Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex

Do new synapses form in the adult cortex to support experience-dependent plasticity? To address this question, we repeatedly imaged individual pyramidal neurons in the mouse barrel cortex over periods of weeks. We found that, although dendritic structure is stable, some spines appear and disappear. Spine lifetimes vary greatly: stable spines, about 50% of the population, persist for at least a month, whereas the remainder are present for a few days or less. Serial-section electron microscopy of imaged dendritic segments revealed retrospectively that spine sprouting and retraction are associated with synapse formation and elimination. Experience-dependent plasticity of cortical receptive fields was accompanied by increased synapse turnover. Our measurements suggest that sensory experience drives the formation and elimination of synapses and that these changes might underlie adaptive remodelling of neural circuits.     

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 alpha-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.     

Opposite effects of fear conditioning and extinction on dendritic spine remodelling

It is generally believed that fear extinction is a form of new learning that inhibits rather than erases previously acquired fear memories. Although this view has gained much support from behavioural and electrophysiological studies, the hypothesis that extinction causes the partial erasure of fear memories remains viable. Using transcranial two-photon microscopy, we investigated how neural circuits are modified by fear learning and extinction by examining the formation and elimination of postsynaptic dendritic spines of layer-V pyramidal neurons in the mouse frontal association cortex. Here we show that fear conditioning by pairing an auditory cue with a footshock increases the rate of spine elimination. By contrast, fear extinction by repeated presentation of the same auditory cue without a footshock increases the rate of spine formation. The degrees of spine remodelling induced by fear conditioning and extinction strongly correlate with the expression and extinction of conditioned fear responses, respectively. Notably, spine elimination and formation induced by fear conditioning and extinction occur on the same dendritic branches in a cue- and location-specific manner: cue-specific extinction causes formation of dendritic spines within a distance of two micrometres from spines that were eliminated after fear conditioning. Furthermore, reconditioning preferentially induces elimination of dendritic spines that were formed after extinction. Thus, within vastly complex neuronal networks, fear conditioning, extinction and reconditioning lead to opposing changes at the level of individual synapses. These findings also suggest that fear memory traces are partially erased after extinction.     

Locally dynamic synaptic learning rules in pyramidal neuron dendrites

Long-term potentiation (LTP) of synaptic transmission underlies aspects of learning and memory. LTP is input-specific at the level of individual synapses, but neural network models predict interactions between plasticity at nearby synapses. Here we show in mouse hippocampal pyramidal cells that LTP at individual synapses reduces the threshold for potentiation at neighbouring synapses. After input-specific LTP induction by two-photon glutamate uncaging or by synaptic stimulation, subthreshold stimuli, which by themselves were too weak to trigger LTP, caused robust LTP and spine enlargement at neighbouring spines. Furthermore, LTP induction broadened the presynaptic-postsynaptic spike interval for spike-timing-dependent LTP within a dendritic neighbourhood. The reduction in the threshold for LTP induction lasted approximately 10 min and spread over approximately 10 microm of dendrite. These local interactions between neighbouring synapses support clustered plasticity models of memory storage and could allow for the binding of behaviourally linked information on the same dendritic branch.     

Repetitive motor learning induces coordinated formation of clustered dendritic spines in vivo

Many lines of evidence suggest that memory in the mammalian brain is stored with distinct spatiotemporal patterns. Despite recent progresses in identifying neuronal populations involved in memory coding, the synapse-level mechanism is still poorly understood. Computational models and electrophysiological data have shown that functional clustering of synapses along dendritic branches leads to nonlinear summation of synaptic inputs and greatly expands the computing power of a neural network. However, whether neighbouring synapses are involved in encoding similar memory and how task-specific cortical networks develop during learning remain elusive. Using transcranial two-photon microscopy, we followed apical dendrites of layer 5 pyramidal neurons in the motor cortex while mice practised novel forelimb skills. Here we show that a third of new dendritic spines (postsynaptic structures of most excitatory synapses) formed during the acquisition phase of learning emerge in clusters, and that most such clusters are neighbouring spine pairs. These clustered new spines are more likely to persist throughout prolonged learning sessions, and even long after training stops, than non-clustered counterparts. Moreover, formation of new spine clusters requires repetition of the same motor task, and the emergence of succedent new spine(s) accompanies the strengthening of the first new spine in the cluster. We also show that under control conditions new spines appear to avoid existing stable spines, rather than being uniformly added along dendrites. However, succedent new spines in clusters overcome such a spatial constraint and form in close vicinity to neighbouring stable spines. Our findings suggest that clustering of new synapses along dendrites is induced by repetitive activation of the cortical circuitry during learning, providing a structural basis for spatial coding of motor memory in the mammalian brain.     

