Dendritic Spine Pathology: Cause or Consequence of Neurological Disorders?

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.     

Automated 3-D Detection of Dendritic Spines from <Emphasis Type="Italic">In Vivo</Emphasis> Two-Photon Image Stacks

Despite the significant advances in the development of automated image analysis algorithms for the detection and extraction of neuronal structures, current software tools still have numerous limitations when it comes to the detection and analysis of dendritic spines. The problem is especially challenging in in vivo imaging, where the difficulty of extracting morphometric properties of spines is compounded by lower image resolution and contrast levels native to two-photon laser microscopy. To address this challenge, we introduce a new computational framework for the automated detection and quantitative analysis of dendritic spines in vivo multi-photon imaging. This framework includes: (i) a novel preprocessing algorithm enhancing spines in a way that they are included in the binarized volume produced during the segmentation of foreground from background; (ii) the mathematical foundation of this algorithm, and (iii) an algorithm for the detection of spine locations in reference to centerline trace and separating them from the branches to whom spines are attached to. This framework enables the computation of a wide range of geometric features such as spine length, spatial distribution and spine volume in a high-throughput fashion. We illustrate our approach for the automated extraction of dendritic spine features in time-series multi-photon images of layer 5 cortical excitatory neurons from the mouse visual cortex.     

A novel computational approach for automatic dendrite spines detection in two-photon laser scan microscopy

BACKGROUND: Recent research has shown that there is a strong correlation between the functional properties of a neuron and its morphologic structure. Current morphologic analyses typically involve a significant component of computer-assisted manual labor, which is very time-consuming and is susceptible to operator bias. The existing semi-automatic approaches largely reduce user efforts. However, some manual interventions, such as setting a global threshold for segmentation, are still needed during image processing. METHODS: We present an automated approach, which can greatly help neurobiologists obtain quantitative morphological information about a neuron and its spines. The automation includes an adaptive thresholding method, which can yield better segment results than the prevalent global thresholding method. It also introduces an efficient backbone extraction method, a SNR based, detached spine component detection method, and an attached spine component detection method based on the estimation of local dendrite morphology. RESULTS: The morphology information obtained both manually and automatically are compared in detail. Using the Kolmogov-Smirnov test, we find a 99.13% probability that the dendrite length distributions are the same for the automatic and manual processing methods. The spine detection results are also compared with other existing semi-automatic approaches. The comparison results show that our approach has 33% fewer false positives and 77% fewer false negatives on average. CONCLUSIONS: Because the proposed detection algorithm requires less user input and performs better than existing algorithms, our approach can quickly and accurately process neuron images without user intervention.     

Repeated stress alters dendritic spine morphology in the rat medial prefrontal cortex

Anatomical alterations in the medial prefrontal cortex (mPFC) are associated with hypothalamopituitary adrenal (HPA) axis dysregulation, altered stress hormone levels, and psychiatric symptoms of stress-related mental illnesses. Functional imaging studies reveal impairment and shrinkage of the mPFC in such conditions, and these findings are paralleled by experimental studies showing dendritic retraction and spine loss following repeated stress in rodents. Here we extend this characterization to how repeated stress affects dendritic spine morphology in mPFC through the utilization of an automated approach that rapidly digitizes, reconstructs three dimensionally, and calculates geometric features of neurons. Rats were perfused after being subjected to 3 weeks of daily restraint stress (6 hours/day), and intracellular injections of Lucifer Yellow were made in layer II/III pyramidal neurons in the dorsal mPFC. To reveal spines in all angles of orientation, deconvolved high-resolution confocal laser scanning microscopy image stacks of dendritic segments were reconstructed and analyzed for spine volume, surface area, and length using a Rayburst-based automated approach (8,091 and 8,987 spines for control and stress, respectively). We found that repeated stress results in an overall decrease in mean dendritic spine volume and surface area, which was most pronounced in the distal portion of apical dendritic fields. Moreover, we observed an overall shift in the population of spines, manifested by a reduction in large spines and an increase in small spines. These results suggest a failure of spines to mature and stabilize following repeated stress and are likely to have major repercussions on function, receptor expression, and synaptic efficacy.     

