects of synaptic plasticity was investigated at the Schaffer-collateral-CA1 pyramidal cell synapse of mouse hippocampus. Low concentrations (0.01-1 microM) of compounds that inhibit actin filament assembly were bath applied to hippocampal slices during extracellular recording of field excitatory postsynaptic potentials. Cytochalasin D, cytochalasin B, and latrunculin A all impaired the maintenance of LTP induced by brief high-frequency stimulation. This effect on LTP maintenance was specific, because none of the compounds affected basal synaptic transmission, paired-pulse facilitation, LTP induction, or post-tetanic potentiation. The effect of cytochalasin B was reversible. The results are consistent with a model in which dynamic actin filaments play an essential role in the molecular mechanisms underlying the early maintenance phase of LTP, such as growth of new synaptic connections or conversion of silent synapses.
During cerebral ischemia, neurons undergo rapid alterations in dendritic structure consisting of focal swelling and spine loss. We used time-lapse microscopy to determine the fate of dendritic spines that disappeared after brief, sublethal hypoxic or excitotoxic exposures. Dendrite and spine morphology were assessed in cultured cortical neurons expressing yellow fluorescent protein or labeled with the fluorescent membrane tracer, DiI. Neurons exposed to NMDA, kainate, or oxygen-glucose deprivation underwent segmental dendritic beading and loss of approximately one-half of dendritic spines. Most spine loss was observed in regions of local dendritic swelling. Despite widespread loss, spines recovered within 2 hr after termination of agonist exposure or oxygen-glucose deprivation and remained stable over the subsequent 24 hr. Recovery was slower after NMDA than AMPA/kainate receptor activation. Time-lapse fluorescence imaging showed that the vast majority of spines reemerged in the same location from which they disappeared. In addition to spine recovery, elaboration of dendritic filopodia was observed in new locations along the dendritic shaft after dendrite recovery. Spine recovery did not depend on actin polymerization because it was not blocked by application of latrunculin-A, which eliminated filamentous actin staining in spines and blocked spine motility. Throughout spine loss and recovery, presynaptic and postsynaptic elements remained in physical proximity. These results suggest that elimination of dendritic spines is not necessarily associated with loss of synaptic contacts. Rapid reestablishment of dendritic spine synapses in surviving neurons may be a substrate for functional recovery after transient cerebral ischemia.
Dendritic spines serve as preferential sites of excitatory synaptic connections and are pleomorphic. To address the structure-function relationship of the dendritic spines, we used two-photon uncaging of glutamate to allow mapping of functional glutamate receptors at the level of the single synapse. Our analyses of the spines of CA1 pyramidal neurons reveal that AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)-type glutamate receptors are abundant (up to 150/spine) in mushroom spines but sparsely distributed in thin spines and filopodia. The latter may be serving as the structural substrates of the silent synapses that have been proposed to play roles in development and plasticity of synaptic transmission. Our data indicate that distribution of functional AMPA receptors is tightly correlated with spine geometry and that receptor activity is independently regulated at the level of single spines.
All coelenterate fluorescent proteins cloned to date display some form of quaternary structure, including the weak tendency of Aequorea green fluorescent protein (GFP) to dimerize, the obligate dimerization of Renilla GFP, and the obligate tetramerization of the red fluorescent protein from Discosoma (DsRed). Although the weak dimerization of Aequorea GFP has not impeded its acceptance as an indispensable tool of cell biology, the obligate tetramerization of DsRed has greatly hindered its use as a genetically encoded fusion tag. We present here the stepwise evolution of DsRed to a dimer and then either to a genetic fusion of two copies of the protein, i.e., a tandem dimer, or to a true monomer designated mRFP1 (monomeric red fluorescent protein). Each subunit interface was disrupted by insertion of arginines, which initially crippled the resulting protein, but red fluorescence could be rescued by random and directed mutagenesis totaling 17 substitutions in the dimer and 33 in mRFP1. Fusions of the gap junction protein connexin43 to mRFP1 formed fully functional junctions, whereas analogous fusions to the tetramer and dimer failed. Although mRFP1 has somewhat lower extinction coefficient, quantum yield, and photostability than DsRed, mRFP1 matures >10 times faster, so that it shows similar brightness in living cells. In addition, the excitation and emission peaks of mRFP1, 584 and 607 nm, are approximately 25 nm red-shifted from DsRed, which should confer greater tissue penetration and spectral separation from autofluorescence and other fluorescent proteins.
Cytoskeleton is believed to contribute to activity-dependent processes underlying neuronal plasticity, such as regulations of cellular morphology and localization of signaling proteins. However, how neuronal activity controls actin cytoskeleton remains obscure. Taking advantage of confocal imaging of enhanced GFP-actin in the primary culture of hippocampal neurons, we show that synaptic activity induces multiple types of actin reorganization, both at the spines and at the somatic periphery. Activation of N-methyl-d-aspartate receptors, accompanied with a local rise in [Ca(2+)]i, was sufficient to trigger a slow and sustained recruitment of actin into dendritic spines. In contrast, opening of voltage-gated Ca(2+) channels rapidly and reversibly enhanced cortical actin at the somatic periphery but not in the spines, in keeping with a high transient rise in somatic [Ca(2+)]i. These data suggest that spatiotemporal dynamics of [Ca(2+)]i, triggered by activation of N-methyl-d-aspartate receptors and voltage-gated Ca(2+) channels, provides the molecular basis for activity-dependent actin remodeling.
