Spine Elimination Research Articles

Novel roles of microglia

AbstractPío del Río Hortega, the father of the study of microglia, referred to microglia with highly branched fine processes in the healthy brain as “resting microglia,” because he thought these cells were quiescent. After brain injury, these resting microglia change their morphology into an activated type, which have phagocytic activity at the sites of neuronal damage and inflammation. At present, approximately 90 years after the discovery of microglia, microglia in the healthy brains of live animals are found to be very active using a two‐photon scanning laser microscope, and are much more active than any other cells in the brain. Beyond their role as brain‐resident macrophages, many lines of evidence have shown that microglia have essential roles in the maturation and maintenance of neuronal circuits in the developing and adult brain through both the elimination and formation of dendritic spines through their processes. Furthermore, the length and structural complexity of highly branched fine processes are regulated by the microglial intrinsic circadian clock. Dysfunction of the dendritic spines and disturbances of the circadian clock system are widely accepted characteristic abnormalities in neuropsychiatric disorders, including autism. Therefore, the growing understanding of the movement and functions of microglial processes might aid in the development of novel pharmacological interventions against neuropsychiatric disorders, which are associated with synapse loss and aberrant neuronal connectivity.

GABA-A receptor-dependent mechanisms prevent excessive spine elimination during postnatal maturation of the mouse cortex in vivo

Dendritic spine dynamics are implicated in the structural plasticity of cognition-related neuroconnectivity. This study utilized the transcranial in vivo imaging approach to investigate spine dynamics in intact brains of living yellow fluorescent protein-expressing mice. A developmental switch in the net spine loss rate occurred at ∼4months of age. The initially rapid rate slowed down ∼6-fold due to substantially reduced spine elimination with minor changes in formation. Furthermore, pharmacological blockade of γ-aminobutyric acid type A (GABA-A) receptors resulted in significantly increased elimination of pre-existing spines without affecting new spine formation. Spine elimination returned to normal levels following treatment cessation. Thus, GABA-A receptor-dependent mechanisms act as “brakes” – keeping spine elimination in check to prevent over-pruning, thereby preserving the integrity of cognition-related cortical circuits.

Loss of mTOR-Dependent Macroautophagy Causes Autistic-like Synaptic Pruning Deficits

Developmental alterations of excitatory synapses are implicated in autism spectrum disorders (ASDs). Here, we report increased dendritic spine density with reduced developmental spine pruning in layer V pyramidal neurons in postmortem ASD temporal lobe. These spine deficits correlate with hyperactivated mTOR and impaired autophagy. In Tsc2 ± ASD mice where mTOR is constitutively overactive, we observed postnatal spine pruning defects, blockade of autophagy, and ASD-like social behaviors. The mTOR inhibitor rapamycin corrected ASD-like behaviors and spine pruning defects in Tsc2 ± mice, but not in Atg7(CKO) neuronal autophagy-deficient mice or Tsc2 ± :Atg7(CKO) double mutants. Neuronal autophagy furthermore enabled spine elimination with no effects on spine formation. Our findings suggest that mTOR-regulated autophagy is required for developmental spine pruning, and activation of neuronal autophagy corrects synaptic pathology and social behavior deficits in ASD models with hyperactivated mTOR.

GluN3A promotes dendritic spine pruning and destabilization during postnatal development.