Early loss of dendritic spines in murine scrapie revealed by confocal analysis

Confocal analysis of dye-filled neurons has revealed a significant early loss of dendritic spines in a murine scrapie model in which neuron loss occurs in the hippocampus. An 18% loss of spines was found at 109 days, > 50 days before neuron loss occurs, and by 126 days a 51% spine loss was found. Spine loss is concurrent with synapse loss, axon terminal degeneration and a decrease in long term potentiation in this model. Preceding these changes is the deposition of disease specific PrP at 70 days, which may initiate the damage to dendritic spines and the subsequent degeneration of synapses. We suggest that these changes underlie the development of clinical disease in the transmissible spongiform encephalopathies.     

Synapse Pathology in Psychiatric and Neurologic Disease

Inhibitory and excitatory synapses play a fundamental role in information processing in the brain. Excitatory synapses usually are situated on dendritic spines, small membrane protrusions that harbor glutamate receptors and postsynaptic density components and help transmit electrical signals. In recent years, it has become evident that spine morphology is intimately linked to synapse function—smaller spines have smaller synapses and support reduced synaptic transmission. The relationship between synaptic signaling, spine shape, and brain function is never more apparent than when the brain becomes dysfunctional. Many psychiatric and neurologic disorders, ranging from mental retardation and autism to Alzheimer’s disease and addiction, are accompanied by alterations in spine morphology and synapse number. In this review, we highlight the structure and molecular organization of synapses and discuss functional effects of synapse pathology in brain disease.     

Simultaneous analysis of dendritic spine density, morphology and excitatory glutamate receptors during neuron maturation in vitro by quantitative immunocytochemistry

Alterations in the density and morphology of dendritic spines are characteristic of multiple cognitive disorders. Elucidating the molecular mechanisms underlying spine alterations are facilitated by the use of experimental and analytical methods that permit concurrent evaluation of changes in spine density, morphology and composition. Here, an automated and quantitative immunocytochemical method for the simultaneous analysis of changes in the density and morphology of spines and excitatory glutamate receptors was established to analyze neuron maturation, in vitro. In neurons of long-term neuron-glia co-cultures, spine density as measured by drebrin cluster fluorescence, increased from DIV (days in vitro)10 to DIV18 (formation phase), remained stable from DIV18 to DIV21 (maintenance phase), and decreased from DIV21 to DIV26 (loss phase). The densities of spine-localized NMDAR and AMPAR clusters followed a similar trend. Spine head sizes as measured by the fluorescence intensities of drebrin clusters increased from DIV10 to DIV21 and decreased from DIV21 to DIV26. Changes in the densities of NR1-only, GluR2-only, and NR1+GluR2 spines were measured by the colocalizations of NR1 and GluR2 clusters with drebrin clusters. The densities of NR1-only spines remained stable from the maintenance to the loss phases, while GluR2-only and NR1+GluR2 spines decreased during the loss phase, thus suggesting GluR2 loss as a proximal molecular event that may underlie spine alterations during neuron maturation. This study demonstrates a sensitive and quantitative immunocytochemical method for the concurrent analysis of changes in spine density, morphology and composition, a valuable tool for determining molecular events involved in dendritic spine alterations.     

Modeling the integrative properties of dendrites: Application to the striatal spiny neuron

The role of the striatum in the control of movements and in the processing of cortical information has received much attention in the recent years. We set out a simple biophysical model for the medium-spiny neuron (msn), the most abundant cell in striatum. This neuron receives two main kinds of inputs, namely, cortical excitatory inputs and dopaminergic inputs coming from the substantia nigra pars compacta. The msn axon impinges directly onto the globus pallidus and onto the substantia nigra pars reticulata neurons and onto striatal neurons through recurrent branches of the axon. The msn is characterized by spiny dendritic trees with a high density of spines (1 to 4 spines/μ) and the probable existence of dendritic spikes. The model predicts that the neuron can integrate excitable inputs in a linear or a nonlinear mode. In the nonlinear mode, the neuron allows the detection of simultaneous (or almost simultaneous) synaptic inputs; it facilitates either a slowing down or a speeding up of the information transfer between the synaptic input location and the soma and is sensitive to inhibition-excitation pairing. Conversely, in the linear integrative mode, the somatic voltage is determined by a weighted summation of the synaptic inputs. Several geometrical, electrical, or temporal factors can control the switch between these behaviors: the density of excitable dendritic elements, the dendritic radius, the resistance of the spine stem, the membrane resistance, the time between excitations, and the distance between synaptic sites. Finally, the signification of this behavior is discussed in connection with the putative role of dopamine and with the striatal net organization. © 1994 Wiley-Liss, Inc.     

Selective localization of high concentrations of F-actin in subpopulations of dendritic spines in rat central nervous system: a three-dimensional electron microscopic study

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.     

