ultrastructural analysis of spine plasticity · ultrastructural analysis of spine plasticity 13....

Ultrastructural Analysis of Spine Plasticity 11

Ultrastructural Analysis of Spine Plasticity
J N Bourne and K M Harris, Medical College ofGeorgia, Augusta, GA, USA
ã 2009 Elsevier Ltd. All rights reserved.

Introduction

Dendritic spines are small protrusions that serve as themajor site of excitatory synaptic transmission in thebrain. The shape and density of spines can be affectedby numerous factors, including developmental stage,experimental preparation, temperature, exposure toenriched environments, and neuronal pathology. Inaddition, physiological models of learning, includinglong-term potentiation (LTP) and long-term depres-sion (LTD), have indicated that alterations in synapticultrastructure may underlie the storage of informationin the brain.

Spine Structure and Function

Spine Shape

Dendritic spines can assume striking differences insize, shape, and subcellular composition both withinand across brain regions (Figure 1). Most spines areunbranched protrusions that can be classified by theirstubby, thin, or mushroom shapes. Branched spineshave multiple heads which, in some brain regions, areall innervated by a single axonal bouton (e.g., hippo-campal area CA3); in other brain regions the differentheads of a branched spine are innervated by differentaxons (e.g., hippocampal area CA1); and in rare casessome of the heads have no presynaptic partners whileother heads are innervated by different axons (e.g.,cerebellar Purkinje spiny branchlets).

Localization of Organelles

Smooth endoplasmic reticulum (SER) is an organellethat is likely to be involved in sequestering cal-cium. Depending on the particular brain regions, few,many, or most of the dendritic spines contain SER(Figure 1). For example, only about 14% of the hippo-campal CA1 spines contain SER, and most of it occurslaminated with dense-staining material into a structureknown as the spine apparatus (as in Figure 1) in thelarge, complex spines. In contrast, nearly 100% of thecerebellar Purkinje spines contain SER in a tubularnetwork. Polyribosomes are also present in or nearthe base of some dendritic spines (Figure 2(d)), suggest-ing that local protein synthesis of dendritic mRNAscan occur in the spines. In immature neurons, synapticactivity can shift the distribution of polyribosomes

from the shaft to spines. Similarly, endosomal compart-ments including coated pits and vesicles, large vesicles,tubules, and multivesicular bodies are restricted to asubpopulation of dendritic spines that differs fromspines that contain SER. Mitochondria rarely occurin dendritic spines and are usually restricted to thosethat are very large, complex, and highly branched.However, during periods of active synapse formationand remodeling in cultured neurons, mitochondriacan localize to smaller dendritic spines.

Postsynaptic Targets

Evaluation of dendritic spine structure readily revealsthem to be the major postsynaptic target of excitatorysynaptic input (Figure 2(a)). Since all dendritic spineshave at least one excitatory synapse on their head,more spines mean more synapses and accordinglymore point-to-point connections in a neuronalensemble involving spiny neurons. The size of thespine head also influences the amount of synapticinput, because larger spines are more sensitive toglutamate, the major excitatory neurotransmitter inthe brain. Larger spines have larger postsynaptic den-sities (PSDs), which can then anchor more glutamatereceptors. Thus, one function of spines is the preser-vation of the individuality of inputs. Occasionally,inhibitory or modulatory axons also form synapseson the heads, necks, or at the bases of dendriticspines. An inhibitory input on spines could act to‘veto’ or modify the strength of the excitatory input.

Amplification of Voltage in Spine Head

The constriction in dendritic spine necks poses a smallresistive barrier, thereby amplifying the depolariza-tion attained in the immediate vicinity of the synapse,in contrast to that which would be generated if thesynapse occurred directly on the wide dendritic shaft(Figure 2(b)). Computer simulations have revealedthat most of the spine necks are sufficiently wideand short such that charge transfer to the postsynap-tic dendrite is 85–100% complete within 100 ms afterthe initiation of a synaptic event. The time delay incharge transfer is sufficient, however, to provide atransient amplification of voltage at the spine synapse,which may facilitate opening of voltage-dependentchannels in the spine head, such as the calcium channelassociated with the N-methyl-D-aspartate (NMDA)class of glutamate receptors.