Mechanisms of synaptic plasticity in CNS circuits are commonly investigated using in vitro preparations such as brain slices or slice culture. During their preparation, slices are exposed to low temperatures, and electrophysiological measurements are sometimes made below physiological temperature. Because dendritic spines, which occur at the majority of excitatory synapses, are morphologically plastic, we investigated the influence of reduced temperature on their morphology and plasticity using live cell imaging of hippocampal slices from transgenic mice expressing a green fluorescent protein-based neuronal surface marker and electron microscopy of adult brain slices. Our data show that dendritic spines are highly sensitive to reduced temperature with rapid loss of actin-based motility followed at longer times by reversible loss of the entire spine structure. Thus, reduced temperature significantly affects synaptic morphology, which is in turn known to influence several key aspects of synaptic transmission. Evidence that hypothermia potentiates anesthesia and is associated with spine loss in hibernating animals further suggests that spine morphology may have a widespread influence on brain function.
The synapse is a highly organized cellular specialization whose structure and composition are reorganized, both positively and negatively, depending on the strength of input signals. The mechanisms orchestrating these changes are not well understood. A plausible locus for the reorganization of synapse components and structure is actin, because it serves as both cytoskeleton and scaffold for synapses and exists in a dynamic equilibrium between F-actin and G-actin that is modulated bidirectionally by cellular signaling. Using a new FRET-based imaging technique to monitor F-actin/G-actin equilibrium, we show here that tetanic stimulation causes a rapid, persistent shift of actin equilibrium toward F-actin in the dendritic spines of rat hippocampal neurons. This enlarges the spines and increases postsynaptic binding capacity. In contrast, prolonged low-frequency stimulation shifts the equilibrium toward G-actin, resulting in a loss of postsynaptic actin and of structure. This bidirectional regulation of actin is actively involved in protein assembly and disassembly and provides a substrate for bidirectional synaptic plasticity.
Calcium/calmodulin-dependent protein kinase II (CaMKII) is a leading candidate for a synaptic memory molecule because it is persistently activated after long-term potentiation (LTP) induction and because mutations that block this persistent activity prevent LTP and learning. Previous work showed that synaptic stimulation causes a rapidly reversible translocation of CaMKII to the synaptic region. We have now measured green fluorescent protein (GFP)-CaMKIIalpha translocation into synaptic spines during NMDA receptor-dependent chemical LTP (cLTP) and find that under these conditions, translocation is persistent. Using red fluorescent protein as a cell morphology marker, we found that there are two components of the persistent accumulation. cLTP produces a persistent increase in spine volume, and some of the increase in GFP-CaMKIIalpha is secondary to this volume change. In addition, cLTP results in a dramatic increase in the bound fraction of GFP-CaMKIIalpha in spines. To further study the bound pool, immunogold electron microscopy was used to measure CaMKIIalpha in the postsynaptic density (PSD), an important regulator of synaptic function. cLTP produced a persistent increase in the PSD-associated pool of CaMKIIalpha. These results are consistent with the hypothesis that CaMKIIalpha accumulation at synapses is a memory trace of past synaptic activity.
Spines may undergo rapid, activity-dependent changes in shape and size, reflecting reorganization of the actin cytoskeleton. This remodeling is implicated in development and also in the late phase of long-term potentiation. However, the cellular mechanisms that convert activity into morphological change remain poorly understood, and little is known about the anatomical distribution of the actin-regulating proteins that mediate this remodeling. Using immunocytochemistry, we demonstrate here that cortactin (a protein implicated in actin filament nucleation, branching, and stabilization) is concentrated in hippocampal spines, where it colocalizes with F-actin. Cortactin has a Shank-binding domain; recent studies report that synaptic activity may trigger actin remodeling via this interaction with Shank. However, our immunogold electron microscopic data show that cortactin concentrates within the spine core, 100-150 nm away from the postsynaptic density (PSD); only a small fraction of the cortactin in spines lies adjacent to the PSD. These data suggest that the adult dendritic spine contains two functional pools of cortactin: a large pool in the spine core that may help to mediates changes in spine shape and a small synaptic pool that may modify the PSD in response to synaptic activity.
Neural activity induces the remodeling of pre- and postsynaptic membranes, which maintain their apposition through cell adhesion molecules. Among them, N-cadherin is redistributed, undergoes activity-dependent conformational changes, and is required for synaptic plasticity. Here, we show that depolarization induces the enlargement of the width of spine head, and that cadherin activity is essential for this synaptic rearrangement. Dendritic spines visualized with green fluorescent protein in hippocampal neurons showed an expansion by the activation of AMPA receptor, so that the synaptic apposition zone may be expanded. N-cadherin-venus fusion protein laterally dispersed along the expanding spine head. Overexpression of dominant-negative forms of N-cadherin resulted in the abrogation of the spine expansion. Inhibition of actin polymerization with cytochalasin D abolished the spine expansion. Together, our data suggest that cadherin-based adhesion machinery coupled with the actin-cytoskeleton is critical for the remodeling of synaptic apposition zone.