Long-term dendritic spine stability in the adult cortex

The structural dynamics of synapses probably has a crucial role in the development and plasticity of the nervous system. In the mammalian brain, the vast majority of excitatory axo-dendritic synapses occur on dendritic specializations called 'spines'. However, little is known about their long-term changes in the intact developing or adult animal. To address this question we developed a transcranial two-photon imaging technique to follow identified spines of layer-5 pyramidal neurons in the primary visual cortex of living transgenic mice expressing yellow fluorescent protein. Here we show that filopodia-like dendritic protrusions, extending and retracting over hours, are abundant in young animals but virtually absent from the adult. In young mice, within the 'critical period' for visual cortex development, approximately 73% of spines remain stable over a one-month interval; most changes are associated with spine elimination. In contrast, in adult mice, the overwhelming majority of spines (approximately 96%) remain stable over the same interval with a half-life greater than 13 months. These results indicate that spines, initially plastic during development, become remarkably stable in the adult, providing a potential structural basis for long-term information storage.     

Functional mapping of single spines in cortical neurons in vivo

The individual functional properties and spatial arrangement of afferent synaptic inputs on dendrites have a critical role in the processing of information by neurons in the mammalian brain. Although recent work has identified visually-evoked local dendritic calcium signals in the rodent visual cortex, sensory-evoked signalling on the level of dendritic spines, corresponding to individual afferent excitatory synapses, remains unexplored. Here we used a new variant of high-resolution two-photon imaging to detect sensory-evoked calcium transients in single dendritic spines of mouse cortical neurons in vivo. Calcium signals evoked by sound stimulation required the activation of NMDA (N-methyl-D-aspartate) receptors. Active spines are widely distributed on basal and apical dendrites and pure-tone stimulation at different frequencies revealed both narrowly and widely tuned spines. Notably, spines tuned for different frequencies were highly interspersed on the same dendrites: even neighbouring spines were mostly tuned to different frequencies. Thus, our results demonstrate that NMDA-receptor-dependent single-spine synaptic inputs to the same dendrite are highly heterogeneous. Furthermore, our study opens the way for in vivo mapping of functionally defined afferent sensory inputs with single-synapse resolution.     

Analysis of calcium channels in single spines using optical fluctuation analysis

Most synapses form on small, specialized postsynaptic structures known as dendritic spines. The influx of Ca2+ ions into such spines--through synaptic receptors and voltage-sensitive Ca2+ channels (VSCCs)--triggers diverse processes that underlie synaptic plasticity. Using two-photon laser scanning microscopy, we imaged action-potential-induced transient changes in Ca2+ concentration in spines and dendrites of CA1 pyramidal neurons in rat hippocampal slices. Through analysis of the large trial-to-trial fluctuations in these transients, we have determined the number and properties of VSCCs in single spines. Here we report that each spine contains 1-20 VSCCs, and that this number increases with spine volume. We are able to detect the opening of a single VSCC on a spine. In spines located on the proximal dendritic tree, VSCCs normally open with high probability (approximately 0.5) following dendritic action potentials. Activation of GABA(B) receptors reduced this probability in apical spines to approximately 0.3 but had no effect on VSCCs in dendrites or basal spines. Our studies show that the spatial distribution of VSCC subtypes and their modulatory potential is regulated with submicrometre precision.     

Dendritic spine changes associated with hippocampal long-term synaptic plasticity.

Long-term enhancement of synaptic efficacy in the hippocampus is an important model for studying the cellular mechanisms of neuronal plasticity, circuit reorganization, and even learning and memory. Although these long-lasting functional changes are easy to induce, it has been very difficult to demonstrate that they are accompanied or even caused by morphological changes on the subcellular level. Here we combined a local superfusion technique with two-photon imaging, which allowed us to scrutinize specific regions of the postsynaptic dendrite where we knew that the synaptic changes had to occur. We show that after induction of long-lasting (but not short-lasting) functional enhancement of synapses in area CA1, new spines appear on the postsynaptic dendrite, whereas in control regions on the same dendrite or in slices where long-term potentiation was blocked, no significant spine growth occurred.     

