Anthony Holtmaat

Experience-dependent structural synaptic plasticity in the mammalian brain

Synaptic plasticity in adult neural circuits may involve the strengthening or weakening of existing synapses as well as structural plasticity, including synapse formation and elimination. Indeed, long-term in vivo imaging studies are beginning to reveal the structural dynamics of neocortical neurons in the normal and injured adult brain. Although the overall cell-specific morphology of axons and dendrites, as well as of a subpopulation of small synaptic structures, are remarkably stable, there is increasing evidence that experience-dependent plasticity of specific circuits in the somatosensory and visual cortex involves cell type-specific structural plasticity: some boutons and dendritic spines appear and disappear, accompanied by synapse formation and elimination, respectively. This Review focuses on recent evidence for such structural forms of synaptic plasticity in the mammalian cortex and outlines open questions.

Transient and Persistent Dendritic Spines in the Neocortex In Vivo

Dendritic spines were imaged over days to months in the apical tufts of neocortical pyramidal neurons (layers 5 and 2/3) in vivo. A fraction of thin spines appeared and disappeared over a few days, while most thick spines persisted for months. In the somatosensory cortex, from postnatal day (PND) 16 to PND 25 spine retractions exceeded additions, resulting in a net loss of spines. The fraction of persistent spines (lifetime > or = 8 days) grew gradually during development and into adulthood (PND 16-25, 35%; PND 35-80, 54%; PND 80-120, 66%; PND 175-225, 73%), providing evidence that synaptic circuits continue to stabilize even in the adult brain, long after the closure of known critical periods. In 6-month-old mice, spines turn over more slowly in visual compared to somatosensory cortex, possibly reflecting differences in the capacity for experience-dependent plasticity in these brain regions.

Experience-dependent and cell-type-specific spine growth in the neocortex

Functional circuits in the adult neocortex adjust to novel sensory experience, but the underlying synaptic mechanisms remain unknown. Growth and retraction of dendritic spines with synapse formation and elimination could change brain circuits. In the apical tufts of layer 5B (L5B) pyramidal neurons in the mouse barrel cortex, a subset of dendritic spines appear and disappear over days, whereas most spines are persistent for months. Under baseline conditions, new spines are mostly transient and rarely survive for more than a week. Transient spines tend to be small, whereas persistent spines are usually large. Because most excitatory synapses in the cortex occur on spines, and because synapse size and the number of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are proportional to spine volume, the excitation of pyramidal neurons is probably driven through synapses on persistent spines. Here we test whether the generation and loss of persistent spines are enhanced by novel sensory experience. We repeatedly imaged dendritic spines for one month after trimming alternate whiskers, a paradigm that induces adaptive functional changes in neocortical circuits. Whisker trimming stabilized new spines and destabilized previously persistent spines. New-persistent spines always formed synapses. They were preferentially added on L5B neurons with complex apical tufts rather than simple tufts. Our data indicate that novel sensory experience drives the stabilization of new spines on subclasses of cortical neurons. These synaptic changes probably underlie experience-dependent remodelling of specific neocortical circuits.

Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window

To understand the cellular and circuit mechanisms of experience-dependent plasticity, neurons and their synapses need to be studied in the intact brain over extended periods of time. Two-photon excitation laser scanning microscopy (2PLSM), together with expression of fluorescent proteins, enables high-resolution imaging of neuronal structure in vivo. In this protocol we describe a chronic cranial window to obtain optical access to the mouse cerebral cortex for long-term imaging. A small bone flap is replaced with a coverglass, which is permanently sealed in place with dental acrylic, providing a clear imaging window with a large field of view (∼0.8–12 mm2). The surgical procedure can be completed within ∼1 h. The preparation allows imaging over time periods of months with arbitrary imaging intervals. The large size of the imaging window facilitates imaging of ongoing structural plasticity of small neuronal structures in mice, with low densities of labeled neurons. The entire dendritic and axonal arbor of individual neurons can be reconstructed.

Spine growth precedes synapse formation in the adult neocortex in vivo

Dendritic spines appear and disappear in an experience-dependent manner. Although some new spines have been shown to contain synapses, little is known about the relationship between spine addition and synapse formation, the relative time course of these events, or whether they are coupled to de novo growth of axonal boutons. We imaged dendrites in barrel cortex of adult mice over 1 month, tracking gains and losses of spines. Using serial section electron microscopy, we analyzed the ultrastructure of spines and associated boutons. Spines reconstructed shortly after they appeared often lacked synapses, whereas spines that persisted for 4 d or more always had synapses. New spines had a large surface-to-volume ratio and preferentially contacted boutons with other synapses. In some instances, two new spines contacted the same axon. Our data show that spine growth precedes synapse formation and that new synapses form preferentially onto existing boutons.

Cell Type-Specific Structural Plasticity of Axonal Branches and Boutons in the Adult Neocortex

We imaged axons in layer (L) 1 of the mouse barrel cortex in vivo. Axons from thalamus and L2/3/5, or L6 pyramidal cells were identified based on their distinct morphologies. Their branching patterns and sizes were stable over times of months. However, axonal branches and boutons displayed cell type-specific rearrangements. Structural plasticity in thalamocortical afferents was mostly due to elongation and retraction of branches (range, 1-150 microm over 4 days; approximately 5% of total axonal length), while the majority of boutons persisted for up to 9 months (persistence over 1 month approximately 85%). In contrast, L6 axon terminaux boutons were highly plastic (persistence over 1 month approximately 40 %), and other intracortical axon boutons showed intermediate levels of plasticity. Retrospective electron microscopy revealed that new boutons make synapses. Our data suggest that structural plasticity of axonal branches and boutons contributes to the remodeling of specific functional circuits.