Altered dendritic spines are characteristic of traumatized or diseased brain. Two general categories of spine pathology can be distinguished: pathologies of distribution and pathologies of ultrastructure. Pathologies of spine distribution affect many spines along the dendrites of a neuron and include altered spine numbers, distorted spine shapes, and abnormal loci of spine origin on the neuron. Pathologies of spine ultrastructure involve distortion of subcellular organelles within dendritic spines. Spine distributions are altered on mature neurons following traumatic lesions, and in progressive neurodegeneration involving substantial neuronal loss such as in Alzheimer's disease and in Creutzfeldt-Jakob disease. Similarly, spine distributions are altered in the developing brain following malnutrition, alcohol or toxin exposure, infection, and in a large number of genetic disorders that result in mental retardation, such as Down's and fragile-X syndromes. An important question is whether altered dendritic spines are the intrinsic cause of the accompanying neurological disturbances. The data suggest that many categories of spine pathology may result not from intrinsic pathologies of the spiny neurons, but from a compensatory response of these neurons to the loss of excitatory input to dendritic spines. More detailed studies are needed to determine the cause of spine pathology in most disorders and relationship between spine pathology and cognitive deficits.
Schaffer collateral axons form excitatory synapses that are distributed across much of the dendritic arborization of hippocampal CA1 pyramidal neurons. Remarkably, AMPA-receptor-mediated miniature EPSP amplitudes at the soma are relatively independent of synapse location, despite widely different degrees of dendritic filtering. A progressive increase with distance in synaptic conductance is thought to produce this amplitude normalization. In this study we examined the mechanism(s) responsible for spatial scaling by making whole-cell recordings from the apical dendrites of CA1 pyramidal neurons. We found no evidence to suggest that there is any location dependence to the range of cleft glutamate concentrations found at Schaffer collateral synapses. Furthermore, we observed that release probability (Pr), paired-pulse facilitation and the size of the readily releasable vesicular pool are not dependent on synapse location. Thus, there do not appear to be any changes in the fundamental presynaptic properties of Schaffer collateral synapses that could account for distance-dependent scaling. On the other hand, two-photon uncaging of 4-methoxy-7-nitroindolinyl-caged l-glutamate onto isolated dendritic spines shows that the number of postsynaptic AMPA receptors per spine increases with distance from the soma. We conclude, therefore, that the main synaptic mechanism involved in the production of distance-dependent scaling of Schaffer collateral synapses is an elevated postsynaptic AMPA receptor density.
Dendritic spines are sites of synaptic plasticity in the brain and are capable of remodeling their shape and size. However, little is known about the cellular mechanisms that regulate spine morphology and motility. Synaptopodin is a recently described actin-associated protein found in renal podocytes and dendritic spines (Mundel et al. J Cell Biol. [1997] 139:193–204), which is believed to play a role in spine plasticity. The presentstudy analyzed the distribution of synaptopodin in the hippocampal formation. In situ hybridization histochemistry revealed a high constitutive expression of synaptopodin mRNA in the principal cell layers. Light microscopic immunohistochemistry showed that the protein is distributed throughout the hippocampal formation in a region- and lamina-specific manner. Postembedding immunogold histochemistry demonstrated that synaptopodin is exclusively present in dendrites and spines, specifically in the spine neck in close association with the spine apparatus. Spines lacking a spine apparatus are not immunoreactive for synaptopodin. These data suggest that synaptopodin links the spine apparatus to actin and may thus be involved in the actin-based plasticity of spines. J. Comp. Neurol. 418:164–181, 2000. © 2000 Wiley-Liss, Inc.
Dendritic spines differ considerably in their size, shape, and internal organization between brain regions. We examined the actin cytoskeleton in dendritic spines in hippocampus (areas CA1, CA3, and dentate gyrus), neostriatum, and cerebellum at both light and electron microscopic levels by using a novel high-resolution photoconversion method based in the high affinity of phalloidin for filamentous (F)-actin. In all brain regions, labeling was strongest in the heads of dendritic spines, diminishing in the spine neck. The number of labeled spines varied by region. Compared with the cerebellar molecular layer and area CA3, where nearly every dendritic spine was labeled, less than half the spines were labeled in CA1, dentate gyrus, and neostriatum. Serial section reconstructions of spines in these areas indicated that phalloidin labeling was restricted to the largest and most morphologically diverse dendritic spines. The resolution of the photoconversion technique allowed us to examine the localization and organization of actin filaments in the spine. The most intense staining for actin was found in the postsynaptic density and associated with the spines internal membrane system. In mushroom-shaped spines, F-actin staining was particularly strong between the lamellae of the spine apparatus. Three-dimensional reconstruction of labeled spines by using electron tomography showed that the labeled dense material was in continuity with the postsynaptic density. These results highlight differences in the actin cytoskeleton between different spine populations and provide novel information on the organization of the actin cytoskeleton in vivo. J. Comp. Neurol. 435:156–170, 2001. © 2001 Wiley-Liss, Inc.
