Postsynaptic signaling during plasticity of dendritic spines

Dendritic spines, small bulbous postsynaptic compartments emanating from neuronal dendrites, have been thought to serve as basic units of memory storage. Despite their small size (~0.1 femtoliter), thousands of species of proteins exist in the spine, including receptors, channels, scaffolding proteins and signaling enzymes. Biochemical signaling mediated by these molecules leads to morphological and functional plasticity of dendritic spines, and ultimately learning and memory in the brain. Here, we review new insights into the mechanisms underlying spine plasticity brought about by recent advances in imaging techniques to monitor molecular events in single dendritic spines. The activity of each protein displays a specific spatiotemporal pattern, coordinating downstream events at different microdomains to change the function and morphology of dendritic spines.     

Regulation of hippocampal dendritic spines following sleep deprivation

Accumulating evidence supports the role of sleep in synaptic plasticity and memory consolidation. One line of investigation, the synaptic homeostasis hypothesis, has emphasized the increase in synaptic strength during waking, and compensatory downsizing of (presumably less frequently used) synapses during sleep. Conversely, other studies have reported downsizing and loss of dendritic spines following sleep deprivation. We wanted to determine the effect of sleep deprivation on dendritic spines of hippocampal CA1 neurons using genetic methods for fluorescent labeling of dendritic spines. Male Vglut2-Cre mice were injected with an AAV-DIO-ChR2-mCherry reporter in CA1 hippocampus. Gentle handling was used to sleep deprive mice for 5 hr, from lights on (7 am ) to 12 noon. Control and sleep-deprived mice were euthanized at 12 noon and processed for quantification of dendritic spines. We used confocal microscope imaging and three-dimensional (3D) analysis to quantify thin, mushroom, and stubby spines from CA1 dendrites, distinguishing between branch segments. We observed significantly greater density of spines in CA1 of sleep-deprived mice, driven primarily by greater numbers of thin spines, and significantly larger spine volume and head diameter. Branch and region-specific analysis revealed that spine volume was greater in primary dendrites of apical and basal segments, along with proximal segments on both apical and basal dendrites, and spine density was increased in secondary branches and distal segments on apical dendrites following sleep deprivation. Our 3D quantification suggests sleep contributes to region- and branch-specific synaptic downscaling in the hippocampus, supporting the theory of broad but selective synaptic downscaling during sleep.     

On the electrical function of dendritic spines

Dendritic spines mediate most excitatory inputs in the brain, yet their function is still unclear. Imaging experiments have demonstrated their role in biochemical compartmentalization at individual synapses, yet theoretical studies have suggested that they could serve an electrical function in transforming synaptic inputs and transmitting dendritic spikes. Recent data indicate that spines possess voltage-dependent conductances and that these channels can be spine-specific. Although direct experimental investigations of the electrical properties of spines have not yet taken place, spines could play a significant electrical role, greatly influencing dendritic integration and the function of neural circuits.     