Independent regulation of calcium revealed by imaging dendritic spines

The dendritic spine is a basic structural unit of neuronal organization. It is assumed to be a primary locus of synaptic plasticity, and to undergo long-term morphological and functional changes, at least some of which are regulated by intracellular calcium concentrations. It is known that physiological stimuli can cause marked increases in intracellular calcium levels in hippocampal dendritic shafts, but it is completely unknown to what extent such changes in the dendrites would also be seen by calcium-sensing structures within spines. Will calcium levels in all spines change in parallel with the dendrite or will there be a heterogeneous response? This study, through direct visualization and measurement of intracellular calcium concentrations in individual living spines, demonstrates that experimentally evoked changes in calcium concentrations in the dendritic shaft ([Ca2+]d).     

Glutamate induces de novo growth of functional spines in developing cortex

Mature cortical pyramidal neurons receive excitatory inputs onto small protrusions emanating from their dendrites called spines. Spines undergo activity-dependent remodelling, stabilization and pruning during development, and similar structural changes can be triggered by learning and changes in sensory experiences. However, the biochemical triggers and mechanisms of de novo spine formation in the developing brain and the functional significance of new spines to neuronal connectivity are largely unknown. Here we develop an approach to induce and monitor de novo spine formation in real time using combined two-photon laser-scanning microscopy and two-photon laser uncaging of glutamate. Our data demonstrate that, in mouse cortical layer 2/3 pyramidal neurons, glutamate is sufficient to trigger de novo spine growth from the dendrite shaft in a location-specific manner. We find that glutamate-induced spinogenesis requires opening of NMDARs (N-methyl-D-aspartate-type glutamate receptors) and activation of protein kinase A (PKA) but is independent of calcium-calmodulin-dependent kinase II (CaMKII) and tyrosine kinase receptor B (TrkB) receptors. Furthermore, newly formed spines express glutamate receptors and are rapidly functional such that they transduce presynaptic activity into postsynaptic signals. Together, our data demonstrate that early neural connectivity is shaped by activity in a spatially precise manner and that nascent dendrite spines are rapidly functionally incorporated into cortical circuits.     

Activity-dependent dynamics and sequestration of proteasomes in dendritic spines

The regulated degradation of proteins by the ubiquitin proteasome pathway is emerging as an important modulator of synaptic function and plasticity. The proteasome is a large, multi-subunit cellular machine that recognizes, unfolds and degrades target polyubiquitinated proteins. Here we report NMDA (N-methyl-D-aspartate) receptor-dependent redistribution of proteasomes from dendritic shafts to synaptic spines upon synaptic stimulation, providing a mechanism for local protein degradation. Using a proteasome-activity reporter and local perfusion, we show that synaptic stimulation regulates proteasome activity locally in the dendrites. We used restricted photobleaching of individual spines and dendritic shafts to reveal the dynamics that underlie proteasome sequestration, and show that activity modestly enhances the entry rate of proteasomes into spines while dramatically reducing their exit rate. Proteasome sequestration is persistent, reflecting an association with the actin-based cytoskeleton. Together, our data indicate that synaptic activity can promote the recruitment and sequestration of proteasomes to locally remodel the protein composition of synapses.     

Regulation of distinct AMPA receptor phosphorylation sites during bidirectional synaptic plasticity

Bidirectional changes in the efficacy of neuronal synaptic transmission, such as hippocampal long-term potentiation (LTP) and long-term depression (LTD), are thought to be mechanisms for information storage in the brain. LTP and LTD may be mediated by the modulation of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazloe proprionic acid) receptor phosphorylation. Here we show that LTP and LTD reversibly modify the phosphorylation of the AMPA receptor GluR1 subunit. However, contrary to the hypothesis that LTP and LTD are the functional inverse of each other, we find that they are associated with phosphorylation and dephosphorylation, respectively, of distinct GluR1 phosphorylation sites. Moreover, the site modulated depends on the stimulation history of the synapse. LTD induction in naive synapses dephosphorylates the major cyclic-AMP-dependent protein kinase (PKA) site, whereas in potentiated synapses the major calcium/calmodulin-dependent protein kinase II (CaMKII) site is dephosphorylated. Conversely, LTP induction in naive synapses and depressed synapses increases phosphorylation of the CaMKII site and the PKA site, respectively. LTP is differentially sensitive to CaMKII and PKA inhibitors depending on the history of the synapse. These results indicate that AMPA receptor phosphorylation is critical for synaptic plasticity, and that identical stimulation conditions recruit different signal-transduction pathways depending on synaptic history.     

