Structural Plasticity Of Spines Research Articles

Differential expression of Mical2 and its activator (Sema3a) in Alzheimer’s disease mouse model

AbstractBackgroundMicals are actin regulatory enzymes which can enhance depolymerization of actin filaments (F‐Actin). Activated Micals catalyze the selective oxidation of methionine residues, Met44 and Met47 within the D‐loop of F‐actin. The oxidation of methionine residues disrupts the interaction between G‐actin monomers, thus facilitating F‐actin depolymerization. Dendritic spines are enriched with filamentous actin (F‐actin) where it regulates the formation, elimination and structural plasticity of spines. Early stage of AD is associated with loss of dendritic spines and F‐actin from synapses, leading to memory deficits. However, the potential mechanism involved in the loss of F‐actin remains largely unknown. Therefore, we aimed to elucidate the role of Micals and semaphorins in actin depolymerization at the synapse of AD mouse model.MethodsTotal RNA was isolated from the cortex, hippocampus and entorhinal cortex of adolescent, young, and middle aged wildtype and APP/PS1 mice as well as from the synaptosomes of respective age groups. The cDNA is synthesized and qRT‐PCR is performed using SYBR green reagents with specific primers to Micals and Semaphorins. Data was analyzed using two tailed non parametric test with a p value of <0.05 signifies the result.ResultsThe mRNA expression levels of Micals and Semaphorins in entorhinal cortex, hippocampus, and cortex of wildtype and APP/PS1 as well as in the synaptosomes of age matched animals were measured by qRT‐PCR. We found that Semaphorin3a showed differential mRNA expression levels in entorhinal cortex of adolescence and young adults of APP/PS1 mice in comparison to wildtype littermate controls. Further, we detected that the synapse specific mRNA levels of Mical 2 and Semaphorin3a are significantly decreased in adolescent APP/PS1 mice in comparison to WT mice. However, we did not detect any significant alteration in the mRNA expression levels of Mical1, Mical3 and Semaphorin3f in APP/PS1 mice at different age groups in comparison to WT littermate controls.ConclusionAltered expression levels of actin regulatory proteins like Micals and Semaphorins at the dendritic spines could play critical roles in Aβ mediated F‐actin depolymerization in Alzheimer's disease pathology.

β‐secretase inhibition prevents structural spine plasticity deficits in AppNL‐G‐F mice

AbstractBackgroundBACE1‐inhibitor trials for the treatment of Alzheimer´s Disease (AD) have failed due to insufficient efficacy or side effects like cognitive worsening. However, the scientific evidence to date suggests that BACE1‐inhibition could be an effective preventative measure if applied early enough and in the correct dose. Preclinical studies have associated strong BACE1‐inhibition with decreased dendritic spine density in the cerebral cortex of wildtype mice (GFP‐M) only.MethodWe investigated dose‐dependent effects of BACE1‐inhibition on hippocampal dendritic spine dynamics in the APP knock‐in mouse line for the first time. We conducted in vivo two‐photon microscopy in the stratum oriens of hippocampal CA1 neurons in 3.5‐month‐old AppNL‐G‐FGFP‐M mice over 6 weeks to monitor the effect of Bace1 inhibition (NB‐360) in low and high doses on dendritic spine plasticity.ResultsStructural spine plasticity is already impaired in CA1 neurons in untreated AppNL‐G‐F mice of this early age. Interestingly, prolonged high‐dose but not low‐dose BACE1‐inhibition was found to enhance spine formation and did not cause spine loss as observed in wildtype control mice.ConclusionIn an early stage of amyloid pathology in a transgenic beta‐amyloid mouse model, BACE1‐inhibition successfully holds the progressive changes in dendritic spine plasticity in hippocampal CA1 neurons.

Structural spine plasticity: Learning and forgetting of odor-specific subnetworks in the olfactory bulb.

