Quantitative analysis of electrical properties of dendritic spines

Several sugestions have been made with regard to the functional significance of dendritic spines in connection with synaptic plasticity. We have shown that for a constant synaptic current, when the synaptic resistance is large compared to the spine-stem resistance, a morphological change in the spine does not produce a marked change in the postsynaptic potential (PSP). When the synaptic resistance is comparable to the spine-stem impedance a morphological change in the spine can induce changes in the synaptic current and the PSP due to the so-called nonlinear effect to the synapse (Kawato and Tsukahara, 1983, 1984). Consequently, in a study of the electrical properties of dendritic spines the input impedance of the parent dendrite, the spinestalk conductance and the conductance change associated with synaptic activity must be considered. We quantitatively estimated all three factors. By comparing electrophysiological data with morphological data, we estimated the synaptic conductance which causes corticorubral EPSP. Its maximum amplitude was 43 nS with a time-to-peak value of 0.3 ms. With this value, the effects of the spine were examined using an improved algorithm based on that of Butz and Cowan (1974). It uses a three-dimensional morphology of the rubrospinal (RS) neurons, which was reconstructed from serial sections containing HRP-filled RS cells. As the spine shortens, the amplitude of the EPSP becomes considerably larger, but its time-to-peak value does not markedly change. Moreover, if unitary EPSP in the RS cell is produced by the activation of several synaptic terminals a morphological change of the spine has a smaller effect on the EPSPs.     

Electrical advantages of dendritic spines

Many neurons receive excitatory glutamatergic input almost exclusively onto dendritic spines. In the absence of spines, the amplitudes and kinetics of excitatory postsynaptic potentials (EPSPs) at the site of synaptic input are highly variable and depend on dendritic location. We hypothesized that dendritic spines standardize the local geometry at the site of synaptic input, thereby reducing location-dependent variability of local EPSP properties. We tested this hypothesis using computational models of simplified and morphologically realistic spiny neurons that allow direct comparison of EPSPs generated on spine heads with EPSPs generated on dendritic shafts at the same dendritic locations. In all morphologies tested, spines greatly reduced location-dependent variability of local EPSP amplitude and kinetics, while having minimal impact on EPSPs measured at the soma. Spine-dependent standardization of local EPSP properties persisted across a range of physiologically relevant spine neck resistances, and in models with variable neck resistances. By reducing the variability of local EPSPs, spines standardized synaptic activation of NMDA receptors and voltage-gated calcium channels. Furthermore, spines enhanced activation of NMDA receptors and facilitated the generation of NMDA spikes and axonal action potentials in response to synaptic input. Finally, we show that dynamic regulation of spine neck geometry can preserve local EPSP properties following plasticity-driven changes in synaptic strength, but is inefficient in modifying the amplitude of EPSPs in other cellular compartments. These observations suggest that one function of dendritic spines is to standardize local EPSP properties throughout the dendritic tree, thereby allowing neurons to use similar voltage-sensitive postsynaptic mechanisms at all dendritic locations.     

Structural plasticity of dendritic spines

Dendritic spines are the major targets of excitatory synaptic input. They exist in a wide variety of shapes and sizes, from thin to mushroom-shaped to stubby. One of the striking characteristics of dendritic spines is their motile nature. Spines can undergo various structural modifications such as changes in density, shape, size, and motility. During development, spines are highly dynamic and many spines are formed and eliminated. As animals mature, most spines become stable and the vast majority of them can last throughout life. However, spine morphology can still undergo progressive changes. Structural dynamics of dendritic spines is thought to play important roles in synapse plasticity and information processing. Abnormal spine structures are often associated with malfunction of the nervous system.     

Structural plasticity of dendritic spines

Dendritic spines are small mushroom-like protrusions arising from neurons where most excitatory synapses reside. Their peculiar shape suggests that spines can serve as an autonomous postsynaptic compartment that isolates chemical and electrical signaling. How neuronal activity modifies the morphology of the spine and how these modifications affect synaptic transmission and plasticity are intriguing issues. Indeed, the induction of long-term potentiation (LTP) or depression (LTD) is associated with the enlargement or shrinkage of the spine, respectively. This structural plasticity is mainly controlled by actin filaments, the principal cytoskeletal component of the spine. Here we review the pioneering microscopic studies examining the structural plasticity of spines and propose how changes in actin treadmilling might regulate spine morphology.     

MARCKS for maintenance in dendritic spines

Synapses in the brain must maintain a balance between learning-related plasticity and the stability necessary for reliable function. In this issue of Neuron, Calabrese and Halpain describe cell-transfection experiments implicating MARCKS, a protein that binds to both the cell surface and actin cytoskeleton, in the maintenance of dendritic spines.     