Ca²⁺-calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine protein kinase critically involved in synaptic plasticity in the brain. It is highly concentrated in the postsynaptic density fraction, exceeding the amount of any other signal transduction molecules. Because kinase signaling can be amplified by catalytic reaction, why CaMKII exists in such a large quantity has been a mystery. Here, we provide biochemical evidence that CaMKII is capable of bundling F-actin through a stoichiometric interaction. Consistent with this evidence, in hippocampal neurons, RNAi-mediated down-regulation of CaMKII leads to a reduction in the volume of dendritic spine head that is mediated by F-actin dynamics. An overexpression of CaMKII slowed down the actin turnover in the spine head. This activity was associated with β subunit of CaMKII in a manner requiring its actin-binding and association domains but not the kinase domain. This finding indicates that CaMKII serves as a central signaling molecule in both functional and structural changes during synaptic plasticity.
• cytoskeleton
• plasticity
• synapse
The cytoskeletal architecture of the postsynaptic cytoplasm in the cerebellar cortex of mice and rats was observed by quick-freeze, deep-etch electron microscopy. The postsynaptic cytoplasm was mainly filled with a network of actin filaments (approximately 8 nm in width). The tips of the actin filaments were closely associated with the true inner side of the postsynaptic membranes. However, the organization of the actin filaments was distinct depending on the types of synapses. In axosomatic synapses the actin filaments tended to run randomly and form a network while in the postsynaptic spine, such as Purkinje cell dendritic spines, the actin filaments were mainly arranged parallel to the stalk of the spines. Only a few actin filaments were found in the postsynaptic cytoplasm of some axodendritic synapses such as mossy fiber-granule cell synapses. In most cases a mesh of fine strands (approximately 6 nm in width) and granular substances was observed just underneath the postsynaptic membranes which also associated with actin filaments. The arrangement of actin filaments in the spine does not support the possibility of constriction of spines as a basis for long-term depression (LTD).
Since synaptic plasticity is an important property of the brain, it is timely to try to understand the possible mechanisms underlying this phenomenon. The role of the cytoplasm for neuronal functions has until now been largely overlooked, the main emphases being on the plasma membrane for fast electrical events and on cytoplasmic organelles for the slower metabolic processes. However, recent studies on the cytoplasm of non-muscle cells have stressed the importance of contractile proteins, like actin, on maintaining the cell shape and a number of vital cellular functions, which may be related to the phase transitions in the cytoplasm. The necessary versatility is conferred on the actin networks by actin-associated proteins and by the free cytosolic calcium. In the nervous system, in addition to actin and myosin, a number of actin regulatory proteins was recently isolated, and they were shown to have properties similar to those of other non-muscle cells. Consequently, actin networks in neurons like those in non-muscle cells may be capable of contraction and phase transitions. The phase transitions have a rapid onset, and they may be quickly terminated or they may last over extended periods of time. In this way actin networks may gain control over the state of the cytoplasm and hence over the function of the neuron. Actin may be, therefore, uniquely suited to regulate various plastic reactions. The cytoplasm of growth cones and dendritic spines contains solely actin networks and is devoid of microtubules and neurofilaments. Since both these structures contain myosin and since growth cones are endowed with a considerable motility, dendritic spines also may have a likewise property. The necessary regulation of the levels of free cytosolic calcium may be provided by the spine apparatus in addition to calcium pumps in the plasma membrane and calcium regulatory proteins in the spine cytoplasm. Various types of stimulation which change the level of free cytosolic calcium may induce contraction of the spine actin network which may be responsible for the morphometric changes observed following different experimental interventions and pathological conditions. Although most of the conclusions in this review are rather speculative, they may provide directions for future research in the spine and synaptic plasticity.
Latrunculin A, a toxin purified from the red sea sponge Latrunculia magnifica, was found previously to induce striking reversible changes in the morphology of mammalian cells in culture and to disrupt the organization of their microfilaments. We now provide evidence that latrunculin A affects the polymerization of pure actin in vitro in a manner consistent with the formation of a 1:1 molar complex between latrunculin A and G-actin. The equilibrium dissociation constant (Kd) for the reaction in vitro is about 0.2 microM whereas the effects of the drug on cultured cells are detectable at concentrations in the medium of 0.1-1 microM.
