Spine Motility Research Articles

A computational approach to dendritic spine motility with calcium signaling by the immersed boundary method with advection-electrodiffusion

Dendritic spines are small protrusions from the dendritic branches of neurons. Influenced by internal and external signals and forces, even adult spines are not static but dynamically move. In this paper, we consider actomyosin-based spine motility with calcium signaling. The simulation begins with influx of calcium ions through glutamate receptors. Calcium Induced Calcium Release (CICR) with IP3 (inositol-1,4,5-trisphosphate) dynamics is also considered. The sensitivity of elasticity of actomyosin network is assumed to follow a Hill-type function of Ca2+ concentration. Several combinations in size of spine head and neck, physiology of Endoplasmic Reticulum (ER), and distribution of receptor/channels/exchangers are considered. Different functions of a spine as absorber, pumper and/or diffuser are observed. The computational framework used for these studies is the immersed boundary method with advection-electrodiffusion.

Getting hot under the calyx

Most mammals are homeothermic, and finely tune their core body temperature within a narrow range (36–39°C). Unsurprisingly, brain function is temperature dependent and it is thought that brain cooling can ameliorate damage following trauma, such as stroke. A variety of structural and physiological properties of neurons are influenced by temperature, including spine morphology and motility, vesicular release of neurotransmitter, action potential generation, and the kinetics of neurotransmitter receptors and transporters (Klyachko & Stevens, 2006). However, a full description of the effects of temperature at central synapses is still missing. What is more, the majority of in vitro studies are conducted at room temperature to improve tissue viability and facilitate electrophysiological recordings. A drawback of this is that the interpretation of the results may not hold in the warmer surroundings of the functioning brain.

α5 Integrin Signaling Regulates the Formation of Spines and Synapses in Hippocampal Neurons

The actin-based dynamics of dendritic spines play a key role in synaptic plasticity, which underlies learning and memory. Although it is becoming increasingly clear that modulation of actin is critical for spine dynamics, the upstream molecular signals that regulate the formation and plasticity of spines are poorly understood. In non-neuronal cells, integrins are critical modulators of the actin cytoskeleton, but their function in the nervous system is not well characterized. Here we show that alpha5 integrin regulates spine morphogenesis and synapse formation in hippocampal neurons. Knockdown of alpha5 integrin expression using small interfering RNA decreased the number of dendritic protrusions, spines, and synapses. Expression of constitutively active or dominant negative alpha5 integrin also resulted in alterations in the number of dendritic protrusions, spines, and synapses. alpha5 integrin signaling regulates spine morphogenesis and synapse formation by a mechanism that is dependent on Src kinase, Rac, and the signaling adaptor GIT1. Alterations in the activity or localization of these molecules result in a significant decrease in the number of spines and synapses. Thus, our results point to a critical role for integrin signaling in regulating the formation of dendritic spines and synapses in hippocampal neurons.

Effects of N-cadherin disruption on spine morphological dynamics

Structural changes at synapses are thought to be a key mechanism for the encoding of memories in the brain. Recent studies have shown that changes in the dynamic behavior of dendritic spines accompany bidirectional changes in synaptic plasticity, and that the disruption of structural constraints at synapses may play a mechanistic role in spine plasticity. While the prolonged disruption of N-cadherin, a key synaptic adhesion molecule, has been shown to alter spine morphology, little is known about the short-term regulation of spine morphological dynamics by N-cadherin. With time-lapse, confocal imaging in cultured hippocampal neurons, we examined the progression of structural changes in spines following an acute treatment with AHAVD, a peptide known to interfere with the function of N-cadherin. We characterized fast and slow timescale spine dynamics (minutes and hours, respectively) in the same population of spines. We show that N-cadherin disruption leads to enhanced spine motility and reduced length, followed by spine loss. The structural effects are accompanied by a loss of functional connectivity. Further, we demonstrate that early structural changes induced by AHAVD treatment, namely enhanced motility and reduced length, are indicators for later spine fate, i.e., spines with the former changes are more likely to be subsequently lost. Our results thus reveal the short-term regulation of synaptic structure by N-cadherin and suggest that some forms of morphological dynamics may be potential readouts for subsequent, stimulus-induced rewiring in neuronal networks.