Passive cable properties of dendritic spines and spiny neurons

A cable model of the linear properties of dendritic spines was generated using the Laplace transform technique. Analytical solutions for the voltages generated in the spine by a current impulse at the spine head were used in a numerical procedure for simulating the effect of a synaptic conductance change. The synaptic current produced by the conductance change was used as an input for evaluation of the postsynaptic potential and current injected into the dendrite at the base of the spine. The primary effect of the dendritic spine was to attenuate synaptic current. This effect was produced by the high input impedance at axospinous synapses, which resulted in giant spike-like excitatory postsynaptic potentials (EPSPs) that approached the reversal potential of the synapse and thus reduced the potential gradient driving the synaptic current. Although virtually all of the synaptic current was transferred to the dendrite, it produced much smaller EPSPs there due to the low dendritic input impedance. Very small conductance changes produced near maximal synaptic currents in dendritic spines. The current attenuating effect of the spine was accentuated with brief synaptic transients and reduced with prolonged synaptic conductance changes. The size and shape of the spine head, and the diameter and boundary conditions of the dendrite had little or no effect on current attenuation for spines in the naturally occurring size range. The diameter and length of the spine stalk and the size and location of the spine apparatus were the key morphological factors determining the synaptic currents generated by axospinous synapses. Naturally occurring size and shape differences among dendritic spines produced large differences in synaptic potency when compared in a model spiny neuron based on the neostriatal spiny projection neuron. These differences were comparable to those produced by differences in synaptic-location on the same neuron.     

Spine formation and maturation in the developing rat auditory cortex

The rat auditory cortex is organized as a tonotopic map of sound frequency. This map is broadly tuned at birth and is refined during the first 3 weeks postnatal. The structural correlates underlying tonotopic map maturation and reorganization during development are poorly understood. We employed fluorescent dye ballistic labeling ("DiOlistics") alone, or in conjunction with immunohistochemistry, to quantify synaptogenesis in the auditory cortex of normal hearing rats. We show that the developmental appearance of dendritic protrusions, which include both immature filopodia and mature spines, on layers 2/3, 4, and 5 pyramidal and layer 4 spiny nonpyramidal neurons occurs in three phases: slow addition of dendritic protrusions from postnatal day 4 (P4) to P9, rapid addition of dendritic protrusions from P9 to P19, and a final phase where mature protrusion density is achieved (>P21). Next, we combined DiOlistics with immunohistochemical labeling of bassoon, a presynaptic scaffolding protein, as a novel method to categorize dendritic protrusions as either filopodia or mature spines in cortex fixed in vivo. Using this method we observed an increase in the spine-to-filopodium ratio from P9-P16, indicating a period of rapid spine maturation. Previous studies report mature spines as being shorter in length compared to filopodia. We similarly observed a reduction in protrusion length between P9 and P16, corroborating our immunohistochemical spine maturation data. These studies show that dendritic protrusion formation and spine maturation occur rapidly at a time previously shown to correspond to auditory cortical tonotopic map refinement (P11-P14), providing a structural correlate of physiological maturation.     

Amyloid plaque formation precedes dendritic spine loss

Amyloid-beta plaque deposition represents a major neuropathological hallmark of Alzheimer??s disease. While numerous studies have described dendritic spine loss in proximity to plaques, much less is known about the kinetics of these processes. In particular, the question as to whether synapse loss precedes or follows plaque formation remains unanswered. To address this question, and to learn more about the underlying kinetics, we simultaneously imaged amyloid plaque deposition and dendritic spine loss by applying two-photon in vivo microscopy through a cranial window in double transgenic APPPS1 mice. As a result, we first observed that the rate of dendritic spine loss in proximity to plaques is the same in both young and aged animals. However, plaque size only increased significantly in the young cohort, indicating that spine loss persists even many months after initial plaque appearance. Tracking the fate of individual spines revealed that net spine loss is caused by increased spine elimination, with the rate of spine formation remaining constant. Imaging of dendritic spines before and during plaque formation demonstrated that spine loss around plaques commences at least 4?weeks after initial plaque formation. In conclusion, spine loss occurs, shortly but with a significant time delay, after the birth of new plaques, and persists in the vicinity of amyloid plaques over many months. These findings hence give further hope to the possibility that there is a therapeutic window between initial amyloid plaque deposition and the onset of structural damage at spines.     

