Dendritic Spine Viscoelasticity and Soft-Glassy Nature: Balancing Dynamic Remodeling with Structural Stability Benjamin A. Smith,* Hugo Roy, yz Paul De Koninck, yz Peter Gru ¨tter,* and Yves De Koninck y *Department of Physics, McGill University, Montreal, QC, Canada H3A 2T8; y Neurobiologie Cellulaire, Centre de recherche Universite ´ Laval Robert-Giffard, Quebec, QC, Canada G1J 2G3; and z De ´ partement de biochimie et microbiologie, Faculte ´ des Sciences et de Ge ´ nie, Universite ´ Laval, Quebec, Canada, G1K 7P4 ABSTRACT Neuronal dendritic spines are a key component of brain circuitry, implicated in many mechanisms for plasticity and long-term stability of synaptic communication. They can undergo rapid actin-based activity-dependent shape fluctuations, an intriguing biophysical property that is believed to alter synaptic transmission. Yet, because of their small size (;1 mm or less) and metastable behavior, spines are inaccessible to most physical measurement techniques. Here we employ atomic force microscopy elasticity mapping and novel dynamic indentation methods to probe the biomechanics of dendritic spines in living neurons. We find that spines exhibit 1), a wide range of rigidities, correlated with morphological characteristics, axonal association, and glutamatergic stimulation, 2), a uniquely large viscosity, four to five times that of other cell types, consistent with a high density of solubilized proteins, and 3), weak power-law rheology, described by the soft-glassy model for cellular mechanics. Our findings provide a new perspective on spine functionality and identify key mechanical properties that govern the ability of spines to rapidly remodel and regulate internal protein trafficking but also maintain structural stability. INTRODUCTION Dendritic spines are micrometer-sized cellular structures (Fig. 1) that are the sites of most excitatory synaptic contacts in the central nervous system (1,2) and have been implicated in many forms of postsynaptic plasticity of neuronal commu- nication (3–9). Whereas dramatic changes in spine density or structure are observed in a number of pathological brain disorders (3,7), subtle changes in spine shape and content in the brain have been related to normal cognitive behavior, learning, and memory (3,4,10,11). Postsynaptic plasticity may involve regulation of the recruitment and organization of signal transduction proteins at the postsynaptic density in spines (3,4,6,9). It is unclear at this point what physical mechanisms enable spines to maintain their structure yet allow for plastic remodeling and internal trafficking of their molecular content. In cultures of dissociated primary hippocampal neurons, the early stages of spine formation involve outgrowth of highly dynamic filopodia (finger-like projections) from the surface of dendrites (12–16). These filopodia are thought to search for presynaptic targets on nearby axons (15,17). When contact is made, it is believed that filopodia differen- tiate into spines (13). As spines mature, which may occur in ,60 min (14,16), they adopt a stabilized spherical or mush- room-like shape connected to the dendritic shaft by a narrow neck a few hundred nanometers wide (18). Spine morphol- ogy is likely maintained by continued low-level stimulations from AMPA-type glutamate receptor activation (19). The surface of the spine head is observed to undergo rapid shape fluctuations (nanoscale motions on a time scale of seconds) (20), a motility that is suppressed by the presence of active presynaptic terminals (21–24). As first proposed by Crick (10), this subtle remodeling is believed to dynamically opti- mize transmission by adjusting connectivity and the geom- etry of the synaptic cleft. The dynamic shape of dendritic spines has been proposed to be associated with high actin content (11,22–26). Al- though microtubules are prominent along the entire length of dendrite shafts, they are largely excluded from the actin-rich spines (25,27). Rapid cycling of filamentous actin and actin regulatory proteins has been observed within minutes or less in spines (24,28). Various forms of synaptic stimulation have resulted in rapid as well as long-term remodeling of the postsynaptic actin cytoskeleton (26,29–32), which may be required for long-term modulation of synaptic transmission (33–35). Dynamic actin filaments likely act not only as major