Time-lapse imaging of dendritic spines in vitro

Dendritic spines are small protrusions present postsynaptically at approximately 90% of excitatory synapses in the brain. Spines undergo rapid spontaneous changes in shape that are thought to be important for alterations in synaptic connectivity underlying learning and memory. Visualization of these dynamic changes in spine morphology are especially challenging because of the small size of spines (approximately 1 microm). Here we describe a microscope system, based on a spinning-disk confocal microscope, suitable for imaging mature dendritic spines in brain slice preparations, with a time resolution of seconds. We discuss two commonly used in vitro brain slice preparations and methods for transfecting them. Preparation and transfection require approximately 1 d, after which slices must be cultured for at least 21 d to obtain spines of mature morphology. We also describe imaging and computer analysis routines for studying spine motility. These procedures require in the order of 2 to 4 h.

Amyloid β peptide adversely affects spine number and motility in hippocampal neurons

Elevated levels of amyloid-beta peptide (Aβ) are found in Down’s syndrome patients and alter synaptic function during the early stages of Alzheimer’s disease. Dendritic spines, sites of most excitatory synaptic contacts, are considered to be an important locus for encoding synaptic plasticity. We used time-lapse two-photon imaging of hippocampal pyramidal neurons in organotypic slices to study the effects of Aβ on the development of dendritic spines. We report that exposure of hippocampal neurons to sub-lethal levels of Aβ decreased spine density, increased spine length and subdued spine motility. The effect of Aβ on spine density was reversible. Moreover, Aβ’s effect on dendritic spine density was blocked by rolipram, a phosphodiesterase type IV inhibitor, suggesting the involvement of a cAMP dependent pathway. These findings raise the possibility that Aβ-induced spine alterations could underlie the cognitive defects in Alzheimer’s disease and Down syndrome.

Parapharyngealabszess und Osteomyelitis im Bereich des Dens axis

We report the case of a 55 year old man with osteomyelitic destruction of the odontoid process and a parapharyngeal abscess. The patient was admitted with diagnosis of meningitis and degenerative cervical spine disease without compression of spinal cord or nerve roots but progressive impairment of cervical spine mobility. Inflammatory parameters in serum and cerebrospinal fluid were detected and antibiotic therapy was initiated resulting in subjective improvement of symptoms. When impairment of cervical spine motility persisted and gait disturbance developed, a parapharyngeal abscess and an osteomyelitic destruction of the odontoid process caused by infection with staphylococcus aureus was diagnosed. After cleavage of the abscess and four months of antibiotic therapy the gait disturbance disappeared and mild impairment of cervical spine motility persisted.

Remodeling of Synaptic Structure in Sensory Cortical AreasIn Vivo

Although plastic changes are known to occur in developing and adult cortex, it remains unclear whether these changes require remodeling of cortical circuitry whereby synapses are formed and eliminated or whether they rely on changes in the strength of existing synapses. To determine the structural stability of dendritic spines and axon terminals in vivo, we chose two approaches. First, we performed time-lapse two-photon imaging of dendritic spine motility of layer 5 pyramidal neurons in juvenile [postnatal day 28 (P28)] mice in visual, auditory, and somatosensory cortices. We found that there were differences in basal rates of dendritic spine motility of the same neuron type in different cortices, with visual cortex exhibiting the least structural dynamics. Rewiring visual input into the auditory cortex at birth, however, failed to alter dendritic spine motility, suggesting that structural plasticity rates might be intrinsic to the cortical region. Second, we investigated the persistence of both the presynaptic (axon terminals) and postsynaptic (dendritic spine) structures in young adult mice (P40-P61), using chronic in vivo two-photon imaging in different sensory areas. Both terminals and spines were relatively stable, with >80% persisting over a 3 week period in all sensory regions. Axon terminals were more stable than dendritic spines. These data suggest that changes in network function during adult learning and memory might occur through changes in the strength and efficacy of existing synapses as well as some remodeling of connectivity through the loss and gain of synapses.