Learning to discriminate between different sensory stimuli is essential for survival. In rodents, the olfactory bulb, which contributes to odor discrimination via pattern separation, exhibits extensive structural synaptic plasticity involving the formation and removal of synaptic spines, even in adult animals. The network connectivity resulting from this plasticity is still poorly understood. To gain insight into this connectivity we present here a computational model for the structural plasticity of the reciprocal synapses between the dominant population of excitatory principal neurons and inhibitory interneurons. It incorporates the observed modulation of spine stability by odor exposure. The model captures the striking experimental observation that the exposure to odors does not always enhance their discriminability: while training with similar odors enhanced their discriminability, training with dissimilar odors actually reduced the discriminability of the training stimuli. Strikingly, this differential learning does not require the activity-dependence of the spine stability and occurs also in a model with purely random spine dynamics in which the spine density is changed homogeneously, e.g., due to a global signal. However, the experimentally observed odor-specific reduction in the response of principal cells as a result of extended odor exposure and the concurrent disinhibition of a subset of principal cells arise only in the activity-dependent model. Moreover, this model predicts the experimentally testable recovery of odor response through weak but not through strong odor re-exposure and the forgetting of odors via exposure to interfering odors. Combined with the experimental observations, the computational model provides strong support for the prediction that odor exposure leads to the formation of odor-specific subnetworks in the olfactory bulb.

β-secretase inhibition prevents structural spine plasticity deficits in App NL-G-F mice.

All clinical BACE1-inhibitor trials for the treatment of Alzheimer's Disease (AD) have failed due to insufficient efficacy or side effects like worsening of cognitive symptoms. However, the scientific evidence to date suggests that BACE1-inhibition could be an effective preventative measure if applied prior to the accumulation of amyloid-beta (Aβ)-peptide and resultant impairment of synaptic function. Preclinical studies have associated BACE1-inhibition-induced cognitive deficits with decreased dendritic spine density. Therefore, we investigated dose-dependent effects of BACE1-inhibition on hippocampal dendritic spine dynamics in an APP knock-in mouse line for the first time. We conducted in vivo two-photon microscopy in the stratum oriens layer of hippocampal CA1 neurons in 3.5-month-old AppNL-G-FGFP-M mice over 6 weeks to monitor the effect of potential preventive treatment with a high and low dose of the BACE1-inhibitor NB-360 on dendritic spine dynamics. Structural spine plasticity was severely impaired in untreated AppNL-G-FGFP-M mice, although spines were not yet showing signs of degeneration. Prolonged high-dose BACE1-inhibition significantly enhanced spine formation, improving spine dynamics in the AD mouse model. We conclude that in an early AD stage characterized by low Aβ-accumulation and no irreversible spine loss, BACE1-inhibition could hold the progressive synapse loss and cognitive decline by improving structural spine dynamics.

Cortactin Contributes to Activity-Dependent Modulation of Spine Actin Dynamics and Spatial Memory Formation.

Postsynaptic structures on excitatory neurons, dendritic spines, are actin-rich. It is well known that actin-binding proteins regulate actin dynamics and by this means orchestrate structural plasticity during the development of the brain, as well as synaptic plasticity mediating learning and memory processes. The actin-binding protein cortactin is localized to pre- and postsynaptic structures and translocates in a stimulus-dependent manner between spines and the dendritic compartment, thereby indicating a crucial role for synaptic plasticity and neuronal function. While it is known that cortactin directly binds F-actin, the Arp2/3 complex important for actin nucleation and branching as well as other factors involved in synaptic plasticity processes, its precise role in modulating actin remodeling in neurons needs to be deciphered. In this study, we characterized the general neuronal function of cortactin in knockout mice. Interestingly, we found that the loss of cortactin leads to deficits in hippocampus-dependent spatial memory formation. This impairment is correlated with a prominent dysregulation of functional and structural plasticity. Additional evidence shows impaired long-term potentiation in cortactin knockout mice together with a complete absence of structural spine plasticity. These phenotypes might at least in part be explained by alterations in the activity-dependent modulation of synaptic actin in cortactin-deficient neurons.

Methodological approaches to understand the molecular mechanism of structural plasticity of dendritic spines.

Dendritic spines are tiny protrusions emanating from the neuronal dendrites, typically housing single excitatory postsynapses. Structural plasticity of dendritic spines is considered to be essential for synaptic functional plasticity and also reorganization of neural circuits during learning and memory. Structural plasticity of spines is mediated by complex biochemical signaling with various spatial and temporal scales. A variety of methods based on pharmacological, genetic, molecular, imaging and optical approaches has been developed and applied to dissect the complex signal transduction pathways. In this review, we overview both conventional and new methodological approaches to identify, monitor and manipulate key molecules for structural plasticity of dendritic spines, ultimately aiming to understand the molecular mechanism of learning and memory in behaving animals.