Recent studies have shown high levels of calcium in activated dendritic spines, where the smooth endoplasmic reticulum (SER) is likely to be important for regulating calcium. Here, the dimensions and organization of the SER in hippocampal spines and dendrites were measured through serial electron microscopy and three-dimensional analysis. SER of some form was found in 58% of the immature spines and in 48% of the adult spines. Less than 50% of the small spines at either age contained SER, suggesting that other mechanisms, such as cytoplasmic buffers, regulate ion fluxes within their small volumes. In contrast, >80% of the large mushroom spines of the adult had a spine apparatus, an organelle containing stacks of SER and dense-staining plates. Reconstructed SER occupied 0.001-0.022 microm3, which was only 2-3.5% of the total spine volume; however, the convoluted SER membranes had surface areas of 0.12-2.19 microm2, which were 12 to 40% of the spine surface area. Coated vesicles and multivesicular bodies occurred in some spines, suggesting local endocytotic activity. Smooth vesicles and tubules of SER were found in continuity with the spine plasma membrane and margins of the postsynaptic density (PSD), respectively, suggesting a role for the SER in the addition and recycling of spine membranes and synapses. The amount of SER in the parent dendrites was proportional to the number of spines and synapses originating along their lengths. These measurements support the hypothesis that the SER regulates the ionic and structural milieu of some, but not all, hippocampal dendritic spines.
Dendritic spines have been proposed as primary sites of synaptic plasticity in the brain. Consistent with this hypothesis, spines contain high concentrations of actin, suggesting that they might be motile. To investigate this possibility, we made video recordings from hippocampal neurons expressing actin tagged with green fluorescent protein (GFP-actin). This reagent incorporates into actin-containing structures and allows the visualization of actin dynamics in living neurons. In mature neurons, recordings of GFP fluorescence revealed large actin-dependent changes in dendritic spine shape, similar to those inferred from previous studies using fixed tissues. Visible changes occurred within seconds, suggesting that anatomical plasticity at synapses can be extremely rapid. As well as providing a molecular basis for structural plasticity, the presence of motile actin in dendritic spines implicates the postsynaptic element as a primary site of this phenomenon.
It has been suggested that some glutamatergic synapses lack functional AMPA receptors. We used quantitative immunogold localization to determine the number and variability of synaptic AMPA receptors in the rat hippocampus. Three classes of synapses show distinct patterns of AMPA receptor content. Mossy fiber synapses on CA3 pyramidal spines and synapses on GABAergic interneurons are all immunopositive, have less variability, and contain 4 times as many AMPA receptors as synapses made by Schaffer collaterals on CA1 pyramidal spines and by commissural/ associational (C/A) terminals on CA3 pyramidal spines. Up to 17% of synapses in the latter two connections are immunonegative. After calibrating the immunosignal (1 gold = 2.3 functional receptors) at mossy synapses of a 17-day-old rat, we estimate that the AMPA receptor content of C/A synapses on CA3 pyramidal spines ranges from <3 to 140. A similar range is found in adult Schaffer collateral and C/A synapses.
The central nervous system functions primarily to convert patterns of activity in sensory receptors into patterns of muscle activity that constitute appropriate behavior. At the anatomical level this requires two complementary processes: a set of genetically encoded rules for building the basic network of connections, and a mechanism for subsequently fine tuning these connections on the basis of experience. Identifying the locus and mechanism of these structural changes has long been among neurobiology's major objectives. Evidence has accumulated implicating a particular class of contacts, excitatory synapses made onto dendritic spines, as the sites where connective plasticity occurs. New developments in light microscopy allow changes in spine morphology to be directly visualized in living neurons and suggest that a common mechanism, based on dynamic actin filaments, is involved in both the formation of dendritic spines during development and their structural plasticity at mature synapses.
Dendritic spines have long been known to contain contractile elements and have recently been shown to express apparent spontaneous motility. Using high-resolution imaging of dendritic spines of green-fluorescent protein (GFP)-expressing, patch-clamped hippocampal neurons in dissociated culture, we find that bursts of action potentials, evoked by depolarizing current pulses, cause momentary contractions of dendritic spines. Blocking calcium currents with cobalt prevented these twitches. In additional experiments with neurons loaded via a micropipette with calcium-sensitive and insensitive dyes, spontaneous calcium transients were associated with a rapid contraction of the spine head. The spine twitch was prolonged by tetraethylammonium or bicuculline, which enhance calcium transients, and was blocked by the actin polymerization antagonist latrunculin-B. The spine twitch may be instrumental in modulating reactivity of the NMDA receptor to afferent stimulation, following back-propagating action potentials.
Ca2+/calmodulin-dependent protein kinase II (CaMKII) is localized in the postsynaptic density (PSD) and is necessary for LTP induction. Much has been learned about the autophosphorylation of CaMKII and its dephosphorylation by PSD protein phosphatase-1 (PP1). Here, we show how the CaMKII/PP1 system could function as an energy-efficient, bistable switch that could be activated during LTP induction and remain active despite protein turnover. We also suggest how recently discovered binding interactions could provide a structural readout mechanism: the autophosphorylated state of CaMKII binds tightly to the NMDAR and forms, through CaMKII-actinin-actin-(4.1/SAP97) linkages, additional sites for anchoring AMPARs at synapses. The proposed model has substantial experimental support and elucidates principles by which a local protein complex could produce stable information storage and readout.