structural components in spines but also as substrates for a variety of scaffolding proteins that link to and regulate the postsynaptic density (9,36,37). Trafficking of intracellular molecules by diffusion within dendritic spines may also be of fundamental importance for function and plasticity of synapses (3,38). Large molecules such as actin and actin-associated proteins (29,30), as well as mRNA (39,40), are translocated into spines during periods of extended activation (5–30 min). Glutamatergic receptor chan- nels are redistributed during LTP, incorporated in either mo- bile transport vesicles or within the plasma membrane of the spine (4,9,41). Rapid diffusion of AMPA receptors has been observed, exhibiting location- and activity-dependent mo- tions in dendritic membranes (9). Within the spine head, fast Submitted July 3, 2006, and accepted for publication October 27, 2006. Address reprint requests to Yves De Koninck, Neurobiologie Cellulaire, Centre de recherche Universite ´ Laval Robert-Giffard, 2601 Chemin de la Canardie `re, Quebec, QC G1J 2G3 Canada. Tel.: 418-663-5747 ext. 6885; Fax: 418-663-8756; E-mail: [email protected]. B. A. Smith’s present address is Dept. of Physics and Astronomy, University of British Columbia, Vancouver, BC, Canada, V6T 1Z1. Ó 2007 by the Biophysical Society 0006-3495/07/02/1419/12 $2.00 doi: 10.1529/biophysj.106.092361 Biophysical Journal Volume 92 February 2007 1419–1430 1419
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Dendritic Spine Viscoelasticity and Soft-Glassy Nature: BalancingDynamic Remodeling with Structural Stability
Benjamin A. Smith,* Hugo Roy,yz Paul De Koninck,yz Peter Grutter,* and Yves De Konincky
*Department of Physics, McGill University, Montreal, QC, Canada H3A 2T8; yNeurobiologie Cellulaire, Centre de recherche Universite LavalRobert-Giffard, Quebec, QC, Canada G1J 2G3; and zDepartement de biochimie et microbiologie, Faculte des Sciences et de Genie,Universite Laval, Quebec, Canada, G1K 7P4
ABSTRACT Neuronal dendritic spines are a key component of brain circuitry, implicated in many mechanisms for plasticity andlong-term stability of synaptic communication. They can undergo rapid actin-based activity-dependent shape fluctuations, anintriguing biophysical property that is believed to alter synaptic transmission. Yet, because of their small size (;1 mm or less) andmetastable behavior, spines are inaccessible to most physical measurement techniques. Here we employ atomic force microscopyelasticity mapping and novel dynamic indentation methods to probe the biomechanics of dendritic spines in living neurons. Wefind that spines exhibit 1), a wide range of rigidities, correlated with morphological characteristics, axonal association, andglutamatergic stimulation, 2), a uniquely large viscosity, four to five times that of other cell types, consistent with a high density ofsolubilized proteins, and 3), weak power-law rheology, described by the soft-glassy model for cellular mechanics. Our findingsprovide a new perspective on spine functionality and identify key mechanical properties that govern the ability of spines to rapidlyremodel and regulate internal protein trafficking but also maintain structural stability.
INTRODUCTION
Dendritic spines are micrometer-sized cellular structures
(Fig. 1) that are the sites of most excitatory synaptic contacts
in the central nervous system (1,2) and have been implicated
in many forms of postsynaptic plasticity of neuronal commu-
nication (3–9). Whereas dramatic changes in spine density or
structure are observed in a number of pathological brain
disorders (3,7), subtle changes in spine shape and content in
the brain have been related to normal cognitive behavior,
learning, and memory (3,4,10,11). Postsynaptic plasticity may
involve regulation of the recruitment and organization of
signal transduction proteins at the postsynaptic density in
spines (3,4,6,9). It is unclear at this point what physical
mechanisms enable spines to maintain their structure yet
allow for plastic remodeling and internal trafficking of their
molecular content.
In cultures of dissociated primary hippocampal neurons,
the early stages of spine formation involve outgrowth of
highly dynamic filopodia (finger-like projections) from the
surface of dendrites (12–16). These filopodia are thought to
search for presynaptic targets on nearby axons (15,17).