Cytoplasmic organization in cerebellar dendritic spines

Three sets of filamentous structures were found to be associated with synaptic junctions in slices of cerebellar tissue prepared by rapid- freezing and freeze-etch techniques. The electron-dense fuzz subjacent to postsynaptic membranes corresponds to a web of 4-6-nm-diam filaments that were clearly visualized in rapid-frozen, freeze-etched preparations. Purkinje cell dendritic spines are filled with a meshwork of 5-7-nm filaments that were found to contact the spine membrane everywhere except at the synaptic junction, and extend through the neck of the spine into the parent dendrite. In addition, 8-10-nm microfilaments, possibly actin, were seen to be associated with the postsynaptic web and to extend into the body and neck of the spine. The arrangements and attachments of the filamentous elements in the Purkinje cell dendritic spine may account for its shape.     

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.     

Spatiotemporal maps of CaMKII in dendritic spines

The calcium calmodulin dependent kinase (CaMKII) is important for long-term potentiation at dendritic spines. Photo-activatable GFP (PaGFP) - CaMKII fusions were used to map CaMKII movements between and within spines in dissociated hippocampal neurons. Photo-activated PaGFP (GFP*) generated in the shaft spread uniformly, but was retained for about 1?s in spines. The differential localization of GFP*-CaMKII isoforms was visualized with hundred nanometer precision frame to frame using de-noising algorithms. GFP*-CaMKIIα localized to the tips of mushroom spines. The spatiotemporal profiles of native and kinase defective GFP*-CaMKIIβ, differed markedly from GFP*-CaMKIIα and mutant GFP*-CaMKIIβ lacking the association domain. CaMKIIβ bound to cortical actin in the dendrite and the stable actin network in spine bodies. Glutamate produced a transiently localized GFP*-CaMKIIα fraction and a soluble GFP*-CaMKIIβ fraction in spine bodies. Single molecule simulations of the interplay between diffusion and biochemistry of GFP* species were guided by the spatiotemporal maps and set limits on binding parameters. They highlighted the role of spine morphology in modulating bound CaMKII lifetimes. The long residence times of GFP*-CaMKIIβ relative to GFP*-CaMKIIα followed as consequence of more binding sites on the actin cytoskeleton than the post-synaptic density. These factors combined to retain CaMKII for tens of seconds, sufficient to outlast the calcium transients triggered by glutamate, without invoking complex biochemistry.     

Loss of dendritic spines in Alzheimer's disease

Neurones from five patients with Alzheimer's disease were studied semi-quantitatively on Golgi impregnated samples of cerebral cortex. They showed a dramatic reduction in dendritic spine number of pyramidal and non-pyramidal cells of all neocortical layers, as compared with control samples matched for age. Dendritic spine loss could be an initial phenomenon in Alzheimer's disease pathology.     

Signaling in dendritic spines and spine microdomains

The specialized morphology of dendritic spines creates an isolated compartment that allows for localized biochemical signaling. Recent studies have revealed complexity in the function of the spine head as a signaling domain and indicate that (1) the spine is functionally subdivided into multiple independent microdomains and (2) not all biochemical signals are equally compartmentalized within the spine. Here we review these findings as well as the developments in fluorescence microscopy that are making possible direct monitoring of signaling within spines and, in the future, within sub-spine microdomains.     

Actin filaments and microtubules in dendritic spines

Dendritic spines are small protrusions emerging from their parent dendrites, and their morphological changes are involved in synaptic plasticity. These tiny structures are composed of thousands of different proteins belonging to several subfamilies such as membrane receptors, scaffold proteins, signal transduction proteins, and cytoskeletal proteins. Actin filaments in dendritic spines consist of double helix of actin protomers decorated with drebrin and ADF/cofilin, and the balance of the two is closely related to the actin dynamics, which may govern morphological and functional synaptic plasticity. During development, the accumulation of drebrin‐binding type actin filaments is one of the initial events occurring at the nascent excitatory postsynaptic site, and plays a pivotal role in spine formation as well as small GTPases. It has been recently reported that microtubules transiently appear in dendritic spines in correlation with synaptic activity. Interestingly, it is suggested that microtubule dynamics might couple with actin dynamics. In this review, we will summarize the contribution of both actin filaments and microtubules to the formation and regulation of dendritic spines, and further discuss the role of cytoskeletal deregulation in neurological disorders.     

Calcium regulation of actin dynamics in dendritic spines

Most excitatory synapses in the brain are made on spines, small protrusions from dendrites that exist in many different shapes and sizes. Spines are highly motile, a process that reflects rapid rearrangements of the actin cytoskeleton inside the spine, and can also change shape and size over longer timescales. These different forms of morphological plasticity are regulated in an activity-dependent way, involving calcium influx through glutamate receptors and voltage-gated calcium channels. Many proteins regulating the turnover of filamentous actin (F-actin) are calcium-dependent and might transduce intracellular calcium levels into spine shape changes. On the other hand, the morphology of a spine might affect the function of the synapse residing on it. In particular, the induction of synaptic plasticity is known to require large elevations in the postsynaptic calcium concentration, which depend on the ability of the spine to compartmentalize calcium. Since the actin cytoskeleton is also known to anchor postsynaptic glutamate receptors, changes in the actin polymerization state have the potential to influence synaptic function in a number of ways. Here we review the most prominent types of changes in spine morphology in hippocampal pyramidal cells with regard to their calcium-dependence and discuss their potential impact on synaptic function.     