Sharing of Postsynaptic Potential

A long-standing hypothesis has been that the narrowdimensions of the spine neck would attenuate current

a b

sa

m

t

s

PSDperf

ast

v

m

t

perf PSD

c

1 µm 1 µm

Figure 1 Dendritic spine shape. (a) Electron micrograph of a section through dendritic spines in stratum radiatum of hippocampal area

CA1. (b) The same micrograph that has been color-coded to identify dendrites (yellow), axons (green), and astroglia (purple). In this

fortuitous section, three spines were sectioned parallel to their longitudinal axis revealing spines of the stubby (s), mushroom (m), and thin (t)

morphologies. The postsynaptic density (PSD) occurs on the spine head (see t) immediately adjacent and to a presynaptic axonal bouton

that is filled with round vesicles (v). This t spine contains a small tube of smooth endoplasmic reticulum (ser) in its neck. In the m spine a

spine apparatus (sa) is visible. A perforated PSD (perf PSD) is evident on the head of another mushroom spine. Near to this spine is

a large astrocytic process (ast) identified by the glycogen granules and clear cytoplasm. (c) A 3-D reconstruction of a dendrite showing a

variety of spine and synapse shapes.

12 Ultrastructural Analysis of Spine Plasticity

flow between the spine head and the dendrite. Mor-phological evidence suggests, however, that mostspine necks are not thin and long enough to signifi-cantly reduce the charge transferred to the parentdendrite. Current electrophysiological evidencefrom hippocampal CA1 cells suggests that themean synaptic conductance for a minimal evokedresponse is 0.21� 0.12 ns, such that the current gen-erated by release of 10–20 quanta would likely befully transmitted to the postsynaptic dendrite. Thus,the constriction in the spine neck is not sufficient toprevent addition of voltage changes among co-acti-vated synapses. Other models endow the spine withactive membrane that would further enhance thesharing of postsynaptic potentials among neighbor-ing spines (Figure 2(c)).

Biochemical Compartmentalization

Compartmentalization of calcium has now beendemonstrated in the heads of dendritic spines undera variety of conditions. Two features of spines help toachieve localization of this second messenger in spineheads at least for a short time: (1) the spine neck couldprovide a narrow diffusion path and (2) a rise in spinecalcium could cause release from SER, the intracellu-lar calcium stores thereby amplifying the calciumsignal. Biochemical compartmentalization in spineheads may also serve an important role in restrictingcalcium from the postsynaptic dendrite, thereby pre-venting excitotoxic cell damage such as microtubulebreakdown and mitochondrial swelling. Since den-dritic spines rarely have microtubules or mitochon-dria, high calcium concentrations in the spine head

Modu

Site of >90% excitatory synapsesAnd some others too...

Necks amplify voltagenear synapse

Necks not too thin → Associativity: share some charge with neighbors

I

a b

de

c

Inhib

Excit

V1

V2

*

*

**V3

V4

Synapse specificity through molecularcompartmentalization and local protein synthesis

Figure 2 Dendritic spine function. (a) Spines exist as principal sites of excitatory synaptic transmission. Spines exist to (b) amplify

electrical potential at the synapse and (c) promote associativity among neighboring synapses. Spine shape and resistance of the spine

neck may influence potential (V) generated by synaptic activation. (d) Spines exist as molecular compartments. Smooth endoplasmic

reticulum (tubules), calcium, and a myriad of other signaling mechanisms (stippling) are recruited in response to synaptic activation

(asterisk). (e) Three-dimensional reconstruction of thin spines emerging from a dendrite. Polyribosomes (black dots) are most frequent at

the base of dendritic spines, although they can also occur within them.