When contact is made, it is believed that filopodia differen-
tiate into spines (13). As spines mature, which may occur in
,60 min (14,16), they adopt a stabilized spherical or mush-
room-like shape connected to the dendritic shaft by a narrow
neck a few hundred nanometers wide (18). Spine morphol-
ogy is likely maintained by continued low-level stimulations
from AMPA-type glutamate receptor activation (19). The
surface of the spine head is observed to undergo rapid shape
fluctuations (nanoscale motions on a time scale of seconds)
(20), a motility that is suppressed by the presence of active
presynaptic terminals (21–24). As first proposed by Crick
(10), this subtle remodeling is believed to dynamically opti-
mize transmission by adjusting connectivity and the geom-
etry of the synaptic cleft.
The dynamic shape of dendritic spines has been proposed
to be associated with high actin content (11,22–26). Al-
though microtubules are prominent along the entire length of
dendrite shafts, they are largely excluded from the actin-rich
spines (25,27). Rapid cycling of filamentous actin and actin
regulatory proteins has been observed within minutes or less
in spines (24,28). Various forms of synaptic stimulation have
resulted in rapid as well as long-term remodeling of the
postsynaptic actin cytoskeleton (26,29–32), which may be
required for long-term modulation of synaptic transmission
(33–35). Dynamic actin filaments likely act not only as major
structural components in spines but also as substrates for a
variety of scaffolding proteins that link to and regulate the
postsynaptic density (9,36,37).
Trafficking of intracellular molecules by diffusion within
dendritic spines may also be of fundamental importance for
function and plasticity of synapses (3,38). Large molecules
such as actin and actin-associated proteins (29,30), as well as
mRNA (39,40), are translocated into spines during periods of
Consequence of spine mechanics oninternal diffusion
There is mounting evidence that molecular diffusion within
spines is regulated in parallel with spine motility (38,43).
Thermally driven motion (diffusion) of small (low-inertia)
particles is restricted by the viscoelastic properties of the
surrounding medium (45). Thus, we expect the diffusional
translocation of proteins or small organelles (e.g., vesicles)
within spine heads to be reduced in spines that present larger
viscoelastic resistance than other spines or dendrite shafts.
Indeed, our observations of CaMKII protein translocation in
spines and dendrites (Fig. 6 B) show correlations between
diffusion constants and spine viscoelastic compliance (Fig. 4
A). Furthermore, we use the complex rheology of spines
(Fig. 5) to predict a strong anomalous component of dif-
fusion (Fig. 6 A).
The thermal motion of a particle in a viscoelastic medium
can be estimated using the generalized Stokes-Einstein
relation (45):
ÆDr2ðtÞæ �Z
dv
2pð1� e
�ivtÞ kBT
3pav
G$ðvÞG9
2ðvÞ1 G$2ðvÞ
� �;
(5)
where kB is the Boltzmann constant, T is absolute temper-
ature, and a is the radius of the particle. The approximation
arises because of the use of G*(v) measurements from a
Dendritic Spine Viscoelasticity 1427
Biophysical Journal 92(4) 1419–1430
limited frequency range in the integration over all frequencies.
This relation, based on the fluctuation-dissipation theorem,
assumes thermal equilibrium and a homogeneous medium,
which may not be correct for the cytoplasm of dendritic
spines. On the basis of the complex rheology, we measured
(Fig. 5) and Eq. 5 above, Fig. 6 A shows a calculation of
thermally driven motion of a 10-nm-radius particle, approx-
imately the size of a single synaptic signaling protein in a
spine (for example, CaMKII). We chose CaMKII because of
its multifunctional role in activity-dependent neuronal func-
tion (69) and the observations of its rapid translocations in
spines (42), but the diffusion model is nonspecific, including
only the dependence on particle size and not molecular
structure or specific interactions with other proteins. The two
regimes of viscoelasticity seen in Fig. 4, namely the high-
frequency fluid viscosity and the low-frequency glassy
rheology, predict two types of intracellular motion. In the
regime where Newtonian viscosity dominates (frequencies
.50 Hz or times ,20 ms), intracellular particles would
follow pure Brownian diffusion (ÆDr2ðtÞæ;t). The dashed
line in Fig. 6 A shows the mean-square displacements
predicted using only the Newtonian viscosity term of the
spine rheology, resulting in Brownian motion with a
diffusion constant of 3.6 3 10�3 mm2/s. On longer time
scales, motions would be restricted by the elastic component
of the spine’s mechanical structure. The weak power-law
scaling of complex shear modulus G*(v) ; va implies
that ÆDr2ðtÞæ;ta, referred to as anomalous subdiffusion
when a , 1.
