Remodeling of Synaptic Structure in Sensory Cortical Areas In Vivo

Imaging spine motility in sensory cortex in vivo

We studied the motility of dendritic spines in vivo in visual, somatosensory, and auditory cortices of P28 mice. We concentrated our measurements on distal spines located on the apical tuft of layer 5 pyramidal neurons (Fig. 1A,B). Because this population of spines had been studied previously at this age in primary visual cortex (Majewska and Sur, 2003), we wanted to extend those findings to understand whether basal spine motility is linked to sensory input, cortical area, or a sensory critical period. For consistency with previous studies, spine motility was quantified as the absolute change in length (from dendrite to the tip of the protrusion) per unit time (Lendvai et al., 2000; Majewska and Sur, 2003). Visual, somatosensory, and auditory cortices were identified on the basis of stereotaxic components during in vivo imaging. At the end of the imaging session, the location was further verified by injecting a tracer into the imaged area and fixing the brain. The injections were then identified in fixed sections and compared with the mouse atlas (supplemental Fig. 1 A, available at www.jneurosci.org as supplemental material). In most cases, imaged areas were located in the primary sensory cortex, although occasionally, secondary sensory cortices were imaged. In some animals, imaging in primary cortex was confirmed by allowing the dye to transport for 2 d and observing labeling in the appropriate nucleus of the sensory thalamus (supplemental Fig. 1 B, available at www.jneurosci.org as supplemental material).

Imaged spines in all sensory areas showed dynamic changes in shape and length as described previously in vivo and in vitro (Fischer et al., 1998; Dunaevsky et al., 1999; Lendvai et al., 2000; Majewska and Sur, 2003; Oray et al., 2004). The changes included elongation, retraction, spine shape change and displacement, and filopodial growth. Motile spines were frequently located next to stable spines, suggesting that spine motility is regulated on a spine-by-spine basis (Fig. 1).

Spines are more dynamic in auditory and somatosensory cortices than in visual cortex

Dendritic spines located on the apical tuft of layer 5 neurons in the visual cortex were the most stable of the protrusions studied (0.0215 ± 0.00050 μm/min; n = 261 spines from 6 animals) (Fig. 2A). As observed previously (Majewska and Sur, 2003), at P28, most spines in this population show adult morphologies, with the majority being mushroom spines (62%) and few filopodia (4%) being observed (Fig. 2B). Additionally, stubby and mushroom spines were most stable and abundant, whereas thin spines were significantly more motile (p < 0.05) but comprised <10% of the spine population. Filopodia were rarely found but were always highly motile compared with spines (p < 0.001).

Spines located in the primary somatosensory cortex were significantly more motile at this age than those in visual cortex (0.0250 ± 0.0008 μm/min; n = 221 spines from 4 animals; p < 0.001) (Fig. 2A). Again, most spines in somatosensory cortex were adult-like mushroom spines (55%), but more thin spines (16%) and filopodia (9%) were observed than in visual cortex (Holtmaat et al., 2005) (Fig. 2B). Mushroom spines in somatosensory cortex were more motile than those in visual cortex (visual, 0.0205 ± 0.0005 μm/min; somatosensory, 0.0247 ± 0.0008 μm/min; p < 0.001). There were no significant differences in the motility of stubby spines, thin spines, or filopodia (p > 0.1).

Interestingly, dendritic spines located in the auditory cortex were also significantly more motile than those in visual cortex (0.0271 ± 0.0010 μm/min; n = 236 spines from 6 animals; p < 0.001) (Fig. 2A). This effect was not caused by an overabundance of motile filopodia in the sample, because most protrusions exhibited spine-like morphologies (97%, with 56% classified as mushroom spines), and the incidence of filopodia was comparable with that observed in visual cortex and lower than that observed in somatosensory cortex (Fig. 2B). Additionally, all spine classes (mushroom, stubby, and thin) showed increased motility (p < 0.05) compared with visual cortical spines, apart from filopodia, which were equally motile in all sensory cortices studied (Fig. 2C).

Rewiring visual input into auditory cortex does not downregulate spine motility

The differences observed between the motility of dendritic spines in different sensory cortices could depend on the nature of the sensory input or on factors intrinsic to the cortical area itself. To distinguish between these two possibilities, we imaged spine motility in the same spine population in animals in which retinal input had been routed into the auditory thalamus, providing visual drive to auditory cortex (Sharma et al., 2000; Lyckman et al., 2001). We reasoned that, if the nature of the sensory input were instructive to structural plasticity, then spines in auditory cortex of rewired mice would show increased stability when compared with wild-type mice. Spines in the auditory cortex of rewired mice, however, did not show increased stability (0.0272 ± 0.00057 μm/min; n = 281 spines from 8 animals) (Fig. 2A). In fact, spine motility in auditory cortex of rewired mice was not statistically different from that in normal mice and was statistically elevated from that observed in visual cortex (p < 0.001), suggesting that the modality of sensory input alone is not enough to alter basal spine motility.

Imaging spine and axon terminal turnover in vivo

To determine whether the differences observed in spine motility between different cortices at P28 translate to differences in the remodeling of cortical circuitry in the adult animal, we performed long-term imaging experiments of both postsynaptic dendritic spines and presynaptic axon terminals (Fig. 3) (see Fig. 5). In one set of animals, control images were taken at P40, and the same areas were then reimaged at P47. In another group of animals, control images were taken at P40 and then the same areas were reimaged at P61. Animals were imaged through thinned skull, and the scalp skin was sutured between imaging sessions. At the end of the last imaging session, tracer was injected into the imaged region and the imaged area was later identified on the basis of this injection in fixed sections (supplemental Fig. 1, available at www.jneurosci.org as supplemental material).