are less likely to have these detrimental effects. Notall spine morphologies would be expected to restrictdiffusion, and only some spines have internal stores.A recent study that utilized caged glutamate toactivate NMDA receptors and a low-affinity Ca2þ

indicator (to minimize the perturbation of endoge-nous Ca2þ buffering) indicates that small spines withnarrownecks exhibit large, isolated increases in [Ca2þ]i.However, the necks of larger spines allow for agreater efflux of Ca2þ into the dendritic shaft at thebase of the spine. Perhaps only a subset of spines, oralternatively all spines, but only at a restricted timeduring their history, achieve the compartmentaliza-tion of calcium, whether in spines or along a shortsegment of dendrite and its associated spines.The specific localization of voltage-dependent cal-

cium channels is also an important factor in determin-ing which components of the dendritic arbor willcompartmentalize and utilize relatively high concen-trations of calcium. Whether spines preferentiallysequester other second messengers remains to bedetermined. Certainly those tethered to the PSD areprevalent in spines, but further work is needed to

determine how they are targeted to spines and wheth-er compartmentalization in the spine head is a crucialelement in their regulation. Recent evidence suggeststhat synaptic activity can create a bidirectional bar-rier to the diffusion of proteins, indicating a synapse-specific mechanism to amplify the biochemical signalsnecessary for establishing plasticity (Figure 2(d)). Inaddition, the presence of polyribosomes in some spinescould provide the necessary machinery for generatingplasticity-related proteins (Figure 2(d)).

Enhanced Connectivity

With the evolution of the brain, the increase in syn-aptic density had to be accompanied by a significantreorganization of the neuropil. Dendritic spines per-mit dendrites to synapse with neurons 1–2 mm away,which allows for increased synaptic density in denselypacked neuropil (Figure 3(a)). Even some invertebrateneurons exhibit spinelike structures, indicating thatdendritic spines appeared well before the evolution ofthe complex mammalian brain. Consider the simplecase of an orthogonal relationship between dendritesand axons (Figure 3(b)). There can only be two

Figure 3 Enhanced connectivity between neurons. (a) Spines increase the packing density of synapses. Schematic illustrates a cross-

section through two dendrites (shaded), one without and one with dendritic spines. Convolution and interdigitation of dendrite, axon, and

spine membranes support more synapses. (b) The presence of spines also allows for an increase in synaptic density without increasing

the overall volume of the brain.


synapses on either side of the dendrite in any givenplane without the presence of spines. Dendrites withspines can reach beyond their immediate perimeter toconnect with axons in nearby rows, thereby at leastdoubling the density of possible connections. In addi-tion, the shape of dendritic spines allows efficientinterdigitation between neighboring processes, thusachieving the high synapse packing density in theneuropil.

Synaptic Activity and Spine Organization

Spine Stability and Turnover

The stability of spines largely depends on the level ofsynaptic activity and the developmental stage of theneuron. In immature neurons, spines and filopodiaturn over rapidly as connections are formed and stabi-lized. Only a small amount of glutamate is required tostabilize a spine. Excitotoxicity resulting from toomuch glutamate can result in the disappearance ofspines, while a block of synaptic transmission canresult in robust spinogenesis, particularly on matureneurons following traumatic injury or exposure to coldtemperatures as occurs during hibernation and somesurgical and experimental procedures. The appearanceand disappearance of spines may reflect the capacity ofmature neurons for recuperative synaptogenesis or aform of synaptic scaling, a homeostatic mechanismthat ensures that the output of a postsynaptic cellremains constant despite alterations in synaptic input.In contrast, young neurons do not exhibit spinogenesiswith blocked transmission, but rather insert morereceptors to maintain synaptic homeostasis.Brain regions also differ in the amount of synaptic

turnover that occurs with the modulation of sensoryexperience. Recent technological advances have

allowed dendritic spines of the somatosensory cortexto be imaged in vivo for periods of time ranging froma few hours to a few months. Small spines (thin orfilopodial) appear and disappear in an experience-dependent manner associated with synapse formationor elimination, whereas larger spines persist formonths. The effect of sensory experience on synapsenumber depends on the rates of synapse formationand elimination at different developmental stages,and the percentage of stable spines increases with age.