Lamellipodia are thin, veil-like extensions at the edge of cells that contain a dynamic array of actin filaments. We describe an approach for analyzing spatial regulation of actin polymerization and depolymerization in vivo in which we tracked single molecules of actin fused to the green fluorescent protein. Polymerization and the lifetime of actin filaments in lamellipodia were measured with high spatial precision. Basal polymerization and depolymerization occurred throughout lamellipodia with largely constant kinetics, and polymerization was promoted within one micron of the lamellipodium tip. Most of the actin filaments in the lamellipodium were generated by polymerization away from the tip.
Dendritic spines are motile structures that contain high concentrations of filamentous actin. Using hippocampal neurons expressing fluorescent actin and the method of fluorescence recovery after photobleaching, we found that 85 +/- 2% of actin in the spine was dynamic, with a turnover time of 44.2 +/- 4.0 s. The rapid turnover is not compatible with current models invoking a large population of stable filaments and static coupling of filaments to postsynaptic components. Low-frequency stimulation known to induce long-term depression in these neurons stabilized nearly half the dynamic actin in the spine. This effect depended on the activation of N-methyl-D-aspartate (NMDA) receptors and the influx of calcium. In neurons from mice lacking gelsolin, a calcium-dependent actin-binding protein, activity-dependent stabilization of actin was impaired. Our studies provide new information on the kinetics of actin turnover in spines, its regulation by neural activity and the mechanisms involved in this regulation.
Cerebral ischemia is a major cause of brain dysfunction. Using a model of delayed death induced by a brief, transient oxygen and glucose deprivation, we studied here how this affected the structural organization of hippocampal synaptic networks. We report that brief anoxic-hypoglycemic episodes rapidly modified the structure of synapses. This was characterized, at the electron microscopic level, by a transient increase in the proportion of perforated synapses, followed after 2 hr by an increase in images of multiple synapse boutons. These changes were considerable because 10-20% of all synapses were affected. This structural remodeling was correlated by three kinds of modifications observed using two-photon confocal microscopy: the growth of filopodia, occurring shortly (5-20 min) after anoxia-hypoglycemia, enlargements of existing spines, and formation of new spines, both seen mainly 20-60 min after the insult. All of these structural changes were calcium and NMDA receptor dependent and thus reproduced, to a larger scale, those associated with synaptic plasticity. Concomitantly and related to the severity of anoxia-hypoglycemia, we could also observe spine loss and images of spine, dendrite, or presynaptic terminal swellings that evolved up to membrane disruption. These changes were also calcium dependent and reduced by NMDA receptor antagonists. Thus, short anoxic-hypoglycemic episodes, through NMDA receptor activation and calcium influx, resulted in a profound structural remodeling of synaptic networks, through growth, formation, and elimination of spines and synapses.
We report a photoactivatable variant of theAequorea victoria green fluorescent protein (GFP) that, after intense irradiation with 413-nanometer light, increases fluorescence 100 times
when excited by 488-nanometer light and remains stable for days under aerobic conditions. These characteristics offer a new
tool for exploring intracellular protein dynamics by tracking photoactivated molecules that are the only visible GFPs in the
cell. Here, we use the photoactivatable GFP both as a free protein to measure protein diffusion across the nuclear envelope
and as a chimera with a lysosomal membrane protein to demonstrate rapid interlysosomal membrane exchange.
Throughout the history of neuroscience, dendritic spines have been considered stable structures, but in recent years, imaging techniques have revealed that spines are constantly changing shape. Spine motility is difficult to categorize, has different forms, and possibly even represents multiple phenomena. It is influenced by synaptic transmission, intracellular calcium, and a multitude of ions and other molecules. An actin-based cascade mediates this phenomenon, and while the precise signaling pathways are still unclear, the Rho family of GTPases could well be a "common denominator" controlling spine morphology. One role of spine motility might be to enable a searching function during synaptogenesis, allowing for more efficacious neuronal connectivity in the neuronal thicket. This idea revisits concepts originally formulated by Cajal, who proposed over a hundred years ago that spines might help to increase and modify synaptic connections.
Motile cells extend a leading edge by assembling a branched network of actin filaments that produces physical force as the polymers grow beneath the plasma membrane. A core set of proteins including actin, Arp2/3 complex, profilin, capping protein, and ADF/cofilin can reconstitute the process in vitro, and mathematical models of the constituent reactions predict the rate of motion. Signaling pathways converging on WASp/Scar proteins regulate the activity of Arp2/3 complex, which mediates the initiation of new filaments as branches on preexisting filaments. After a brief spurt of growth, capping protein terminates the elongation of the filaments. After filaments have aged by hydrolysis of their bound ATP and dissociation of the gamma phosphate, ADF/cofilin proteins promote debranching and depolymerization. Profilin catalyzes the exchange of ADP for ATP, refilling the pool of ATP-actin monomers bound to profilin, ready for elongation.