Contributions of dendritic spines and perforated synapses to synaptic plasticity

The dynamic nature of synaptic connections has presented morphologists with considerable problems which, from a structural perspective, have frustrated the development of ideas on synaptic plasticity. Gradually, however, progress has been made on concepts such as the structural remodelling and turnover of synapses. This has been considerably helped by the recent elaboration of unbiased stereological procedures. The major emphasis of this review is on naturally occurring synaptic plasticity, which is regarded as an ongoing process in the postdevelopmental CNS. The focus of attention are PSs, with their characteristically discontinuous synaptic active zone, since there is mounting evidence that this synaptic type is indicative of synaptic remodelling and turnover in the mature CNS. Since the majority of CNS synapses can only be considered in terms of their relationship to dendritic spines, the contribution of these spines to synaptic plasticity is discussed initially. Changes in the configuration of these spines appears to be crucial for the plasticity, and these can be viewed in terms of the significance of the cytoskeleton, of various dendritic organelles, and also of the biophysical properties of spines. Of the synaptic characteristics that may play a role in synaptic plasticity, the PSD, synaptic curvature, the spinule, coated vesicles, polyribosomes, and the spine apparatus have all been implicated. Each of these is assessed. Especial emphasis is placed on PSs because of their ever-increasing significance in discussions of synaptic plasticity. The possibility of their being artefacts is dismissed on a number of grounds, including consideration of the results of serial section studies. Various roles, other than one in synaptic plasticity have been put forward in discussing PSs. Although relevant to synaptic plasticity, these include a role in increasing synaptic efficacy, as a more permanent type of synaptic connection, or as a route for the intercellular exchange of metabolites or membrane components. The consideration of many estimates of synaptic density, and of PS frequency, have proved misleading, since studies have reported diverse and sometimes low figures. A recent reassessment of PS frequency, using unbiased stereological procedures, has provided evidence that in some brain regions PSs may account for up to 40% of all synapses. All ideas that have been put forward to date regarding the role of PSs are examined, with particular attention being devoted to the major models of Nieto-Sampedro and co-workers, Carlin and Siekevitz, and Dyson and Jones. New ideas based on recent analysis of quantitative data and of 3-dimensional reconstructions of PSs are discussed. According to these, PSs constitute a separate, and more enduring, component of the synaptic population than NPSs, and have a major role in the maintenance of synaptic efficacy in the mature CNS.     

Postpubertal decrease in hippocampal dendritic spines of female rats

Hippocampal dendritic spine and synapse numbers in female rats vary across the estrous cycle and following experimental manipulation of hormone levels in adulthood. Based on behavioral studies demonstrating that learning patterns are altered following puberty, we hypothesized that dendritic spine number in rat hippocampal CA1 region would change postpubertally. Female Sprague-Dawley rats were divided into prepubertal (postnatal day (P) 22), peripubertal (P35) and postpubertal (P49) groups, with the progression of puberty evaluated by vaginal opening, and estrous cyclicity subsequently assessed by daily vaginal smears. Spinophilin immunoreactivity in dendritic spines was used as an index of spinogenesis in area CA1 stratum radiatum (CA1sr) of hippocampus. First, electron microscopy analyses confirmed the presence of spinophilin specifically in dendritic spines of CA1sr, supporting spinophilin as a reliable marker of hippocampal spines in young female rats. Second, stereologic analysis was performed to assess the total number of spinophilin-immunoreactive puncta (i.e. spines) and CA1sr volume in developing rats. Our results indicated that the number of spinophilin-immunoreactive spines in CA1sr was decreased 46% in the postpubertal group compared to the two younger groups, whereas the volume of the hippocampus underwent an overall increase during this same developmental time frame. Third, to determine a potential role of estradiol in this process, an additional group of rats was ovariectomized (OVX) prepubertally at P22, then treated with estradiol or vehicle at P35, and spinophilin quantified as above in rats perfused on P49. No difference in spinophilin puncta number was found in OVX rats between the two hormone groups, suggesting that this developmental decrease is independent of peripheral estradiol. These changes in spine density coincident with puberty may be related to altered hippocampal plasticity and synaptic consolidation at this phase of maturity.     

Homeostatic plasticity during alcohol exposure promotes enlargement of dendritic spines