Rac1 is a downstream effector of PKC\u03b1 in structural synaptic plasticity

Structural and functional plasticity of dendritic spines is the basis of animal learning. The rapid remodeling of actin cytoskeleton is associated with spine enlargement and shrinkage, which are essential for structural plasticity. The calcium-dependent protein kinase C isoform, PKCα, has been suggested to be critical for this actin-dependent plasticity. However, mechanisms linking PKCα and structural plasticity of spines are unknown. Here, we examine the spatiotemporal activation of actin regulators, including small GTPases Rac1, Cdc42 and Ras, in the presence or absence of PKCα during single-spine structural plasticity. Removal of PKCα expression in the postsynapse attenuated Rac1 activation during structural plasticity without affecting Ras or Cdc42 activity. Moreover, disruption of a PDZ binding domain within PKCα led to impaired Rac1 activation and deficits in structural spine remodeling. These results demonstrate that PKCα positively regulates the activation of Rac1 during structural plasticity.

Cyclin-Dependent Kinase 5 Dysfunction Contributes to Depressive-like Behaviors in Huntington’s Disease by Altering the DARPP-32 Phosphorylation Status in the Nucleus Accumbens

BackgroundDepression is the most common psychiatric condition in Huntington’s disease (HD), with rates more than twice those found in the general population. At the present time, there is no established molecular evidence to use as a basis for depression treatment in HD. Indeed, in some patients, classic antidepressant drugs exacerbate chorea or anxiety. Cyclin-dependent kinase 5 (Cdk5) has been involved in processes associated with anxiety and depression. This study evaluated the involvement of Cdk5 in the development and prevalence of depressive-like behaviors in HD and aimed to validate Cdk5 as a target for depression treatment. MethodsWe evaluated the impact of pharmacological inhibition of Cdk5 in depressive-like and anxiety-like behaviors in Hdh+/Q111 knock-in mutant mice by using a battery of behavioral tests. Biochemical and morphological studies were performed to define the molecular mechanisms acting downstream of Cdk5 activation. A double huntingtin/DARPP-32 (dopamine- and cAMP-regulated phosphoprotein 32) knock-in mutant mouse was generated to analyze the role of DARPP-32 in HD depression. ResultsWe found that Hdh+/Q111 mutant mice exhibited depressive-like, but not anxiety-like, behaviors starting at 2 months of age. Cdk5 inhibition by roscovitine infusion prevented depressive-like behavior and reduced DARPP-32 phosphorylation at Thr75 in the nucleus accumbens. Hdh+/Q111 mice heterozygous for DARPP-32 Thr75Ala point mutation were resistant to depressive-like behaviors. We identified β-adducin phosphorylation as a Cdk5 downstream mechanism potentially mediating structural spine plasticity changes in the nucleus accumbens and depressive-like behavior. ConclusionsThese results point to Cdk5 in the nucleus accumbens as a critical contributor to depressive-like behaviors in HD mice by altering DARPP-32/β-adducin signaling and disrupting the dendritic spine cytoskeleton.

Caldendrin Directly Couples Postsynaptic Calcium Signals to Actin Remodeling in Dendritic Spines

Compartmentalization of calcium-dependent plasticity allows for rapid actin remodeling in dendritic spines. However, molecular mechanisms for the spatio-temporal regulation of filamentous actin (F-actin) dynamics by spinous Ca2+-transients are still poorly defined. We show that the postsynaptic Ca2+ sensor caldendrin orchestrates nano-domain actin dynamics that are essential for actin remodeling in the early phase of long-term potentiation (LTP). Steep elevation in spinous [Ca2+]i disrupts an intramolecular interaction of caldendrin and allows cortactin binding. The fast on and slow off rate of this interaction keeps cortactin in an active conformation, and protects F-actin at the spine base against cofilin-induced severing. Caldendrin gene knockout results in higher synaptic actin turnover, altered nanoscale organization of spinous F-actin, defects in structural spine plasticity, LTP, and hippocampus-dependent learning. Collectively, the data indicate that caldendrin-cortactin directly couple [Ca2+]i to preserve a minimal F-actin pool that is required for actin remodeling in the early phase of LTP.