Synaptic rearrangements during critical periods of postnatal brain development rely on the correct formation, strengthening, and elimination of synapses and associated dendritic spines to form functional networks. The correct balance of these processes is thought to be regulated by synapse-specific changes in the subunit composition of NMDA-type glutamate receptors (NMDARs). Among these, the nonconventional NMDAR subunit GluN3A has been suggested to play a role as a molecular brake in synaptic maturation. We tested here this hypothesis using confocal time-lapse imaging in rat hippocampal organotypic slices and assessed the role of GluN3A-containing NMDARs on spine dynamics. We found that overexpressing GluN3A reduced spine density over time, increased spine elimination, and decreased spine stability. The effect of GluN3A overexpression could be further enhanced by using an endocytosis-deficient GluN3A mutant and reproduced by silencing the adaptor protein PACSIN1, which prevents the endocytosis of endogenous GluN3A. Conversely, silencing of GluN3A reduced spine elimination and favored spine stability. Moreover, reexpression of GluN3A in more mature tissue reinstated an increased spine pruning and a low spine stability. Mechanistically, the decreased stability in GluN3A overexpressing neurons could be linked to a failure of plasticity-inducing protocols to selectively stabilize spines and was dependent on the ability of GluN3A to bind the postsynaptic scaffold GIT1. Together, these data provide strong evidence that GluN3A prevents the activity-dependent stabilization of synapses thereby promoting spine pruning, and suggest that GluN3A expression operates as a molecular signal for controlling the extent and timing of synapse maturation.

Spatiotemporal dynamics of dendritic spines in the living brain.

Dendritic spines are ubiquitous postsynaptic sites of most excitatory synapses in the mammalian brain, and thus may serve as structural indicators of functional synapses. Recent works have suggested that neuronal coding of memories may be associated with rapid alterations in spine formation and elimination. Technological advances have enabled researchers to study spine dynamics in vivo during development as well as under various physiological and pathological conditions. We believe that better understanding of the spatiotemporal patterns of spine dynamics will help elucidate the principles of experience-dependent circuit modification and information processing in the living brain.

Local Pruning of Dendrites and Spines by Caspase-3-Dependent and Proteasome-Limited Mechanisms

Synapse loss occurs normally during development and pathologically during neurodegenerative disease. Long-term depression, a proposed physiological correlate of synapse elimination, requires caspase-3 and the mitochondrial pathway of apoptosis. Here, we show that caspase-3 activity is essential--and can act locally within neurons--for regulation of spine density and dendrite morphology. By photostimulation of Mito-KillerRed, we induced caspase-3 activity in defined dendritic regions of cultured neurons. Within the photostimulated region, local elimination of dendritic spines and dendrite retraction occurred in a caspase-3-dependent manner without inducing cell death. However, pharmacological inhibition of inhibitor of apoptosis proteins or proteasome function led to neuronal death, suggesting that caspase activation is spatially restricted by these "molecular brakes" on apoptosis. Caspase-3 knock-out mice have increased spine density and altered miniature EPSCs, confirming a physiological involvement of caspase-3 in the regulation of spines in vivo.

Long-term depression: a cell biological view

Recent studies of the molecular mechanisms of long-term depression (LTD) suggest a crucial role for the signalling pathways of apoptosis (programmed cell death) in the weakening and elimination of synapses and dendritic spines. With this backdrop, we suggest that LTD can be considered as the electrophysiological aspect of a larger cell biological programme of synapse involution, which uses localized apoptotic mechanisms to sculpt synapses and circuits without causing cell death.

Steady-state dynamics and experience-dependent plasticity of dendritic spines of layer 4/5a pyramidal neurons in somatosensory cortex

Steady-state dynamics and experience-dependent plasticity of dendritic spines of layer 4/5a pyramidal neurons in somatosensory cortex

Dendritic Arborization and Spine Dynamics Are Abnormal in the Mouse Model ofMECP2Duplication Syndrome

MECP2 duplication syndrome is a childhood neurological disorder characterized by intellectual disability, autism, motor abnormalities, and epilepsy. The disorder is caused by duplications spanning the gene encoding methyl-CpG-binding protein-2 (MeCP2), a protein involved in the modulation of chromatin and gene expression. MeCP2 is thought to play a role in maintaining the structural integrity of neuronal circuits. Loss of MeCP2 function causes Rett syndrome and results in abnormal dendritic spine morphology and decreased pyramidal dendritic arbor complexity and spine density. The consequences of MeCP2 overexpression on dendritic pathophysiology remain unclear. We used in vivo two-photon microscopy to characterize layer 5 pyramidal neuron spine turnover and dendritic arborization as a function of age in transgenic mice expressing the human MECP2 gene at twice the normal levels of MeCP2 (Tg1; Collins et al., 2004). We found that spine density in terminal dendritic branches is initially higher in young Tg1 mice but falls below control levels after postnatal week 12, approximately correlating with the onset of behavioral symptoms. Spontaneous spine turnover rates remain high in older Tg1 animals compared with controls, reflecting the persistence of an immature state. Both spine gain and loss rates are higher, with a net bias in favor of spine elimination. Apical dendritic arbors in both simple- and complex-tufted layer 5 Tg1 pyramidal neurons have more branches of higher order, indicating that MeCP2 overexpression induces dendritic overgrowth. P70S6K was hyperphosphorylated in Tg1 somatosensory cortex, suggesting that elevated mTOR signaling may underlie the observed increase in spine turnover and dendritic growth.