Two key hypotheses about the structural basis of long-term potentiation (LTP) are evaluated in light of new findings from immature rat hippocampal slices. First, it is shown why dendritic spines do not split during LTP. Instead a small number of spine-like dendritic protrusions may emerge to enhance connectivity with potentiated axons. These 'same dendrite multiple synapse boutons' provide less than a 3% increase in connectivity and do not account for all of LTP or memory, as they do not accumulate during maturation. Second, polyribosomes in dendritic spines served to identify which of the existing synapses enlarged to sustain more than a 30% increase in synaptic strength. Thus, both enhanced connectivity and enlarged synapses result during LTP, with synapse enlargement being the greater effect.
Rho family GTPases have been implicated in neuronal growth cone guidance; however, the underlying cytoskeletal mechanisms are unclear. We have used multimode fluorescent speckle microscopy (FSM) to directly address this problem. We report that actin arcs that form in the transition zone are incorporated into central actin bundles in the C domain. These actin structures are Rho/Rho Kinase (ROCK) effectors. Specifically, LPA mediates growth cone retraction by ROCK-dependent increases in actin arc and central actin bundle contractility and stability. In addition, these treatments had marked effects on MT organization as a consequence of strong MT-actin arc interactions. In contrast, LPA or constitutively active Rho had no effect on P domain retrograde actin flow or filopodium bundle number. This study reveals a novel mechanism for domain-specific spatial control of actin-based motility in the growth cone with implications for understanding chemorepellant growth cone responses and nerve regeneration.
Dendritic spines of pyramidal neurons in the cerebral cortex undergo activity-dependent structural remodelling that has been proposed to be a cellular basis of learning and memory. How structural remodelling supports synaptic plasticity, such as long-term potentiation, and whether such plasticity is input-specific at the level of the individual spine has remained unknown. We investigated the structural basis of long-term potentiation using two-photon photolysis of caged glutamate at single spines of hippocampal CA1 pyramidal neurons. Here we show that repetitive quantum-like photorelease (uncaging) of glutamate induces a rapid and selective enlargement of stimulated spines that is transient in large mushroom spines but persistent in small spines. Spine enlargement is associated with an increase in AMPA-receptor-mediated currents at the stimulated synapse and is dependent on NMDA receptors, calmodulin and actin polymerization. Long-lasting spine enlargement also requires Ca2+/calmodulin-dependent protein kinase II. Our results thus indicate that spines individually follow Hebb's postulate for learning. They further suggest that small spines are preferential sites for long-term potentiation induction, whereas large spines might represent physical traces of long-term memory.
More dendritic spine synapses occur on mature neurons in hippocampal slices by 2 h of incubation in vitro, than in perfusion-fixed hippocampus. What conditions initiate this spinogenesis and how rapidly do the spines begin to proliferate on mature neurons? To address these questions, CA1 field of the hippocampus neurons expressing green fluorescent protein in living slices from mature mice were imaged with two-photon microscopy. Spines disappeared and dendrites were varicose immediately after slice preparation in ice-cold artificial cerebrospinal fluid (ACSF). Electron microscopy (EM) revealed disrupted dendritic cytoplasm, enlarged or free-floating postsynaptic densities, and excessive axonal endocytosis. Upon warming dendritic varicosities shrank and spines rapidly reappeared within a few minutes illustrating the remarkable resilience of mature hippocampal neurons in slices. When membrane impermeant sucrose was substituted for NaCl in ACSF dendrites remained spiny at ice-cold temperatures and EM revealed less disruption. Nevertheless, spine number and length increased within 30 min in warm ACSF even when the extracellular calcium concentration was zero and synaptic transmission was blocked. When slices were first recovered for several hours and then chilled in 6 degrees C ACSF many spines disappeared and the dendrites became varicose. Upon re-warming varicosities shrank and spines reemerged in the same position from which they disappeared. In addition, new spines formed and spines were longer suggesting that chilling, not the initial injury from slicing, caused the spines to disappear while re-warming triggered the spine proliferation on mature neurons. The new spines might be a substrate for neuronal recovery of function, when neurons have been chilled or exposed to other traumatic conditions that disrupt ionic homeostasis.
LTP and LTD, the long-term potentiation and depression of excitatory synaptic transmission, are widespread phenomena expressed at possibly every excitatory synapse in the mammalian brain. It is now clear that "LTP" and "LTD" are not unitary phenomena. Their mechanisms vary depending on the synapses and circuits in which they operate. Here we review those forms of LTP and LTD for which mechanisms have been most firmly established. Examples are provided that show how these mechanisms can contribute to experience-dependent modifications of brain function.