In the visual cortex of adult mice, in vivo imagingreveals that the majority of spines (75–90%) are stableunder baseline conditions. In another study, rats rearedin the dark until postnatal day 30 have a significantlylower spine density compared to light-reared controlsand also have shorter, rounder spines. Although anincrease in spine head diameter is observed in dark-reared animals that are exposed to light for 10days,the low spine density is not reversed. Presuming thatan increase in the number of receptors present in thePSD accompanies the increase in spine head diameter,the parent dendrite could still preserve the level ofsynaptic input even in the absence of spinogenesis.

In the neocortex, cerebellum, and hippocampus,localization of different afferent inputs on the samedendritic arbor is dictated by the laminar structure ofthese brain areas. However, in the lateral nucleus of theamygdala, inputs from the cortex and thalamus areinterspersed on the same dendritic branches and affer-ent specificity is determined by spine morphology.Large spines are contacted by thalamic afferents andexhibit larger Ca2þ transients following activationthan smaller spines contacted by cortical afferents.The increased Ca2þ signaling observed in large spinesis mediated by R-type voltage-dependent Ca2þ chan-nels that are preferentially localized to thalamic inputs.Thus, in the amygdala, organization of afferent inputs


can be regulated at the level of an individual spine byaltering its morphological and molecular properties.

Perisynaptic Astroglia and Spine Structure

Traditionally, astroglia have been thought of as ‘sup-port’ cells for neurons. However, recent evidencesuggests that astroglia may play a much larger role inthe maintenance and plasticity of synapses. Releaseof the cytokine tumor necrosis factor alpha (TNFa)by glial cells enhances excitatory synaptic strengthby increasing the surface expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)receptors on dendritic spines while reducing inhibi-tory synaptic strength by causing the endocytosis ofg-aminobutyric acid (GABAA) receptors. Since astro-glial processes can project extensively throughoutthe neuropil, astroglia are in a position to scalesynaptic responses at the level of entire neuronalnetworks. In addition to affecting the physiologicalproperties of synapses, astroglia can also regulate thestructure of dendritic spines in response to synapticactivity. Transient interactions between cell surfacemolecules such as the ephrinA3 ligand and theEphA4 receptor regulate the structure of excitatorysynaptic connections through neuro-glial cross-talk.Activation of EphA4 by ephrin-A3 induces spineretraction, and inhibiting ephrin/EphA4 interactionsdistort spine shape and organization. In addition toEphs and ephrins, astrocytic membranes containother contact-mediated factors that influence synap-tic maturation. Astroglial processes tend to be foundaround larger and presumably more mature synapses,while areas that have undergone recent spinogenesistend to be devoid of astroglia. Thus in the maturebrain, astroglia may be involved in enhancing thestability of spines and preventing the extension ofnew dendritic protrusions. Interactions between cellsurface molecules and the release of various solublefactors by astroglia may be of crucial importanceto the turnover and structural alteration of spinesobserved with synaptic plasticity.

Structural Synaptic Plasticity atDendritic Spines

Long-Term Potentiation

LTP is a cellular model of learning and memory thathas been extensively investigated regarding changesin spine and synapse structure. Two key structuralchanges would contribute to a potentiated synapticresponse: an increase in the number of spines or anincrease in the size of synapses with LTP. Severalstudies in the hippocampus over the last 30 yearshave attempted to link structural alterations of the