Accelerated Experience-Dependent Pruning of Cortical Synapses in Ephrin-A2 Knockout Mice

Refinement of mammalian neural circuits involves substantial experience-dependent synapse elimination. Using invivo two-photon imaging, we found that experience-dependent elimination of postsynaptic dendritic spines in the cortex was accelerated in ephrin-A2 knockout (KO) mice, resulting in fewer adolescent spines integrated into adult circuits. Such increased spine removal in ephrin-A2 KOs depended on activation of glutamate receptors, as blockade of the N-methyl-D-aspartate (NMDA) receptors eliminated the difference in spine loss between wild-type and KO mice. We also showed that ephrin-A2 in the cortex colocalized with glial glutamate transporters, which were significantly downregulated in ephrin-A2 KOs. Consistently, glial glutamate transport was reduced in ephrin-A2 KOs, resulting in an accumulation of synaptic glutamate. Finally, inhibition of glial glutamate uptake promoted spine elimination in wild-type mice, resembling the phenotype of ephrin-A2 KOs. Together, our results suggest that ephrin-A2 regulates experience-dependent, NMDA receptor-mediated synaptic pruning through glial glutamate transport during maturation of the mouse cortex.

GABA promotes the competitive selection of dendritic spines by controlling local Ca2+ signaling

Activity-dependent competition of synapses plays a key role in neural organization and is often promoted by GABA; however, its cellular bases are poorly understood. Excitatory synapses of cortical pyramidal neurons are formed on small protrusions known as dendritic spines, which exhibit structural plasticity. We used two-color uncaging of glutamate and GABA in rat hippocampal CA1 pyramidal neurons and found that spine shrinkage and elimination were markedly promoted by the activation of GABAA receptors shortly before action potentials. GABAergic inhibition suppressed bulk increases in cytosolic Ca(2+) concentrations, whereas it preserved the Ca(2+) nanodomains generated by NMDA-type receptors, both of which were necessary for spine shrinkage. Unlike spine enlargement, spine shrinkage spread to neighboring spines (<15 μm) and competed with their enlargement, and this process involved the actin-depolymerizing factor ADF/cofilin. Thus, GABAergic inhibition directly suppresses local dendritic Ca(2+) transients and strongly promotes the competitive selection of dendritic spines.

Long Lasting Protein Synthesis- and Activity-Dependent Spine Shrinkage and Elimination after Synaptic Depression

Neuronal circuits modify their response to synaptic inputs in an experience-dependent fashion. Increases in synaptic weights are accompanied by structural modifications, and activity dependent, long lasting growth of dendritic spines requires new protein synthesis. When multiple spines are potentiated within a dendritic domain, they show dynamic structural plasticity changes, indicating that spines can undergo bidirectional physical modifications. However, it is unclear whether protein synthesis dependent synaptic depression leads to long lasting structural changes. Here, we investigate the structural correlates of protein synthesis dependent long-term depression (LTD) mediated by metabotropic glutamate receptors (mGluRs) through two-photon imaging of dendritic spines on hippocampal pyramidal neurons. We find that induction of mGluR-LTD leads to robust and long lasting spine shrinkage and elimination that lasts for up to 24 hours. These effects depend on signaling through group I mGluRs, require protein synthesis, and activity. These data reveal a mechanism for long lasting remodeling of synaptic inputs, and offer potential insights into mental retardation.