Modifications of the size, shape and number of dendritic spines is thought to be an important component of activity-dependent changes of neuronal circuits, and may play an important role in the plasticity of drug addiction. The present study examined whether homeostatic increases in synaptic N-methyl-d-aspartate (NMDA) receptors in response to chronic ethanol exposure is associated with corresponding morphological changes in dendritic spines. Prolonged exposure of rat hippocampal cultures to either the NMDA receptor antagonist d(-)-2-amino-5-phosphono-pentanoic acid or to ethanol increased punctate staining of F-actin and the postsynaptic density protein-95 (PSD-95). The increase in dendritic F-actin occurred only with clusters that co-localized with PSD-95 clusters, indicating that these actin structures likely represent dendritic spines. The ethanol-induced increases in PSD-95 and F-actin clusters were activity-dependent and reversible. Finally, inhibition of protein palmitoylation prevented ethanol-induced increases in synaptic NMDA receptor clustering and F-actin without altering the basal clustering of either F-actin or PSD-95. These observations support a model in which chronic ethanol exposure induces homeostatic increases of NR2B-containing NMDA receptors and PSD-95 to the postsynaptic density. This in turn may provide a scaffolding platform for the subsequent recruitment of actin signaling cascades that alter actin cycling and promote spine enlargement.     

Dynamic distribution of endoplasmic reticulum in hippocampal neuron dendritic spines

The role of the endoplasmic reticulum (ER) localized in dendritic spines has become a subject of intense interest because of its potential functions in local protein synthesis and signal transduction. Although it is recognized from electron microscopic studies that not all spines contain ER, little is know of its dynamic regulation or turnover. Here, we report a surprising degree of turnover of ER within spines. Using confocal microscopy imaging we observed continuity of spine-ER with dendritic ER in hippocampal primary neurons. Over 24 h, less than 50% of spine ER was stable. Despite this high degree of turn over, we identified a significant subset of spines that maintained ER for at least 4 days. These results indicate that within a single neuron, the organelle composition of a spine is unexpectedly dynamic and may explain aspects of the spine-to-spine variation in calcium spike magnitude and localized protein synthesis and trafficking.     

Three-dimensional structure of dendritic spines in the rat neostriatum

Dendritic spines of rat neostriatal neurons were examined by light microscopy and high voltage stereo electron microscopy (HVEM) following selective staining by intracellular microinjection of horseradish peroxidase. Conventionally prepared material also was used for quantitative analysis of dendritic spines from serial thin sections of neostriatum. Stereo electron microscopy of semithin sections from rat neostriatum fixed using a protocol designed to preserve cytoskeletal integrity was employed to examine the organization of the dendritic spine cytoplasm. Light microscopic and HVEM examination of spiny dendrites and quantitative analysis of serial thin sections from normal material revealed no distinct spine types but rather continuous and independent variation of spine head diameter, stalk diameter, and stalk length. Likewise, there was no systematic relationship between any of these spine dimensions and dendritic diameter. Spine head membrane surface area was directly related to the area of the synaptic junctional membrane of the spine head. In semithin sections, the cytoplasm of the spine contained membranous saccules of spine apparatus and a delicate cytoskeletal network composed of microfilaments and a set of finer and more variable cytoskeletal filaments. It is proposed that this cytoskeletal network together with the spine apparatus is responsible for the maintenance and alteration of spine shape and in this way controls the effectiveness of axospinous synapses.     

Synaptopodin maintains the neural activity-dependent enlargement of dendritic spines in hippocampal neurons

Synaptopodin (SYNPO) is an F-actin interacting protein expressed in dendritic spines and upregulated during the late-phase of long-term potentiation. Here, we investigated whether SYNPO regulates spine morphology through interactions with F-actin, the major cytoskeletal element of spines. In primary hippocampal neuron cultures, both endogenous and exogenous SYNPO localized preferentially in large spines under basal conditions. SYNPO overexpression did not affect the number or volume of spines in unstimulated neurons. Pharmacological activation of synaptic NMDA receptors transiently increased spine volume in control neurons, while the increase was persistent in neurons overexpressing SYNPO. In addition, exogenous SYNPO in PtK2 cells suppressed staurosporine-dependent disruption of F-actin stress fibers, suggesting that SYNPO protected F-actin from disruption. These results suggest that SYNPO stabilized activity-dependent increases in spine volume and imply that late-phase changes in spine morphology involve SYNPO.     