Epac2-mediated dendritic spine remodeling: implications for disease

In the mammalian forebrain, most glutamatergic excitatory synapses occur on small dendritic protrusions called dendritic spines. Dendritic spines are highly plastic and can rapidly change morphology in response to numerous stimuli. This dynamic remodeling of dendritic spines is thought to be critical for information processing, memory and cognition. Conversely, multiple studies have revealed that neuropathologies such as autism spectrum disorders (ASDs) are linked with alterations in dendritic spine morphologies and miswiring of neural circuitry. One compelling hypothesis is that abnormal dendritic spine remodeling is a key contributing factor for this miswiring. Ongoing research has identified a number of mechanisms that are critical for the control of dendritic spine remodeling. Among these mechanisms, regulation of small GTPase signaling by guanine-nucleotide exchange factors (GEFs) is emerging as a critical mechanism for integrating physiological signals in the control of dendritic spine remodeling. Furthermore, multiple proteins associated with regulation of dendritic spine remodeling have also been implicated with multiple neuropathologies, including ASDs. Epac2, a GEF for the small GTPase Rap, has recently been described as a novel cAMP (yet PKA-independent) target localized to dendritic spines. Signaling via this protein in response to pharmacological stimulation or cAMP accumulation, via the dopamine D1/5 receptor, results in Rap activation, promotes structural destabilization, in the form of dendritic spine shrinkage, and functional depression due to removal of GluR2/3-containing AMPA receptors. In addition, Epac2 forms macromolecular complexes with ASD-associated proteins, which are sufficient to regulate Epac2 localization and function. Furthermore, rare non-synonymous variants of the EPAC2 gene associated with the ASD phenotype alter protein function, synaptic protein distribution, and spine morphology. We review here the role of Epac2 in the remodeling of dendritic spines under normal conditions, the mechanisms that underlie these effects, and the implications these disease-associated variants have on our understanding of the pathophysiology of ASD.     

Estrogen selectively regulates spine density within the dendritic arbor of rat ventromedial hypothalamic neurons.

Estrogen acts in the hypothalamic ventromedial nucleus (VMH) to promote female sexual behavior. One potential mechanism through which estrogen may facilitate this behavior is by reconfiguring synaptic connections within the VMH. Estrogen treatment increases the number of synapses and dendritic spines in the VMH, but how this remodeling occurs within the context of the local, behaviorally relevant microcircuitry is unknown. The goal of this study was to localize estrogen-induced changes in spine density within the VMH and relate these to dendritic morphology and the presence of nuclear estrogen receptor. The hypothalami from ovariectomized rats, treated with either vehicle or estradiol, were lightly fixed, and VMH neurons were iontophoretically filled with Lucifer yellow. Confocal microscopy was used to examine neuronal morphology. Estrogen treatment increased dendritic spine density by 48% in the ventrolateral VMH but had no effect on spine density in the dorsal VMH. The primary dendrites of VMH neurons were differentially affected by estrogen. Estrogen treatment increased spine density twofold on the short primary dendrites but did not affect spine density on long primary dendrites. Immunocytochemical staining showed that none of the filled neurons expressed estrogen receptor-alpha. Thus, although the effect of estrogen on spine density is localized to a VMH subdivision where estrogen receptor is expressed, estrogen treatment induces spines on neurons that lack estrogen receptor. Taken together, our results suggest that the effect of estrogen on ventrolateral VMH spines is selective within the dendritic arbor of a neuron and may be mediated by an indirect, possibly transynaptic, mechanism.     

Regulation of hippocampal synapse remodeling by epileptiform activity

We examined the regulation of dendritic spines and synapses by epileptiform activity (EA) in rat hippocampal slice cultures. EA, which was induced by a GABA(A) receptor inhibitor, gabazine, reduced pyramidal neuron spine density by approximately 50% after 48 h and also caused an increase in the average length of remaining spines. To directly determine the effects of EA on synapses, we used fluorescent protein-tagged PSD95, which marks postsynaptic densities. EA induced a net loss of synapses on spines but not shafts; conversely, activity blockade (TTX) induced a loss of shaft synapses. Time-lapse confocal imaging in live tissue slices revealed that EA (1) shifts the balance of synapse gain and loss in dendrites leading to a net loss of spine synapses and (2) induces the formation of new filopodia-like dendritic structures having abnormally slow motility. These results identify EA-induced changes in the density and distribution of synaptic structures on dendrites.     