Distinct Roles for Somatically and Dendritically Synthesized Brain-Derived Neurotrophic Factor in Morphogenesis of Dendritic Spines

Dendritic spines undergo the processes of formation, maturation, and pruning during development. Molecular mechanisms controlling spine maturation and pruning remain largely unknown. The gene for brain-derived neurotrophic factor (BDNF) produces two pools of mRNA, with either a short or long 3' untranslated region (3' UTR). Our previous results show that short 3' UTR Bdnf mRNA is restricted to cell bodies, whereas long 3' UTR Bdnf mRNA is also trafficked to dendrites for local translation. Mutant mice lacking long 3' UTR Bdnf mRNA display normal spines at 3 weeks of age, but thinner and denser spines in adults compared to wild-type littermates. These observations suggest that BDNF translated from long 3' UTR Bdnf mRNA, likely in dendrites, is required for spine maturation and pruning. In this study, using rat hippocampal neuronal cultures, we found that knocking down long 3' UTR Bdnf mRNA blocked spine head enlargement and spine elimination, whereas overexpressing long 3' UTR Bdnf mRNA had the opposite effect. The effect of long 3' UTR Bdnf mRNA on spine head enlargement and spine elimination was diminished by a human single-nucleotide polymorphism (SNP, rs712442) in its 3' UTR that inhibited dendritic localization of Bdnf mRNA. Furthermore, we found that overexpression of either Bdnf mRNA increased spine density at earlier time points. Spine morphological alterations were associated with corresponding changes in density, size, and function of synapses. These results indicate that somatically synthesized BDNF promotes spine formation, whereas dendritically synthesized BDNF is a key regulator of spine head growth and spine pruning.

Bacterial cytolysin during meningitis disrupts the regulation of glutamate in the brain, leading to synaptic damage.

Streptococcus pneumoniae (pneumococcal) meningitis is a common bacterial infection of the brain. The cholesterol-dependent cytolysin pneumolysin represents a key factor, determining the neuropathogenic potential of the pneumococci. Here, we demonstrate selective synaptic loss within the superficial layers of the frontal neocortex of post-mortem brain samples from individuals with pneumococcal meningitis. A similar effect was observed in mice with pneumococcal meningitis only when the bacteria expressed the pore-forming cholesterol-dependent cytolysin pneumolysin. Exposure of acute mouse brain slices to only pore-competent pneumolysin at disease-relevant, non-lytic concentrations caused permanent dendritic swelling, dendritic spine elimination and synaptic loss. The NMDA glutamate receptor antagonists MK801 and D-AP5 reduced this pathology. Pneumolysin increased glutamate levels within the mouse brain slices. In mouse astrocytes, pneumolysin initiated the release of glutamate in a calcium-dependent manner. We propose that pneumolysin plays a significant synapto- and dendritotoxic role in pneumococcal meningitis by initiating glutamate release from astrocytes, leading to subsequent glutamate-dependent synaptic damage. We outline for the first time the occurrence of synaptic pathology in pneumococcal meningitis and demonstrate that a bacterial cytolysin can dysregulate the control of glutamate in the brain, inducing excitotoxic damage.

Peripheral elevation of TNF-α leads to early synaptic abnormalities in the mouse somatosensory cortex in experimental autoimmune encephalomyelitis

Sensory abnormalities such as numbness and paresthesias are often the earliest symptoms in neuroinflammatory diseases including multiple sclerosis. The increased production of various cytokines occurs in the early stages of neuroinflammation and could have detrimental effects on the central nervous system, thereby contributing to sensory and cognitive deficits. However, it remains unknown whether and when elevation of cytokines causes changes in brain structure and function under inflammatory conditions. To address this question, we used a mouse model for experimental autoimmune encephalomyelitis (EAE) to examine the effect of inflammation and cytokine elevation on synaptic connections in the primary somatosensory cortex. Using in vivo two-photon microscopy, we found that the elimination and formation rates of dendritic spines and axonal boutons increased within 7 d of EAE induction--several days before the onset of paralysis--and continued to rise during the course of the disease. This synaptic instability occurred before T-cell infiltration and microglial activation in the central nervous system and was in conjunction with peripheral, but not central, production of TNF-α. Peripheral administration of a soluble TNF inhibitor prevented abnormal turnover of dendritic spines and axonal boutons in presymptomatic EAE mice. These findings indicate that peripheral production of TNF-α is a key mediator of synaptic instability in the primary somatosensory cortex and may contribute to sensory and cognitive deficits seen in autoimmune diseases.