Morphological development of dendritic spines on rat cerebellar Purkinje cells

The posterior cerebellum is strongly involved in motor coordination and its maturation parallels the development of motor control. Climbing and mossy fibers from the spinal cord and inferior olivary complex, respectively, provide excitatory afferents to cerebellar Purkinje neurons. From post-natal day 19 climbing fibers form synapses with thorn-like spines located on the lower primary and secondary dendrites of Purkinje cells. By contrast, mossy fibers transmit synaptic information to Purkinje cells trans-synaptically through granule cells. This communication occurs via excitatory synapses between the parallel fibers of granule cells and spines on the upper dendritic branchlets of Purkinje neurons that are first evident at post-natal day 21. Dendritic spines influence the transmission of synaptic information through plastic changes in their distribution, density and geometric shape, which may be related to cerebellar maturation. Thus, spine density and shape was studied in the upper dendritic branchlets of rat Purkinje cells, at post-natal days 21, 30 and 90. At 90 days the number of thin, mushroom and thorn-like spines was greater than at 21 and 30 days, while the filopodia, stubby and wide spines diminished. Thin and mushroom spines are associated with increased synaptic strength, suggesting more efficient transmission of synaptic impulses than stubby or wide spines. Hence, the changes found suggest that the development of motor control may be closely linked to the distinct developmental patterns of dendritic spines on Purkinje cells, which has important implications for future studies of cerebellar dysfunctions.     

Is the function of dendritic spines to concentrate calcium?

Although dendritic spines are thought to play an important role in synaptic transmission and plasticity, their function remains unknown. Theoretical investigations of spine function have focused on the large electrical resistance provided by the narrow constriction of the spine neck. However, this narrow constriction is also thought to provide a large diffusional resistance. The importance of this diffusional resistance was investigated theoretically with models. When calcium currents were activated on dendritic spines, peak spine head Ca²⁺ concentration was an order of magnitude larger in ‘long-thin’ spines than in ‘mushroom-shaped’ or ‘stubby’ spines. The same currents activated on dendrites produced even smaller local Ca²⁺ concentration changes. Although the diffusional resistance of the spine neck was important for producing these differences in [Ca²⁺], the amplitude and duration of the Ca²⁺ current relative to the number of Ca²⁺ binding sites determined whether Ca²⁺ would be concentrated near synapses. Given the importance of Ca²⁺ for long-term potentiation, the ability of spines to concentrate Ca²⁺ may play a key role in processes leading to learning and memory storage.     

Perforated synapses on double-headed dendritic spines: a possible structural substrate of synaptic plasticity

Examination of axospinous synapses in serial sections obtained from the middle molecular layer of the rat dentate gyrus has revealed that some of them involve double-healed dendritic spines. Each spine head is apposed by a separate axon terminal with which it always forms a perforated synaptic contact distinguished by a discontinuous postsynaptic density. The number of perforated synapses on double-headed spines was estimated as a synapse-to-neuron ratio with the aid of the disector technique and found to be significantly increased in rats kindled via medial perforant path stimulation. These results support the notion that perforated synapses involving double-headed dendritic spines represent a structural modification related to enhanced synaptic efficacy.     

Actin filament organization within dendrites and dendritic spines during development

The myosin S-1 subfragment was used to label actin filaments in the developing rat brain. The results show actin filaments present throughout the dendritic region with highest concentrations within growth cones and regions of spine development. Between 6 and 25 days postnatal, spines became more complex and actin filaments within them increased in number and formed a complex network. The observed organization of actin supports the hypothesis that actin has a role in the protrusion of spines from the dendrite during development.     