Estrogen Alters Spine Number and Morphology in Prefrontal Cortex of Aged Female Rhesus Monkeys

Long-term cyclic treatment with 17beta-estradiol reverses age-related impairment in ovariectomized rhesus monkeys on a test of cognitive function mediated by the prefrontal cortex (PFC). Here, we examined potential neurobiological substrates of this effect using intracellular loading and morphometric analyses to test the possibility that the cognitive benefits of hormone treatment are associated with structural plasticity in layer III pyramidal cells in PFC area 46. 17beta-Estradiol did not affect several parameters such as total dendritic length and branching. In contrast, 17beta-estradiol administration increased apical and basal dendritic spine density, and induced a shift toward smaller spines, a response linked to increased spine motility, NMDA receptor-mediated activity, and learning. These results document that, although the aged primate PFC is vulnerable in the absence of factors such as circulating estrogens, it remains responsive to long-term cyclic 17beta-estradiol treatment, and that increased dendritic spine density and altered spine morphology may contribute to the cognitive benefits of such treatment.

A Critical Role for Myosin IIB in Dendritic Spine Morphology and Synaptic Function

Dendritic spines show rapid motility and plastic morphology, which may mediate information storage in the brain. It is presently believed that polymerization/depolymerization of actin is the primary determinant of spine motility and morphogenesis. Here, we show that myosin IIB, a molecular motor that binds and contracts actin filaments, is essential for normal spine morphology and dynamics and represents a distinct biophysical pathway to control spine size and shape. Myosin IIB is enriched in the postsynaptic density (PSD) of neurons. Pharmacologic or genetic inhibition of myosin IIB alters protrusive motility of spines, destabilizes their classical mushroom-head morphology, and impairs excitatory synaptic transmission. Thus, the structure and function of spines is regulated by an actin-based motor in addition to the polymerization state of actin.

Physiological roles of spine motility: development, plasticity and disorders.

The vast majority of excitatory connections in the hippocampus are made on dendritic spines. Both dendritic spines and molecules within the membrane are able to move, but the physiological role of these movements is unclear. In the developing brain, spines show highly dynamic behaviour thought to facilitate new synaptic connections. Dynamic movements also occur in adults but the role of this movement is unclear. We have studied the effects of the most important excitatory neurotransmitter, glutamate, and found receptor activation to enhance movement of molecules within the spine membrane. This action of glutamate may be important in regulating the trafficking of neurotransmitter receptors that mediate change in synaptic function. In addition, we have studied the dynamic interactions between pre- and postsynaptic structures labelled with FM 4-64 and a membrane-targeted GFP (green fluorescent protein), respectively, in hippocampal slice cultures under conditions of increased activity, such as epilepsy. Our findings suggest a novel form of activity-dependent synaptic plasticity where spontaneous glutamate release is sufficient to trigger changes in the hippocampal microcircuitry by attracting neighbouring spines responsive to an enhanced level of extracellular glutamate.

Physiological roles of spine motility: development, plasticity and disorders

The vast majority of excitatory connections in the hippocampus are made on dendritic spines. Both dendritic spines and molecules within the membrane are able to move, but the physiological role of these movements is unclear. In the developing brain, spines show highly dynamic behaviour thought to facilitate new synaptic connections. Dynamic movements also occur in adults but the role of this movement is unclear. We have studied the effects of the most important excitatory neurotransmitter, glutamate, and found receptor activation to enhance movement of molecules within the spine membrane. This action of glutamate may be important in regulating the trafficking of neurotransmitter receptors that mediate change in synaptic function. In addition, we have studied the dynamic interactions between pre- and postsynaptic structures labelled with FM 4-64 and a membrane-targeted GFP (green fluorescent protein), respectively, in hippocampal slice cultures under conditions of increased activity, such as epilepsy. Our findings suggest a novel form of activity-dependent synaptic plasticity where spontaneous glutamate release is sufficient to trigger changes in the hippocampal microcircuitry by attracting neighbouring spines responsive to an enhanced level of extracellular glutamate.