synapse to the increase in synaptic strength, but theresults remain controversial, particularly in the adultbrain. Numerous studies involving in vivo inductionof LTP in the dentate gyrus of adult rats have led todiffering conclusions about the amount of structuralplasticity that occurs. Early studies describe an input-specific increase in spine volume and a shortening ofspines accompanied by an enlargement of the spineneck. Overall spine density and branched spinesin particular are increased 30min after the in vivoinduction of LTP in the dentate gyrus, although theincrease in branched spines may be due to high-frequency stimulation alone, even in the absence ofLTP. While there has been some speculation thatbranched spines might arise from the splitting of indi-vidual potentiated spines, careful three-dimensional(3-D) reconstructions of the neuropil have revealedthat branched spines never synapse on the same axo-nal bouton. In addition, the number of neuronal anddendritic processes that pass between the branchesargues against the idea of spine splitting. An alterna-tive hypothesis suggests that branched spines arisefrom the formation of new protrusions called filopo-dia which can be initiated by the enhanced synapticactivity, navigate between neighboring processes tofind a presynaptic partner, and then become dendriticspines. In any case, the majority of morphologicalalterations that have been attributed to in vivo induc-tion of LTP have included more subtle changes suchas increases in the concavity of spine heads and anincrease in PSD area, as well as an increase in the ratioof perforated to nonperforated PSDs. Becauseperforated PSDs tend to occur in mushroom spines,this change could indicate an alteration in spine mor-phology if potentiation results in a shift from thinspines to mushroom spines.

Uncertainty also exists about the extent of struc-tural plasticity that occurs following the induction ofLTP in area CA1 of the adult rat hippocampus. Earlystudies that analyzed single EM images suggestthat there is an increase in shaft and sessile synapsesfollowing induction of LTP. However, it was notspecified whether these synapses were symmetric(inhibitory) or asymmetric (excitatory). More recentstudies specifically aimed at detecting changes in syn-aptic density failed to detect a reliable increase insynapse number after induction of LTP. Althoughchanges in synaptic density have been difficult todetect, recent evidence suggests that spines can getbigger with LTP.When LTP is induced via stimulationof the Schaffer collaterals, a small fraction of spinestransiently expand within minutes of tetanic or thetaburst stimulation, but return to their initial size withina few minutes. The percentage of expanding spinesvaries directly with stimulus strength, presumably


reflecting an increase in number of presynapticfibers recruited. Even with a stimulation intensitythat evokes a maximal field excitatory postsynapticpotential (fEPSP), no more than 10% of spinesrespond. Thus, the relatively small fraction of expand-ing spines that synapse with the presynaptic terminalsactivated by field stimulation reflects the challengespresent in detecting plasticity-related changes in theadult brain. Even serial section electron microscopyand 3-D reconstruction of dendrites 2 h post-LTP didnot reveal net changes in the density, shape, or size ofspines. One possibility is that synaptic proliferationoccurs immediately after LTP induction, but withtime, competition between recently potentiatedspines and their nonpotentiated neighbors results inthe elimination of the nonpotentiated spines; thus, thedendrite maintains its optimal number of synapticconnections. Under such a scenario, individual spinescould exhibit dramatic plasticity while the overallpopulation number and average size of dendriticspines would appear unchanged.Although the mature brain presents several chal-

lenges to describing the morphological changes thatunderlie synaptic plasticity, the immature nervoussystem has provided more consistent results. Dynamicchanges in local dendritic segments that were targetedfor potentiation-induced changes have been observedin organotypic slice cultures. Significant local out-growth of filopodia-like dendritic processes is seen20min after delivery of a tetanic stimulus, and someof the activity-induced filopodia later turn into spine-like structures. When LTP is induced by pairing post-synaptic depolarizations with single presynapticstimuli and transmitter release is blocked everywherebut in the small area being imaged, two to nine newspines are formed in the nonblocked region, empha-sizing the localized specificity of structural changesassociated with LTP. Similarly, stimulation of individ-ual spines with uncaged glutamate induces a localizedincrease in spine head diameter that is apparentwithin 1–5min, and 30% of the stimulated spinesremain enlarged 70–100min after stimulation. Themagnitude of potentiation correlates with early orlong-lasting spine enlargement, suggesting a highlevel of fidelity between physiological and morpho-logical changes. Spine and PSD enlargement are alsoassociated with the translocation of polyribosomesinto spines following the induction of LTP in acutePN15 hippocampal slices. Given that the mainte-nance of LTP is dependent on the synthesis of newproteins, the presence of polyribosomes in a subset ofspines may provide an indication of which spinesunderwent potentiation.Although quite a bit of uncertainty still surrounds