NMDA Receptors Regulate the Structural Plasticity of Spines and Axonal Boutons in Hippocampal Interneurons

N-methyl-D-aspartate receptors (NMDARs) are present in both pyramidal neurons and interneurons of the hippocampus. These receptors play an important role in the adult structural plasticity of excitatory neurons, but their impact on the remodeling of interneurons is unknown. Among hippocampal interneurons, somatostatin-expressing cells located in the stratum oriens are of special interest because of their functional importance and structural characteristics: they display dendritic spines, which change density in response to different stimuli. In order to understand the role of NMDARs on the structural plasticity of these interneurons, we have injected acutely MK-801, an NMDAR antagonist, to adult mice which constitutively express enhanced green fluorescent protein (EGFP) in these cells. We have behaviorally tested the animals, confirming effects of the drug on locomotion and anxiety-related behaviors. NMDARs were expressed in the somata and dendritic spines of somatostatin-expressing interneurons. Twenty-four hours after the injection, the density of spines did not vary, but we found a significant increase in the density of their en passant boutons (EPB). We have also used entorhino-hippocampal organotypic cultures to study these interneurons in real-time. There was a rapid decrease in the apparition rate of spines after MK-801 administration, which persisted for 24 h and returned to basal levels afterwards. A similar reversible decrease was detected in spine density. Our results show that both spines and axons of interneurons can undergo remodeling and highlight NMDARs as regulators of this plasticity. These results are specially relevant given the importance of all these players on hippocampal physiology and the etiopathology of certain psychiatric disorders.

The Role of APP in Structural Spine Plasticity.

Amyloid precursor protein (APP) is a transmembrane protein highly expressed in neurons. The full-length protein has cell-adhesion and receptor-like properties, which play roles in synapse formation and stability. Furthermore, APP can be cleaved by several proteases into numerous fragments, many of which affect synaptic function and stability. This review article focuses on the mechanisms of APP in structural spine plasticity, which encompasses the morphological alterations at excitatory synapses. These occur as changes in the number and morphology of dendritic spines, which correspond to the postsynaptic compartment of excitatory synapses. Both overexpression and knockout (KO) of APP lead to impaired synaptic plasticity. Recent data also suggest a role of APP in the regulation of astrocytic D-serine homeostasis, which in turn regulates synaptic plasticity.

Rho GTPase complementation underlies BDNF-dependent homo- and heterosynaptic plasticity.

The Rho GTPase proteins Rac1, RhoA and Cdc42 have a central role in regulating the actin cytoskeleton in dendritic spines, thereby exerting control over the structural and functional plasticity of spines and, ultimately, learning and memory. Although previous work has shown that precise spatiotemporal coordination of these GTPases is crucial for some forms of cell morphogenesis, the nature of such coordination during structural spine plasticity is unclear. Here we describe a three-molecule model of structural long-term potentiation (sLTP) of murine dendritic spines, implicating the localized, coincident activation of Rac1, RhoA and Cdc42 as a causal signal of sLTP. This model posits that complete tripartite signal overlap in spines confers sLTP, but that partial overlap primes spines for structural plasticity. By monitoring the spatiotemporal activation patterns of these GTPases during sLTP, we find that such spatiotemporal signal complementation simultaneously explains three integral features of plasticity: the facilitation of plasticity by brain-derived neurotrophic factor (BDNF), the postsynaptic source of which activates Cdc42 and Rac1, but not RhoA; heterosynaptic facilitation of sLTP, which is conveyed by diffusive Rac1 and RhoA activity; and input specificity, which is afforded by spine-restricted Cdc42 activity. Thus, we present a form of biochemical computation in dendrites involving the controlled complementation of three molecules that simultaneously ensures signal specificity and primes the system for plasticity.