Actin Redistribution Underlies the Sparing Effect of Mild Hypothermia on Dendritic Spine Morphology after in Vitro Ischemia

Brain hypothermia is at present the most effective neuroprotective treatment against brain ischemia in man. Ischemia induces a redistribution of proteins involved in synaptic functions, which is markedly diminished by therapeutic hypothermia (33 degrees C). Dendritic spines at excitatory synapses are motile and show both shape changes and rearrangement of synaptic proteins as a consequence of neuronal activity. We investigated the effect of reduced temperature (33 degrees C and 27 degrees C compared with 37 degrees C), on spine motility, length and morphology by studying the distribution of GFP-actin before, during and after induction of in vitro ischemia. Because high-concentration actin filaments are located inside spines, dissociated hippocampal neurons (7-11 DIV) from transgenic mice expressing GFP-actin were used in this study. The movement of the spines and the distribution of GFP-actin were recorded using time-lapse fluorescence microscopy. Under normal conditions rapid rearrangement of GFP-actin was seen in dendritic spines, indicating highly motile spines at 37 degrees C. Decreasing the incubation temperature to 33 degrees C or 27 degrees C, dramatically reduces actin dynamics (spine motility) by approximately 50% and 70%, respectively. In addition, the length of the spine shaft was reduced by 20%. We propose that decreasing the temperature from 37 degrees C to 33 degrees C during ischemia decreases the neuronal actin polymerization rate, which reduces spine calcium kinetics, disrupts detrimental cell signaling and protects neurons against damage.

Effects of Synaptic Activity on Dendritic Spine Motility of Developing Cortical Layer V Pyramidal Neurons

It is increasingly clear that dendritic spines play an important role in compartmentalizing post-synaptic signals and that their dynamic morphological properties have functional consequences. Here, we examine this issue using two-photon microscopy to characterize spine motility on layer V pyramidal neurons in acute slices of the developing mouse cortex. In this system, all spine classes except filopodia become less dynamic as development proceeds. General manipulations of activity (TTX or KCl treatment) do not alter spine dynamics, although increased glutamatergic transmission (AMPA or NMDA treatment) stabilizes developing cortical spines. These effects on spine dynamics do not appear to be related to AMPA or NMDA receptor expression as assessed with immunolabeling, as there is no correlation between spine motility and AMPA (GluR1/2) or NMDA (NR1/NR2B) receptor subunit expression on a spine by spine basis. These results indicate that activity through glutamatergic synapses is important for regulating spine motility in the developing mouse cortex, and that the relative complement of receptors, while different across morphological classifications, cannot account for differences in dynamic structural changes in dendritic spines.

The Rho-Specific GEF Lfc Interacts with Neurabin and Spinophilin to Regulate Dendritic Spine Morphology

Neurabin and spinophilin are homologous protein phosphatase 1 and actin binding proteins that regulate dendritic spine function. A yeast two-hybrid analysis using the coiled-coil domain of neurabin revealed an interaction with Lfc, a Rho GEF. Lfc was highly expressed in brain, where it interacted with either neurabin or spinophilin. In neurons, Lfc was largely found in the shaft of dendrites in association with microtubules but translocated to spines upon neuronal stimulation. Moreover, expression of Lfc resulted in reduction in spine length and size. Both the translocation and the effect on spine morphology depended on the coiled-coil domain of Lfc. Coexpression of neurabin or spinophilin with Lfc resulted in their clustering together with F-actin, a process that depended on Rho activity. Thus, interaction between Lfc and neurabin/spinophilin selectively regulates Rho-dependent organization of F-actin in spines and is a link between the microtubule and F-actin cytoskeletons in dendrites.

Calcium dynamics in dendritic spines, modeling and experiments

Dendritic spines are microstructures, about one femtoliter in volume, where excitatory synapses are made with incoming afferents, in most neurons of the vertebrate brain. The spine contains all the molecular constituents of the postsynaptic side of the synapse, as well as a contractile element that can cause its movement in space. It also contains calcium handling machineries to allow fast buffering of excess calcium that influx through voltage and NMDA gated channels. The spine is connected to the dendrite through a thin neck that serves as a variable barrier between the spine head and the parent dendrite. We review a novel modeling approach that is more suitable for the accurate description of the stochastic behavior of individual molecules in microstructures. Using this approach, we predict the calcium handling ability of the spine in complex situations associated with synaptic activity, spine motility and plasticity.