Calcium signaling in dendritic spines of hippocampal neurons

Intracellular Ca²⁺ dynamics have been measured using imaging techniques in dendrites and spines of CA3 hippocampal neurons in brain slice under both acute and tissue culture conditions. In response to presynatic stimulation, micromolar levels of Ca²⁺ are rapidly reached in spines of distal dendrites. If stimulus parameters are chosen judiciously so as to minimize postsynaptic firing, then the dendrite shaft increases are far less. Spine Ca²⁺ increases are largely dependent upon activation of NMDA receptors. At the large mossy fiber synapses, presynaptic stimuli also produce large Ca²⁺ increases but the differences in shaft-spine Ca²⁺ levels are much less; often they are insignificant. Also at these locations, postsynaptic firing, without presynaptic stimulation is sufficient to produce large increase in spine Ca²⁺ levels. 1994 John Wiley & Sons, Inc.     

Structure and molecular organization of dendritic spines

Dendritic spines mediate most excitatory synapses in the CNS and are therefore likely to be of major importance for neural processing. We review the structural aspects of dendritic spines, with particular emphasis on recent advances in the characterization of their molecular components. Spine morphology is very diverse and spine size is correlated with the strength of the synaptic transmission. In addition, the spine neck biochemically isolates individual synapses. Therefore, spine morphology directly reflects its function. A large number of molecules have been described in spines, involving several biochemical families. Considering the small size of a spine, the variety of molecules found is astounding, suggesting that spines are paramount examples of biological nanotechnology. Single-molecular studies appear necessary for future progress. The purpose of this rich molecular diversity is still mysterious but endows synapses with a diverse and flexible biochemical machinery.     

Ca(2+) signaling in dendritic spines

Dendritic spines are cellular microcompartments that are isolated from their parent dendrites and neighboring spines. Recently, imaging studies of spine Ca(2+) dynamics have revealed that Ca(2+) can enter spines through voltage-sensitive and ligand-activated channels, as well as through Ca(2+) release from intracellular stores. Relationships between spine Ca(2+) signals and induction of various forms of synaptic plasticity are beginning to be elucidated. Measurements of spine Ca(2+) concentration are also being used to probe the properties of single synapses and even individual calcium channels in their native environment.     

Ca(2+) signaling in dendritic spines

Recent studies have revealed that Ca(2+) signals evoked by action potentials or by synaptic activity within individual dendritic spines are regulated at multiple levels. Ca(2+) influx through glutamate receptors and voltage-sensitive Ca(2+) channels located on spines depends on the channel subunit composition, the activity of kinases and phosphatases, the local membrane potential and past patterns of activity. Furthermore, sources of spine Ca(2+) interact nonlinearly such that activation of one Ca(2+) channel can enhance or depress the activity of others. These studies have revealed that each spine is a complex and partitioned Ca(2+) signaling domain capable of autonomously regulating the electrical and biochemical consequences of synaptic activity.     

Calcium dynamics in dendritic spines and spine motility

A dendritic spine is an intracellular compartment in synapses of central neurons. The role of the fast twitching of spines, brought about by a transient rise of internal calcium concentration above that of the parent dendrite, has been hitherto unclear. We propose an explanation of the cause and effect of the twitching and its role in the functioning of the spine as a fast calcium compartment. Our molecular model postulates that rapid spine motility is due to the concerted contraction of calcium-binding proteins. The contraction induces a stream of cytoplasmic fluid in the direction of the dendritic shaft, thus speeding up the time course of spine calcium dynamics, relative to pure diffusion. Simulations indicate that chemical reaction rate theory at the molecular level can explain spine motility. They reveal two time periods in calcium dynamics, as measured recently by other researchers. It appears that rapid motility in dendritic spines increases the efficiency of calcium conduction to the dendrite and speeds up the emptying of the spine. This could play a major role in the induction of synaptic plasticity. A prediction of the model is that alteration of spine motility will modify the time course of calcium in the dendritic spine and could be tested experimentally.     

LOTOS-based two-photon calcium imaging of dendritic spines in vivo

Neurons in the mammalian brain receive thousands of synaptic inputs on their dendrites. In many types of neurons, such as cortical pyramidal neurons, excitatory synapses are formed on fine dendritic protrusions called spines. Usually, an individual spine forms a single synaptic contact with an afferent axon. In this protocol, we describe a recently established experimental procedure for measuring intracellular calcium signals from dendritic spines in cortical neurons in vivo by using a combination of two-photon microscopy and whole-cell patch-clamp recordings. We have used mice as an experimental model system, but the protocol may be readily adapted to other species. This method involves data acquisition at high frame rates and low-excitation laser power, and is termed low-power temporal oversampling (LOTOS). Because of its high sensitivity of fluorescence detection and reduced phototoxicity, LOTOS allows for prolonged and stable calcium imaging in vivo. Key aspects of the protocol, which can be completed in 5-6 h, include the use of a variant of high-speed two-photon imaging, refined surgery procedures and optimized tissue stabilization.