Structural plasticity of spines at giant mossy fiber synapses

The granule cells of the dentate gyrus give rise to thin unmyelinated axons, the mossy fibers. They form giant presynaptic boutons impinging on large complex spines on the proximal dendritic portions of hilar mossy cells and CA3 pyramidal neurons. While these anatomical characteristics have been known for some time, it remained unclear whether functional changes at mossy fiber synapses such as long-term potentiation (LTP) are associated with structural changes. Since subtle structural changes may escape a fine-structural analysis when the tissue is fixed by using aldehydes and is dehydrated in ethanol, rapid high-pressure freezing (HPF) of the tissue was applied. Slice cultures of hippocampus were prepared and incubated in vitro for 2 weeks. Then, chemical LTP (cLTP) was induced by the application of 25 mM tetraethylammonium (TEA) for 10 min. Whole-cell patch-clamp recordings from CA3 pyramidal neurons revealed a highly significant potentiation of mossy fiber synapses when compared to control conditions before the application of TEA. Next, the slice cultures were subjected to HPF, cryosubstitution, and embedding in Epon for a fine-structural analysis. When compared to control tissue, we noticed a significant decrease of synaptic vesicles in mossy fiber boutons and a concomitant increase in the length of the presynaptic membrane. On the postsynaptic side, we observed the formation of small, finger-like protrusions, emanating from the large complex spines. These short protrusions gave rise to active zones that were shorter than those normally found on the thorny excrescences. However, the total number of active zones was significantly increased. Of note, none of these cLTP-induced structural changes was observed in slice cultures from Munc13-1 deficient mouse mutants showing severely impaired vesicle priming and docking. In conclusion, application of HPF allowed us to monitor cLTP-induced structural reorganization of mossy fiber synapses.

Potential Synaptic Connectivity of Different Neurons onto Pyramidal Cells in a 3D Reconstruction of the Rat Hippocampus

Most existing connectomic data and ongoing efforts focus either on individual synapses (e.g., with electron microscopy) or on regional connectivity (tract tracing). An individual pyramidal cell (PC) extends thousands of synapses over macroscopic distances (∼cm). The contrasting requirements of high-resolution and large field of view make it too challenging to acquire the entire synaptic connectivity for even a single typical cortical neuron. Light microscopy can image whole neuronal arbors and resolve dendritic branches. Analyzing connectivity in terms of close spatial appositions between axons and dendrites could thus bridge the opposite scales, from synaptic level to whole systems. In the mammalian cortex, structural plasticity of spines and boutons makes these “potential synapses” functionally relevant to learning capability and memory capacity. To date, however, potential synapses have only been mapped in the surrounding of a neuron and relative to its local orientation rather than in a system-level anatomical reference. Here we overcome this limitation by estimating the potential connectivity of different neurons embedded into a detailed 3D reconstruction of the rat hippocampus. Axonal and dendritic trees were oriented with respect to hippocampal cytoarchitecture according to longitudinal and transversal curvatures. We report the potential connectivity onto PC dendrites from the axons of a dentate granule cell, three CA3 PCs, one CA2 PC, and 13 CA3b interneurons. The numbers, densities, and distributions of potential synapses were analyzed in each sub-region (e.g., CA3 vs. CA1), layer (e.g., oriens vs. radiatum), and septo-temporal location (e.g., dorsal vs. ventral). The overall ratio between the numbers of actual and potential synapses was ∼0.20 for the granule and CA3 PCs. All potential connectivity patterns are strikingly dependent on the anatomical location of both pre-synaptic and post-synaptic neurons.

Structural Plasticity Can Produce Metaplasticity

BackgroundSynaptic plasticity underlies many aspect of learning memory and development. The properties of synaptic plasticity can change as a function of previous plasticity and previous activation of synapses, a phenomenon called metaplasticity. Synaptic plasticity not only changes the functional connectivity between neurons but in some cases produces a structural change in synaptic spines; a change thought to form a basis for this observed plasticity. Here we examine to what extent structural plasticity of spines can be a cause for metaplasticity. This study is motivated by the observation that structural changes in spines are likely to affect the calcium dynamics in spines. Since calcium dynamics determine the sign and magnitude of synaptic plasticity, it is likely that structural plasticity will alter the properties of synaptic plasticity.Methodology/Principal FindingsIn this study we address the question how spine geometry and alterations of N-methyl-D-aspartic acid (NMDA) receptors conductance may affect plasticity. Based on a simplified model of the spine in combination with a calcium-dependent plasticity rule, we demonstrated that after the induction phase of plasticity a shift of the long term potentiation (LTP) or long term depression (LTD) threshold takes place. This induces a refractory period for further LTP induction and promotes depotentiation as observed experimentally. That resembles the BCM metaplasticity rule but specific for the individual synapse. In the second phase, alteration of the NMDA response may bring the synapse to a state such that further synaptic weight alterations are feasible. We show that if the enhancement of the NMDA response is proportional to the area of the post synaptic density (PSD) the plasticity curves most likely return to the initial state.Conclusions/SignificanceUsing simulations of calcium dynamics in synaptic spines, coupled with a biophysically motivated calcium-dependent plasticity rule, we find under what conditions structural plasticity can form the basis of synapse specific metaplasticity.