Dynamics of dendritic spines and their afferent terminals: spines are more motile than presynaptic boutons

Previous work has established that dendritic spines, sites of excitatory input in CNS neurons, can be highly dynamic, in later development as well as in mature brain. Although spine motility has been proposed to facilitate the formation of new synaptic contacts, we have reported that spines continue to be dynamic even if they bear synaptic contacts. An outstanding question related to this finding is whether the presynaptic terminals that contact dendritic spines are as dynamic as their postsynaptic targets. Using multiphoton time-lapse microscopy of GFP-labeled Purkinje cells and DiI-labeled granule cell parallel fiber afferents in cerebellar slices, we monitored the dynamic behavior of both presynaptic terminals and postsynaptic dendritic spines in the same preparation. We report that while spines are dynamic, the presynaptic terminals they contact are quite stable. We confirmed the relatively low levels of presynaptic terminal motility by imaging parallel fibers in vivo. Finally, spine motility can occur when a functional presynaptic terminal is apposed to it. These analyses further call into question the function of spine motility, and to what extent the synapse breaks or maintains its contact during the movement of the spine.

Imaging the motility of dendritic protrusions and axon terminals: roles in axon sampling and synaptic competition

Dendritic spines and filopodia display actin-based morphological plasticity. The function of this rapid motility is unknown. Its ubiquitous expression during development has led to the hypothesis that motility plays a role in synaptogenesis. We investigated this by simultaneously imaging presynaptic boutons and dendritic protrusions in acute hippocampal slices from GFP-M transgenic mice loaded with FM 1-43 followed by immunostaining. Postsynaptic motility was inversely correlated with the presence of stable synaptic contacts. Filopodia were highly motile and made transient interactions, whereas spines were less motile and had stable contacts, although they could still move together with a synaptic terminal. "Head morphing" of spines was associated with interactions with more than one presynaptic terminal. Our data indicate that filopodia motility could serve to transiently sample the surrounding neuropil, while the motility of established spines could mediate interactions with two axonal terminals. Spine "morphing" could therefore be the morphological signature for synaptic input competition in central synapses.

Induction of spine growth and synapse formation by regulation of the spine actin cytoskeleton.

We explored the relationship between regulation of the spine actin cytoskeleton, spine morphogenesis, and synapse formation by manipulating expression of the actin binding protein NrbI and its deletion mutants. In pyramidal neurons of cultured rat hippocampal slices, NrbI is concentrated in dendritic spines by binding to the actin cytoskeleton. Expression of one NrbI deletion mutant, containing the actin binding domain, dramatically increased the density and length of dendritic spines with synapses. This hyperspinogenesis was accompanied by enhanced actin polymerization and spine motility. Synaptic strengths were reduced to compensate for extra synapses, keeping total synaptic input per neuron constant. Our data support a model in which synapse formation is promoted by actin-powered motility.

Influx of extracellular calcium regulates actin-dependent morphological plasticity in dendritic spines

Dendritic spines contain a specialized cytoskeleton composed of dynamic actin filaments capable of producing rapid changes in their motility and morphology. Transient changes in Ca 2+ levels in the spine cytoplasm have been associated with the modulation of these effects in a variety of ways. To characterize the contribution of Ca 2+ fluxes originating through different pathways to these phenomena, we used time-lapse imaging of cultured hippocampal neurons expressing GFP-actin to follow the influence of postsynaptic neurotransmitter receptors, voltage-activated Ca 2+ channels and release from internal Ca 2+ stores on spine actin dynamics. Stimulation of AMPA receptors produced a rapid blockade of actin-dependent spine motility that was immediately reversible when AMPA was removed. Stimulation of NMDA receptors also blocked spine motility but in this case suppression of actin dynamics was delayed by up to 30 min depending on NMDA concentration and motility was never seen to recover when NMDA was removed. These effects could be mimicked by depolarizing neurons under appropriate circumstances demonstrating the involvement of voltage-activated Ca 2+ channels in AMPA receptor-mediated effects and the receptor associated Ca 2+ channel in the effects of NMDA. Caffeine, an agent that releases Ca 2+ from internal stores, had no immediate effect on spine actin, a result compatible with the lack of caffeine-releasable Ca 2+ in cultured hippocampal neurons under resting conditions. Blocking internal store function by thapsigargin led to a delayed suppression of spine actin dynamics that was dependent on extracellular Ca 2+. Together these results indicate the common involvement of changes in Ca 2+ levels in modulating actin-dependent effects on dendritic spine motility and morphology through several modes of electrophysiological activation.