Opioids block long-term potentiation of inhibitory synapses

Excitatory brain synapses are strengthened or weakened in response to specific patterns of synaptic activation, and these changes in synaptic strength are thought to underlie persistent pathologies such as drug addiction, as well as learning. In contrast, there are few examples of synaptic plasticity of inhibitory GABA (gamma-aminobutyric acid)-releasing synapses. Here we report long-term potentiation of GABA(A)-mediated synaptic transmission (LTP(GABA)) onto dopamine neurons of the rat brain ventral tegmental area, a region required for the development of drug addiction. This novel form of LTP is heterosynaptic, requiring postsynaptic NMDA (N-methyl-d-aspartate) receptor activation at glutamate synapses, but resulting from increased GABA release at neighbouring inhibitory nerve terminals. NMDA receptor activation produces nitric oxide, a retrograde signal released from the postsynaptic dopamine neuron. Nitric oxide initiates LTP(GABA) by activating guanylate cyclase in GABA-releasing nerve terminals. Exposure to morphine both in vitro and in vivo prevents LTP(GABA). Whereas brief treatment with morphine in vitro blocks LTP(GABA) by inhibiting presynaptic glutamate release, in vivo exposure to morphine persistently interrupts signalling from nitric oxide to guanylate cyclase. These neuroadaptations to opioid drugs might contribute to early stages of addiction, and may potentially be exploited therapeutically using drugs targeting GABA(A) receptors.     

Postsynaptic NMDA receptor-mediated calcium accumulation in hippocampal CA1 pyramidal cell dendrites

In the CA1 hippocampal region, intracellular calcium is a putative second messenger for the induction of long-term potentiation (LTP), a persistent increase of synaptic transmission produced by high frequency afferent fibre stimulation. Because LTP in this region is blocked by the NMDA (N-methyl-D-aspartate) receptor antagonist AP5 (DL-2-amino-5-phosphonovaleric acid) and the calcium permeability of NMDA receptors is controlled by a voltage-dependent magnesium block, a model has emerged that suggests that the calcium permeability of NMDA receptor-coupled ion channels is the biophysical basis for LTP induction. We have performed microfluorometric measurements in individual CA1 pyramidal cells during stimulus trains that induce LTP. In addition to a widespread component of postsynaptic calcium accumulation previously described, we now report that brief high frequency stimulus trains produce a transient component spatially localized to dendritic areas near activated afferents. This localized component is blocked by the NMDA receptor antagonist AP5. The results directly confirm the calcium rise predicted by NMDA receptor models of LTP induction.     

Synaptic calcium transients in single spines indicate that NMDA receptors are not saturated.

At excitatory synapses in the central nervous system, the number of glutamate molecules released from a vesicle is much larger than the number of postsynaptic receptors. But does release of a single vesicle normally saturate these receptors? Answering this question is critical to understanding how the amplitude and variability of synaptic transmission are set and regulated. Here we describe the use of two-photon microscopy to image transient increases in Ca2+ concentration mediated by NMDA (N-methyl-D-aspartate) receptors in single dendritic spines of CA1 pyramidal neurons in hippocampal slices. To test for NMDA-receptor saturation, we compared responses to stimulation with single and double pulses. We find that a single release event does not saturate spine NMDA receptors; a second release occurring 10 ms later produces approximately 80% more NMDA-receptor activation. The amplitude of spine NMDA-receptor-mediated [Ca2+] transients (and the synaptic plasticity which depends on this) may thus be sensitive to the number of quanta released by a burst of action potentials and to changes in the concentration profile of glutamate in the synaptic cleft.     