Learning to deal with life's ups and downs

A study shows that circadian glucocorticoid oscillations have dual roles in dendritic spine plasticity, controlling spine formation and elimination through distinct mechanisms important for motor learning.

Learning with peaks and troughs

Circadian corticosterone fluctuations support learning and memory by promoting learning-associated spine formation and elimination.

Circadian glucocorticoid oscillations promote learning-dependent synapse formation and maintenance

Excessive glucocorticoid exposure during chronic stress causes synapse loss and learning impairment. Under normal physiological conditions, glucocorticoid activity oscillates in synchrony with the circadian rhythm. Whether and how endogenous glucocorticoid oscillations modulate synaptic plasticity and learning is unknown. Here we show that circadian glucocorticoid peaks promote postsynaptic dendritic spine formation in the mouse cortex after motor skill learning, whereas troughs are required for stabilizing newly formed spines that are important for long-term memory retention. Conversely, chronic and excessive exposure to glucocorticoids eliminates learning-associated new spines and disrupts previously acquired memories. Furthermore, we show that glucocorticoids promote rapid spine formation through a non-transcriptional mechanism by means of the LIM kinase-cofilin pathway and increase spine elimination through transcriptional mechanisms involving mineralocorticoid receptor activation. Together, these findings indicate that tightly regulated circadian glucocorticoid oscillations are important for learning-dependent synaptic formation and maintenance. They also delineate a new signaling mechanism underlying these effects.

Corticosteroid-Induced Neural Remodeling Predicts Behavioral Vulnerability and Resilience

Neurons in distinct brain regions remodel in response to postnatal stressor exposure, and structural plasticity may underlie stress-related modifications in behavioral outcomes. Given the persistence of stress-related diseases such as depression, a critical next step in identifying the contributions of neural structure to psychopathology will be to identify brain circuits and cell types that fail to recover from stressor exposure. We enumerated dendritic spines during and after chronic stress hormone exposure in hippocampal CA1, deep-layer prefrontal cortex, and the basal amygdala and also reconstructed dendritic arbors of CA1 pyramidal neurons. Corticosterone modified dendritic spine density in these regions, but with the exception of the orbitofrontal cortex, densities normalized with a recovery period. Dendritic retraction of hippocampal CA1 neurons and anhedonic-like insensitivity to a sucrose solution also persisted despite a recovery period. Using mice with reduced gene dosage of p190rhogap, a cytoskeletal regulatory protein localized to dendritic spines, we next isolated structural correlates of both behavioral vulnerability (spine elimination) and resilience (spine proliferation) to corticosterone within the orbital cortex. Our findings provide novel empirical support for the perspective that stress-related structural reorganization of certain neuron populations can persist despite a "recovery" period from stressor exposure and that these modifications may lay a structural foundation for stressor vulnerability-or resiliency-across the lifespan.

Mef2 promotes spine elimination in absence of δ-catenin

δ-Catenin is a component of the cadherin–catenin cell adhesion complex and its loss has been implicated in the mental retardation associated with the Cri du chat syndrome. We have previously demonstrated that loss of δ-catenin in a murine model during development results in excessive spine and synaptic density and function. In order to examine the role of potential molecules that might cooperate with δ-catenin to regulate spine density, we focused on Mef2. Our data demonstrate that while loss of δ-catenin does not alter the expression levels of endogenous Mef2, expression of Mef2 in neurons that are knocked down for δ-catenin promotes spine elimination. These results establish a molecular mechanism by which excessive spines in the absence of δ-catenin may be eliminated and may point toward pharmacological therapy for the Cri du chat syndrome.