Quantitatively, the results of Fig. 6 A are quite small com-
pared to reported values of CaMKII diffusion in nonneuronal
cultured cells (;1 mm2/s) (70). However, our FRAP mea-
surements of GFP-CaMKII diffusion in hippocampal den-
drites (Fig. 6 B) show that diffusion is much slower in spines:
t ¼ 99 6 5 s mean recovery time, corresponding to a dif-
fusion constant Ds � 10�2 mm2/s using the geometric model
for diffusion into spines (see Methods). Particle-tracking
experiments in other cell types have observed anomalous
mean-square displacements similar to those we calculate
(subdiffusion at ;10�4 mm2 in 1 s for a particle of radius
;100 nm) (71), but others report motions two to three orders
of magnitude faster (72–74). Thus, our prediction of intra-
cellular diffusion within spines is at the lower end of the
spectrum of observed diffusion rates in cells. As revealed in
this study, the viscosity of dendritic spines is four to five
times that of other cells, which supports impaired diffusion
in spines.
The ratio of diffusion constants from Fig. 6 B reveals that
diffusion is two to seven times slower in spines relative to
dendrites. This reduced diffusion is in agreement with our
results of enhanced viscoelastic resistance in spines relative
to dendrites (relative stiffness of 2.0 6 0.3 from Fig. 3 A for
axon-associated spines). A recent study showed that mem-
brane-linked diffusion is also slower in spines than in
dendrite shafts and that use of an anomalous subdiffusion
model significantly improved fits to these FRAP data,
although the motion was two-dimensional and the power-
law exponents were a ¼ 0.7–0.8 (43). Finally, our results of
increased viscoelastic resistance in stimulated spines (Fig. 4)
may help to explain recent observations that neuronal
activity reduces diffusion into spines (38) without the need
for a large variation in the cross-sectional area of the spine
neck.
CONCLUSION
We have demonstrated a novel approach to study the
mechanical properties of dendritic spines at the submicrom-
eter scale in live neurons. Furthermore, the viscoelastic char-
acterization presented here provides an entirely new perspective
on how to describe the functional state of dendritic spines.
Our results show that the soft-glassy materials description of
cellular mechanics is an appropriate model of spine visco-
elasticity and extends its previous success in the larger-scale
cytoskeletal dynamics of cells such as smooth muscle cells
(49,52,54). Within this framework, the concepts of activity-
dependent structural plasticity, metastability, and congestion
in the cytoplasm of spines are gauged by only a few mea-
surable parameters. Most importantly, the effective noise
temperature, which is an integrative factor reflecting the level
of molecular agitations, acts as the primary determinant of
not only viscoelasticity, striking the delicate balance between
solid-like and fluid-like properties, but also the degree to
which spines are capable of remodeling and maintaining
structural stability. We therefore form the characterization of
mechanically soft, malleable spines, likely with the mor-
phological plasticity necessary for learning in the brain, as
hot spines with elevated noise temperature. More rigid,
stable spines, with properties likely associated with memory
retention, are characterized as cold spines with reduced noise
temperature. This new perspective adds viscoelasticity to the
list of properties of dendritic spines that is of central
importance to their function.
SUPPLEMENTARY MATERIAL
An online supplement to this article can be found by visiting
BJ Online at http://www.biophysj.org.
We thank Francine Nault and Salma Behna for their expert technical
assistance, Greg McDonald for preliminary results with FRAP measure-
ments and discussions, Helen Bourque and Eric LeBel for fruitful dis-
cussions. We thank Chun Seow and R. Anne McKinney for providing
comments on previous versions of this manuscript.
We thank the Natural Science and Engineering Research Council of Canada
(NSERC: grants to P.G., Y.D.K., and P.D.K.; postgraduate scholarships
to B.S. and H.R.), the Canadian Institutes of Health Research (CIHR)
through a new emerging team (NET) grant (P.G., Y.D.K., and P.D.K.), and
the Neurophysics Strategic Training Grant for partial support of B.S., and
the Canadian Foundation for Innovation for their financial support.
1428 Smith et al.
Biophysical Journal 92(4) 1419–1430
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