the set of structural changes associated with LTP, it

appears likely that the effect is selective to poten-tiated synapses and that changes in a tissue will becorrelated with the numbers of stimulated axons anddendrites. Nevertheless, there is evidence that LTP-inducing stimuli can result in increases in spine sizeand number and may involve the remodeling of thePSD and the translocation of organelles into activatedspines. All of these changes could form the basis oflong-lasting changes in synaptic strength.

Long-Term Depression

In contrast to LTP, LTD is a long-lasting reduction insynaptic transmission that may also play an integralrole in the processing and retention of information.Although the literature concerning structural changesassociated with LTD is much less abundant, theredoes appear to be a correlation between a reductionin synaptic response and a decrease in the number andsize of dendritic spines. Low-frequency stimulation(900 pulses at 1Hz) results in the shrinkage ofdendritic spines in acute hippocampal slices and theretraction of spines in organotypic hippocampal slicecultures. While these results indicate a strong linkbetween structural and synaptic plasticity, muchwork remains to be done to fully elucidate the mor-phological correlates of LTD.

See also: Developmental Synaptic Plasticity: LTP, LTD,

and Synapse Formation and Elimination; Endocytic

Traffic in Spines; Eph Receptor Signaling and Spine

Morphology; LIM Kinase and Actin Regulation of Spines;

Long-Term Depression: Cerebellum; Long-Term

Potentiation and Long-Term Depression in Experience-

Dependent Plasticity; Long-Term Potentiation (LTP).

Further Reading

Bliss T, Collingridge G, and Morris R (eds.) (2004) Long-TermPotentiation: Enhancing Neuroscience for 30 Years. NewYork: Oxford University Press.

Ethell IM and Pasquale EB (2005) Molecular mechanisms of

dendritic spine development and remodeling. Progress inNeurobiology 75: 161–205.

Harris KM and Kater SB (1994) Dendritic spines: Cellular specia-

lizations imparting both stability and flexibility to synaptic

function. Annual Review of Neuroscience 17: 341–371.Harris KM and Stevens JK (1988) Dendritic spines of rat cerebellar

Purkinje cells: Serial electron microscopy with reference to

their biophysical characteristics. Journal of Neuroscience 8:

4455–4469.Harris KM and Stevens JK (1989) Dendritic spines of CA1 pyrami-

dal cells in the rat hippocampus: Serial electron microscopy

with reference to their biophysical characteristics. Journal ofNeuroscience 9: 2982–2997.

Nimchinsky EA, Sabatini BL, and Svoboda K (2002) Structure and

function of dendritic spines. Annual Review of Physiology 64:

313–353.


Peters A, Palay SL, and Webster HDF (1991) The Fine Structure ofthe Nervous System: Neurons and Their Supporting Cells, 3rdedn. New York: Oxford University Press.

Sorra KE and Harris KM (2000) Overview on the structure, com-position, function, development, and plasticity of hippocampal

dendritic spines. In: Eichenbaum HB (ed.) with Harris KM

and Sorra KE (special issue eds.) Dendritic Spines of theHippocampus. Hippocampus 10: 501–511.

Stuart G, Spruston N, and Hausser M (eds.) (2001) Dendrites.New York: Oxford University Press.

Yuste R and Bonhoeffer T (2001) Morphological changes in den-

dritic spines associated with long-term synaptic plasticity.

Annual Review of Neuroscience 24: 1071–1089.

Relevant Website

http://synapses.clm.utexas.edu – synapse web.

http://synapses.clm.utexas.edu

ultrastructural analysis of spine plasticity · ultrastructural analysis of spine plasticity 13....

Documents