Chronic ethanol consumption and hippocampal neuron dendritic spines: A morphometric and physiological analysis

The effects of chronic ethanol administration on hippocampal pyramidal neuron dendritic spines were examined in Long-Evans rats. The density and shape of spines did not differ between ethanol-treated animals and pair-fed controls. Data from both groups were similar to those reported previously in untreated animals. Spine function was studied electrophysiologically. No change in population spikes or EPSPs, evoked by Schaffer-commissural stimulation, was seen between alcohol-treated and pair-fed control rats. Moreover, the properties of long-term potentiation were similar in the two groups. Taken together, these data strongly suggest that chronic ethanol administration does not reduced hippocampal dendritic spine numbers or function. Given the previously reported ethanol-induced abnormalities using Golgi impregnation, however, it is possible that chronic alcohol treatment alters membrane properties of the spines.     

Neuronal plasticity and dendritic spines: effect of environmental enrichment on intact and postischemic rat brain.

The authors compared the influence of environmental enrichment on intact and lesioned brain, and tested the hypothesis that postischemic exposure to an enriched environment can alter dendritic spine density in pyramidal neurons contralateral to a cortical infarct. The middle cerebral artery was occluded distal to the striatal branches in spontaneously hypertensive rats postoperatively housed either in a standard or in an enriched environment. Intact rats were housed in the same environment. Three weeks later the brains were perfused in situ. The dendritic and spine morphology was studied with three-dimensional confocal laser scanning microscopy after microinjection of Lucifer yellow in pyramidal neurons in layers II/III and V/VI in the somatosensory cortex. In intact rats, the number of dendritic spines was significantly higher in the enriched group than in the standard group in all layers ( P < 0.05). Contralateral to the infarct, pyramidal neurons in layers II/III, which have extensive intracortical connections that may play a role in cortical plasticity, had significantly more spines in the enriched group than in the standard group ( P < 0.05). No difference was observed in layers V/VI. They conclude that housing rats in an enriched environment significantly increases spine density in superficial cortical layers in intact and lesioned brain, but in deeper layers of intact brain.     

Actin dynamics in dendritic spines: a form of regulated plasticity at excitatory synapses

Dendritic spines form the postsynaptic element at most excitatory synapses in the brain. The spine cytoskeleton consists of actin filaments which, in time-lapse recordings of living neurons expressing actin labeled with green fluorescent protein, can be seen to undergo rapid, dynamic changes. Because actin dynamics are associated with changes in cell shape, these cytoskeletal rearrangements may form a molecular basis for the morphological plasticity at brain synapses. The rapidity of these dynamic events in dendritic spines raises new questions. First, do the changes in actin cytoskeleton that are visible by light microscopy really correspond to changes in spine morphology, or do they represent changes in the relationship between actin and its many binding partners at postsynaptic sites? Second, how are these changes regulated by synaptic transmission? Third, to what extent do these changes occur in organized brain tissue? Answers to these questions are now beginning to emerge.     

Postnatal development of dendritic spines on olfactory bulb granule cells in rats

Postnatal morphological changes in granule cell dendritic spines and filopodia (collectively referred to as "spines/filopodia") were examined in the rat main olfactory bulb to characterize the development of the neural circuitry for olfaction. Granule cells were labeled with a membrane dye and confocal laser scanning microscope images of labeled spines/filopodia were acquired in the following three dendritic domains: apical dendrites in the external plexiform layer, those in the granule cell layer, and basal dendrites. In all three domains the proportion of typical spines slightly increased during development, with a concomitant decrease in the proportion of "stubby" spines lacking a neck; the proportion of filopodia remained unchanged, accounting for 20-40% of all protrusions. The mean diameter and length of the spine/filopodium population were nearly constant throughout development. On the other hand, the developmental pattern of the spine/filopodium density varied markedly, depending on the domain of the dendrites. In the external plexiform layer, the density did not change remarkably during development. The density in apical dendrites in the granule cell layer increased during the initial 2 postnatal weeks, then gradually decreased. The spine/filopodium density in basal dendrites, however, continued to increase until 4 weeks of age, and then began to decrease. These results suggest that a substantial amount of input-specific synaptic remodeling occurs in granule cells during development, which proceeds from superficial dendritic domains to deeper ones, occurring most prominently in the basal dendrites.