Schaffer collateral axons form excitatory synapses that are distributed across much of the dendritic arborization of hippocampal CA1 pyramidal neurons. Remarkably, AMPA-receptor-mediated miniature EPSP amplitudes at the soma are relatively independent of synapse location, despite widely different degrees of dendritic filtering. A progressive increase with distance in synaptic conductance is thought to produce this amplitude normalization. In this study we examined the mechanism(s) responsible for spatial scaling by making whole-cell recordings from the apical dendrites of CA1 pyramidal neurons. We found no evidence to suggest that there is any location dependence to the range of cleft glutamate concentrations found at Schaffer collateral synapses. Furthermore, we observed that release probability (Pr), paired-pulse facilitation and the size of the readily releasable vesicular pool are not dependent on synapse location. Thus, there do not appear to be any changes in the fundamental presynaptic properties of Schaffer collateral synapses that could account for distance-dependent scaling. On the other hand, two-photon uncaging of 4-methoxy-7-nitroindolinyl-caged L-glutamate onto isolated dendritic spines shows that the number of postsynaptic AMPA receptors per spine increases with distance from the soma. We conclude, therefore, that the main synaptic mechanism involved in the production of distance-dependent scaling of Schaffer collateral synapses is an elevated postsynaptic AMPA receptor density.
Activity-induced modification of neuronal connections is essential for the development of the nervous system and may also underlie learning and memory functions of mature brain. Previous studies have shown an increase in dendritic spine density and/or enlargement of spines after the induction of long-term potentiation (LTP). Using two-photon time-lapse imaging of dendritic spines in acute hippocampal slices from neonatal rats, we found that the induction of long-term depression (LTD) by low-frequency stimulation is accompanied by a marked shrinkage of spines, which can be reversed by subsequent high-frequency stimulation that induces LTP. The spine shrinkage requires activation of NMDA receptors and calcineurin, similar to that for LTD. However, spine shrinkage is mediated by cofilin, but not by protein phosphatase 1 (PP1), which is essential for LTD, suggesting that different downstream pathways are involved in spine shrinkage and LTD. This activity-induced spine shrinkage may contribute to activity-dependent elimination of synaptic connections.
Dendritic spines were imaged over days to months in the apical tufts of neocortical pyramidal neurons (layers 5 and 2/3) in vivo. A fraction of thin spines appeared and disappeared over a few days, while most thick spines persisted for months. In the somatosensory cortex, from postnatal day (PND) 16 to PND 25 spine retractions exceeded additions, resulting in a net loss of spines. The fraction of persistent spines (lifetime > or = 8 days) grew gradually during development and into adulthood (PND 16-25, 35%; PND 35-80, 54%; PND 80-120, 66%; PND 175-225, 73%), providing evidence that synaptic circuits continue to stabilize even in the adult brain, long after the closure of known critical periods. In 6-month-old mice, spines turn over more slowly in visual compared to somatosensory cortex, possibly reflecting differences in the capacity for experience-dependent plasticity in these brain regions.
Synapse formation and elimination occur throughout life, but the magnitude of such changes at distinct developmental stages remains unclear. Using transgenic mice overexpressing yellow fluorescent protein and transcranial two-photon microscopy, we repeatedly imaged dendritic spines on the apical dendrites of layer 5 pyramidal neurons. In young adolescent mice (1-month-old), 13%-20% of spines were eliminated and 5%-8% formed over 2 weeks in barrel, motor, and frontal cortices, indicating a cortical-wide spine loss during this developmental period. As animals mature, there is also a substantial loss of dendritic filopodia involved in spinogenesis. In adult mice (4-6 months old), 3%-5% of spines were eliminated and formed over 2 weeks in various cortical regions. Over 18 months, only 26% of spines were eliminated and 19% formed in adult barrel cortex. Thus, after a concurrent loss of spines and spine precursors in diverse regions of young adolescent cortex, spines become stable and a majority of them can last throughout life.
This report covers the two-photon activation and excitation properties of the PA-GFP, a photoactivatable variant of the Aequorea victoria green fluorescent protein in the spectral region from 720 to 920 nm. It is known from this special form of the molecule that it has an increased level of fluorescence emission when excited at 488 nm after irradiation at lambda approximately 413 nm, under single-photon excitation conditions. Here, we show that upon two-photon irradiation, PA-GFP yields activation in the spectral region from 720 to 840 nm. After photoactivation, the excitation spectrum shifts maintaining the very same emission spectrum of the single-photon case for the native and photoactivated protein. Additionally, when comparing the conventional photoactivation at lambda = 405 nm with a two-photon one, a sharper and better controllable three-dimensional volume of activation is obtained.
Increases in cytosolic Ca2+ concentration ([Ca2+]i) mediated by NMDA-sensitive glutamate receptors (NMDARs) are important for synaptic plasticity. We studied a wide variety of dendritic spines on rat CA1 pyramidal neurons in acute hippocampal slices. Two-photon uncaging and Ca2+ imaging revealed that NMDAR-mediated currents increased with spine-head volume and that even the smallest spines contained a significant number of NMDARs. The fate of Ca2+ that entered spine heads through NMDARs was governed by the shape (length and radius) of the spine neck. Larger spines had necks that permitted greater efflux of Ca2+ into the dendritic shaft, whereas smaller spines manifested a larger increase in [Ca2+]i within the spine compartment as a result of a smaller Ca2+ flux through the neck. Spine-neck geometry is thus an important determinant of spine Ca2+ signaling, allowing small spines to be the preferential sites for isolated induction of long-term potentiation.