Structural Plasticity Underlies Experience-Dependent Functional Plasticity of Cortical Circuits

The stabilization of new spines in the barrel cortex is enhanced after whisker trimming, but its relationship to experience-dependent plasticity is unclear. Here we show that in wild-type mice, whisker potentiation and spine stabilization are most pronounced for layer 5 neurons at the border between spared and deprived barrel columns. In homozygote αCaMKII-T286A mice, which lack experience-dependent potentiation of responses to spared whiskers, there is no increase in new spine stabilization at the border between barrel columns after whisker trimming. Our data provide a causal link between new spine synapses and plasticity of adult cortical circuits and suggest that αCaMKII autophosphorylation plays a role in the stabilization but not formation of new spines.

Activity-Dependent Structural Plasticity of Perisynaptic Astrocytic Domains Promotes Excitatory Synapse Stability

BACKGROUND Excitatory synapses in the CNS are highly dynamic structures that can show activity-dependent remodeling and stabilization in response to learning and memory. Synapses are enveloped with intricate processes of astrocytes known as perisynaptic astrocytic processes (PAPs). PAPs are motile structures displaying rapid actin-dependent movements and are characterized by Ca(2+) elevations in response to neuronal activity. Despite a debated implication in synaptic plasticity, the role of both Ca(2+) events in astrocytes and PAP morphological dynamics remain unclear. RESULTS In the hippocampus, we found that PAPs show extensive structural plasticity that is regulated by synaptic activity through astrocytic metabotropic glutamate receptors and intracellular calcium signaling. Synaptic activation that induces long-term potentiation caused a transient PAP motility increase leading to an enhanced astrocytic coverage of the synapse. Selective activation of calcium signals in individual PAPs using exogenous metabotropic receptor expression and two-photon uncaging reproduced these effects and enhanced spine stability. In vivo imaging in the somatosensory cortex of adult mice revealed that increased neuronal activity through whisker stimulation similarly elevates PAP movement. This in vivo PAP motility correlated with spine coverage and was predictive of spine stability. CONCLUSIONS This study identifies a novel bidirectional interaction between synapses and astrocytes, in which synaptic activity and synaptic potentiation regulate PAP structural plasticity, which in turn determines the fate of the synapse. This mechanism may represent an important contribution of astrocytes to learning and memory processes.

Sensory-evoked LTP driven by dendritic plateau potentials in vivo

Long-term synaptic potentiation (LTP) is thought to be a key process in cortical synaptic network plasticity and memory formation. Hebbian forms of LTP depend on strong postsynaptic depolarization, which in many models is generated by action potentials that propagate back from the soma into dendrites. However, local dendritic depolarization has been shown to mediate these forms of LTP as well. As pyramidal cells in supragranular layers of the somatosensory cortex spike infrequently, it is unclear which of the two mechanisms prevails for those cells in vivo. Using whole-cell recordings in the mouse somatosensory cortex in vivo, we demonstrate that rhythmic sensory whisker stimulation efficiently induces synaptic LTP in layer 2/3 (L2/3) pyramidal cells in the absence of somatic spikes. The induction of LTP depended on the occurrence of NMDAR (N-methyl-d-aspartate receptor)-mediated long-lasting depolarizations, which bear similarities to dendritic plateau potentials. In addition, we show that whisker stimuli recruit synaptic networks that originate from the posteromedial complex of the thalamus (POm). Photostimulation of channelrhodopsin-2 expressing POm neurons generated NMDAR-mediated plateau potentials, whereas the inhibition of POm activity during rhythmic whisker stimulation suppressed the generation of those potentials and prevented whisker-evoked LTP. Taken together, our data provide evidence for sensory-driven synaptic LTP in vivo, in the absence of somatic spiking. Instead, LTP is mediated by plateau potentials that are generated through the cooperative activity of lemniscal and paralemniscal synaptic circuitry.

The Relationship between PSD-95 Clustering and Spine Stability In Vivo

The appearance and disappearance of dendritic spines, accompanied by synapse formation and elimination may underlie the experience-dependent reorganization of cortical circuits. The exact temporal relationship between spine and synapse formation in vivo remains unclear, as does the extent to which synapse formation enhances the stability of newly formed spines and whether transient spines produce synapses. We used in utero electroporation of DsRedExpress- and eGFP-tagged postsynaptic density protein 95 (PSD-95) to investigate the relationship between spine and PSD stability in mouse neocortical L2/3 pyramidal cells in vivo. Similar to previous studies, spines and synapses appeared and disappeared, even in naive animals. Cytosolic spine volumes and PSD-95-eGFP levels in spines covaried over time, suggesting that the strength of many individual synapses continuously changes in the adult neocortex. The minority of newly formed spines acquired PSD-95-eGFP puncta. Spines that failed to acquire a PSD rarely survived for more than a day. Although PSD-95-eGFP accumulation was associated with increased spine lifetimes, most new spines with a PSD did not convert into persistent spines. This indicates that transient spines may serve to produce short-lived synaptic contacts. Persistent spines that were destined to disappear showed, on average, reduced PSD-95-eGFP levels well before the actual pruning event. Altogether, our data indicate that the PSD size relates to spine stability in vivo.