Neurotrophic regulation of synapse development and plasticity

Neurotrophic factors are traditionally thought to be secretory proteins that regulate long-tern survival and differe, ntiation of neurons. Recent studies have revealed a previously unexpected role for these factors in synaptie de velopment ami plasticity in diverse neuronal populations. Here we review experimeuts carried oul in our own laboratory in the last few years.. We have made two important discoveries.First,we were among the first to report that brain-derived. neurotrophie faclor (BDNF) facilitates hippocampal hmg-term potentiation (LTP), a form of synaptic plaslicity believed to be involved in learning and memory. BDNF modulates LTP al CAI synapses by enhaneing synaptic responses to high frequency, tetanic slimulalion. This is achieved primafily by facilitating synaptie vesicle doeking, possibly due to an in crease in the levels of the vesicle prolein synaptobrevin and synaptoplysin in the nerve terminals. Gene knockout study demonstrates thai the effects of BDNF are primarily mediated through presynaptic mechanisms. Second, we demonstrated a form of long-term, neurotrophin-mediated synaptic regulation. We showed that long-term treatment of the neuromuscu lar synapses with neurotrophin-3 (NT3) resulted in an enhancement of both spontaneous and evoked synaptic currcuts, as well as profound changes in thc number of synaptic varicosities and syuaptic vesicle proteins in motoneurons, all of which are indicative of more mature synapses. Our current work addresses the following issues:(i) activity-dependent trafficking of neurotrophin receptors, and its role in synapse-specific modulation; (ii) signal transduction mechanisms medialing the acute enhancement of synaplic transmission by neurotrophins; (iii) acute and long-tenn synaptie actions of the GDNF family; (iv) role of BDNF in late-phase LTP and in the development of hippocampal circuit.     

Temporally distinct pre- and post-synaptic mechanisms maintain long-term potentiation

Long-term potentiation (LTP) in the hippocampus is widely studied as the mechanisms involved in its induction and maintenance are believed to underlie fundamental properties of learning and memory in vertebrates. Most synapses that exhibit LTP use an excitatory amino-acid neurotransmitter that acts on two types of receptor, the N-methyl-D-aspartate (NMDA) and quisqualate receptors. The quisqualate receptor mediates the fast synaptic response evoked by low-frequency stimulation, whereas the NMDA receptor system is activated transiently by tetanic stimulation, leading to the induction of LTP. The events responsible for maintaining LTP once it is established are not known. We now demonstrate that the sensitivity of CA1 neurons in hippocampal slices to ionophoretically-applied quisqualate receptor ligands slowly increases following the induction of LTP. This provides direct evidence for a functional post-synaptic change and suggests that pre-synaptic mechanisms also contribute, but in a temporally distinct manner, to the maintenance of LTP.     

Induction of dendritic spines by an extracellular domain of AMPA receptor subunit GluR2

Synaptic transmission from excitatory nerve cells in the mammalian brain is largely mediated by AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors located at the surface of dendritic spines. The abundance of postsynaptic AMPA receptors correlates with the size of the synapse and the dimensions of the dendritic spine head. Moreover, long-term potentiation is associated with the formation of dendritic spines as well as synaptic delivery of AMPA receptors. The molecular mechanisms that coordinate AMPA receptor delivery and spine morphogenesis are unknown. Here we show that overexpression of the glutamate receptor 2 (GluR2) subunit of AMPA receptors increases spine size and density in hippocampal neurons, and more remarkably, induces spine formation in GABA-releasing interneurons that normally lack spines. The extracellular N-terminal domain (NTD) of GluR2 is responsible for this effect, and heterologous fusion proteins of the NTD of GluR2 inhibit spine morphogenesis. We propose that the NTD of GluR2 functions at the cell surface as part of a receptor-ligand interaction that is important for spine growth and/or stability.     

Kainate receptors are involved in synaptic plasticity

The ability of synapses to modify their synaptic strength in response to activity is a fundamental property of the nervous system and may be an essential component of learning and memory. There are three classes of ionotropic glutamate receptor, namely NMDA (N-methyl-D-aspartate), AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid) and kainate receptors; critical roles in synaptic plasticity have been identified for two of these. Thus, at many synapses in the brain, transient activation of NMDA receptors leads to a persistent modification in the strength of synaptic transmission mediated by AMPA receptors. Here, to determine whether kainate receptors are involved in synaptic plasticity, we have used a new antagonist, LY382884 ((3S, 4aR, 6S, 8aR)-6-((4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-decahydro isoquinoline-3-carboxylic acid), which antagonizes kainate receptors at concentrations that do not affect AMPA or NMDA receptors. We find that LY382884 is a selective antagonist at neuronal kainate receptors containing the GluR5 subunit. It has no effect on long-term potentiation (LTP) that is dependent on NMDA receptors but prevents the induction of mossy fibre LTP, which is independent of NMDA receptors. Thus, kainate receptors can act as the induction trigger for long-term changes in synaptic transmission.