Morphological changes in dendritic spines of Purkinje cells associated with motor learning

Experience-dependent changes of spine structure and number may contribute to long-term memory storage. Although several studies demonstrated structural spine plasticity following associative learning, there is limited evidence associating motor learning with alteration of spine morphology. Here, we investigated this issue in the cerebellar Purkinje cells using high voltage electron microscopy (HVEM). Adult rats were trained in an obstacle course, demanding significant motor coordination to complete. Control animals either traversed an obstacle-free runway or remained sedentary. Quantitative analysis of spine morphology showed that the density and length of dendritic spines along the distal dendrites of Purkinje cells were significantly increased in the rats that learned complex motor skills compared to active or inactive controls. Classification of spines into shape categories indicated that the increased spine density and length after motor learning was mainly attributable to an increase in thin spines. These findings suggest that motor learning induces structural spine plasticity in the cerebellar Purkinje neurons, which may play a crucial role in acquiring complex motor skills.

EphA4 Signaling Regulates Phospholipase Cγ1 Activation, Cofilin Membrane Association, and Dendritic Spine Morphology

Specialized postsynaptic structures known as dendritic spines are the primary sites of glutamatergic innervation at synapses of the CNS. Previous studies have shown that spines rapidly remodel their actin cytoskeleton to modify their shape and this has been associated with changes in synaptic physiology. However, the receptors and signaling intermediates that restructure the actin network in spines are only beginning to be identified. We reported previously that the EphA4 receptor tyrosine kinase regulates spine morphology. However, the signaling pathways downstream of EphA4 that induce spine retraction on ephrin ligand binding remain poorly understood. Here, we demonstrate that ephrin stimulation of EphA4 leads to the recruitment and activation of phospholipase Cgamma1 (PLCgamma1) in heterologous cells and in hippocampal slices. This interaction occurs through an Src homology 2 domain of PLCgamma1 and requires the EphA4 juxtamembrane tyrosines. In the brain, PLCgamma1 is found in multiple compartments of synaptosomes and is readily found in postsynaptic density fractions. Consistent with this, PLC activity is required for the maintenance of spine morphology and ephrin-induced spine retraction. Remarkably, EphA4 and PLC activity modulate the association of the actin depolymerizing/severing factor cofilin with the plasma membrane. Because cofilin has been implicated previously in the structural plasticity of spines, this signaling may enable cofilin to depolymerize actin filaments and restructure spines at sites of ephrin-EphA4 contact.

Activity-Dependent Dendritic Spine Structural Plasticity Is Regulated by Small GTPase Rap1 and Its Target AF-6

Activity-dependent remodeling of dendritic spines is essential for neural circuit development and synaptic plasticity, but the mechanisms that coordinate synaptic structural and functional plasticity are not well understood. Here we investigate the signaling pathways that enable excitatory synapses to undergo activity-dependent structural modifications. We report that activation of NMDA receptors in cultured cortical neurons induces spine morphogenesis and activation of the small GTPase Rap1. Rap1 bimodally regulates spine morphology: activated Rap1 recruits the PDZ domain-containing protein AF-6 to the plasma membrane and induces spine neck elongation, while inactive Rap1 dissociates AF-6 from the membrane and induces spine enlargement. Rap1 also regulates spine content of AMPA receptors: thin spines induced by Rap1 activation have reduced GluR1-containing AMPA receptor content, while large spines induced by Rap1 inactivation are rich in AMPA receptors. These results identify a signaling pathway that regulates activity-dependent synaptic structural plasticity and coordinates it with functional plasticity.