Dendritic spine dynamics regulate the long-term stability of synaptic plasticity

Long-term synaptic plasticity requires postsynaptic influx of Ca2? and is accompanied by changes in dendritic spine size. Unless Ca2? influx mechanisms and spine volume scale proportionally, changes in spine size will modify spine Ca2? concentrations during subsequent synaptic activation. We show that the relationship between Ca2? influx and spine volume is a fundamental determinant of synaptic stability. If Ca2? influx is undercompensated for increases in spine size, then strong synapses are stabilized and synaptic strength distributions have a single peak. In contrast, overcompensation of Ca2? influx leads to binary, persistent synaptic strengths with double-peaked distributions. Biophysical simulations predict that CA1 pyramidal neuron spines are undercompensating. This unifies experimental findings that weak synapses are more plastic than strong synapses, that synaptic strengths are unimodally distributed, and that potentiation saturates for a given stimulus strength. We conclude that structural plasticity provides a simple, local, and general mechanism that allows dendritic spines to foster both rapid memory formation and persistent memory storage.     

Persistence of excitatory shaft synapses adjacent to newly emerged dendritic protrusions

In the early postnatal hippocampus, the first synapses to appear on excitatory pyramidal neurons are formed directly on dendritic shafts. Very few dendritic spines are present at this time. By adulthood, however, the overwhelming majority of synapses are located at the tips of dendritic spines. Several models have been proposed to account for the transition from mostly shaft to mostly spinous synapses but none has been demonstrated conclusively. To investigate the cellular mechanism underlying the shaft-to-spinous synapse transition, we designed live imaging experiments to directly observe the dynamics of shaft and spinous synapses on developing dendrites. Immunofluorescent synaptic labeling of GFP-filled neurons showed that the shaft-to-spinous synapse transition in dissociated culture mirrors that in vivo. Along with electron microscopy, the fluorescent labeling also showed that veritable shaft synapses are abundant in dissociated culture and that shaft synapses are frequently adjacent to spines or other dendritic protrusions, a configuration previously observed in vivo by others. We used live long-term time lapse confocal microscopy of GFP-filled dendrites and VAMP2-DsRed-labeled boutons to record the fate of shaft synapses and associated dendritic protrusions and boutons with images taken hourly for up to 31 continuous hours. Inspection of the time lapse imaging series revealed that shaft synapses can persist adjacent to either existing or newly grown dendritic protrusions. Alternatively, a shaft synapse bouton can redistribute to contact an adjacent dendritic protrusion. However, we never observed shaft synapses transforming themselves into spines or any type of dendritic protrusions. We conclude that repeated iterations of dendritic protrusion or spine outgrowth adjacent to shaft synapses is very likely to be a critical component of the shaft-to-spinous synapse transition during CNS development.     

Rapid Ultrastructural Changes in the PSD and Surrounding Membrane after Induction of Structural LTP in Single Dendritic Spines

The structural plasticity of dendritic spines is considered to be an important basis of synaptic plasticity, learning, and memory. Here, we induced input-specific structural LTP (sLTP) in single dendritic spines in organotypic hippocampal slices from mice of either sex and performed ultrastructural analyses of the spines using efficient correlative light and electron microscopy. We observed reorganization of the PSD nanostructure, such as perforation and segmentation, at 2–3, 20, and 120 min after sLTP induction. In addition, PSD and nonsynaptic axon–spine interface (nsASI) membrane expanded unevenly during sLTP. Specifically, the PSD area showed a transient increase at 2–3 min after sLTP induction. The PSD growth was to a degree less than spine volume growth at 2–3 min and 20 min after sLTP induction but became similar at 120 min. On the other hand, the nsASI area showed a profound and lasting expansion, to a degree similar to spine volume growth throughout the process. These rapid ultrastructural changes in PSD and surrounding membrane may contribute to rapid electrophysiological plasticity during sLTP.SIGNIFICANCE STATEMENT To understand the ultrastructural changes during synaptic plasticity, it is desired to efficiently image single dendritic spines that underwent structural plasticity in electron microscopy. We induced structural long-term potentiation (sLTP) in single dendritic spines by two-photon glutamate uncaging. We then identified the same spines at different phases of sLTP and performed ultrastructural analysis by using an efficient correlative light and electron microscopy method. We found that postsynaptic density undergoes dramatic modification in its structural complexity immediately after sLTP induction. Meanwhile, the nonsynaptic axon–spine interface area shows a rapid and sustained increase throughout sLTP. Our results indicate that the uneven modification of synaptic and nonsynaptic postsynaptic membrane might contribute to rapid electrophysiological plasticity during sLTP.