Dendritic spines are equally persistent in visual, somatosensory, and auditory cortices

Previous studies showed contradictory results regarding spine persistence: one study yielded high turnover rates in somatosensory cortex (Trachtenberg et al., 2002), whereas another study showed highly stable spines in visual cortex (Grutzendler et al., 2002). Because we had already established differing rates of spine dynamics in different sensory areas in juvenile (P28) animals, we wondered whether the different sensory regions analyzed in the two studies could account for these discrepancies in turnover. To address this issue, we measured dendritic spine turnover in visual, somatosensory, and auditory cortices.

Turnover measurements were performed over a 1–3 week period in young adult animals (starting at P40) to aid in comparison with previous studies (Fig. 3). Cortical spines were remarkably stable over 1 week (stable spines, 89.0 ± 5.4% of all protrusions; n = 1490 in 13 animals) as well as over 3 weeks (stable spines, 84.4 ± 9.7% of all protrusions; n = 1154 in 15 animals). At these ages, spine loss (13.4 ± 7.1%) was greater than spine gain (8.8 ± 7.4%; p < 0.005). These data suggest that there is a small (∼15%) subpopulation of synapses that remodel over a period of 1 week in young adult animals. The number of spines that turn over is, however, an overestimate given that any changes in imaging conditions might allow some spines to fall below or above the detection threshold on different imaging days.

Turnover rates were comparable in all three sensory areas studied (Fig. 3C). Approximately 90% of spines persisted over a period of 1 week (visual, 93.2 ± 1.77%, n = 282 spines in 3 animals; somatosensory, 88.8 ± 3.96%, n = 345 in 3 animals; auditory, 91.4 ± 4.5%, n = 286 spines in 4 animals). Dendritic spines were also persistent over a period of 3 weeks in all sensory areas studied (Fig. 3C). Eighty to 90% of the same spines could be reidentified after a period of 3 weeks (visual, 78.2 ± 12.1%, n = 450 spines in 6 animals; somatosensory, 87.5 ± 5.4%, n = 309 spines in 3 animals; auditory, 88.5 ± 7.3, n = 290 spines in 4 animals). This suggests that there is either a large population of spines that remains stable during early adulthood and a smaller population that turns over or that there is a limited amount of turnover after the animal is older than P40.

Dendritic spine types and turnover

At the ages studied (P28–P61), most spines had adult mushroom-like morphologies (supplemental Fig. 2, available at www.jneurosci.org as supplemental material). Between P28 and P40, there was a general decrease in the number of thin spines and filopodia in all sensory modalities (Zuo et al., 2005) and an increase in the number of stubby spines. After P40, there was little change in spine class composition, and the types of spines present did not differ significantly between the different sensory modalities studied (p > 0.05).

Mushroom spines were most common at these ages and were therefore responsible for both the stability and the limited turnover observed. Mushroom spines made up ∼60% of all stable protrusions and ∼40% of both lost and new protrusions (Fig. 4A). Thin spines were less common (∼15% of the total spine population) but were less likely to be stable than either mushroom or stubby spines. A total of 90% of all mushroom and stubby spines were stable over 3 weeks, whereas only 72% of thin spines persisted during this time (Fig. 4B), suggesting that thin spines may comprise a transient population that is susceptible to remodeling (Holtmaat et al., 2005). We cannot rule out that some of the thin spines we observed were filopodia in the process of extension or retraction.

To determine whether changes in spine shape occurred in stable synapses in early adulthood, we classified spines based on morphology in the first and second imaging sessions. We found that most spine types, excluding filopodia, maintained their classification throughout the imaging interval. Thus, >50% of all mushroom, thin, and stubby spines could be reidentified with similar morphologies 1 or 3 weeks later (Fig. 4C, Table 1). Mushroom spines were least likely to change morphology, however, with >80% maintaining a mushroom-like structure over 3 weeks. Thin spines were significantly more likely to undergo morphological changes (p < 0.005), with only 55% maintaining a thin-like structure over 3 weeks, again suggesting that thin spines are more susceptible to remodeling. Additionally, more remodeling was observed in the 3 versus 1 week imaging period (Fig. 4C) in all spine types, suggesting that morphological changes are ongoing in the cortex and may reflect activity-dependent plasticity. This type of analysis captures only large changes in spine morphology, and it is likely that more subtle changes in spine size are more prevalent. These data were pooled from all cortical areas, and no significant differences were observed between visual, auditory, and somatosensory cortices.

Axon terminals are persistent in visual, somatosensory, and auditory cortices

To determine the stability of presynaptic structures, we investigated the rates of axon terminal turnover in the same images used for dendritic spine turnover analysis. Axon terminals were identified on the basis of their morphology, as thick round structures or finger, spine-like protrusions on axons (Fig. 5A,B). In fixed sections, such structures colocalized with synapsin staining, indicating that they are likely to be functional presynaptic sites (supplemental Fig. 1C, available at www.jneurosci.org as supplemental material). Axon terminals were highly stable in all cortices studied (Fig. 5C). After a 1 week period, >90% of axon terminals could be reidentified (visual, 90.0 ± 0.8%, n = 258 in 3 animals; somatosensory, 92.9 ± 3.4%, n = 231 in 3 animals; auditory, 94.9 ± 3.3%, n = 220 in 4 animals).

In a 3 week imaging period, axon terminals were also persistent, with 80–90% of the same terminals being present in both imaging sessions (visual, 88.3 ± 5.44%, n = 425 in 6 animals; somatosensory, 91.8 ± 3.5%, n = 133 in 3 animals; auditory, 94.8 ± 3.9%, n = 236 in 4 animals) (Fig. 5C). Again, rates of terminal turnover were low in 3 weeks of imaging, indicating that either a small population of terminals turns over or that turnover becomes less prevalent after P40. Terminals in the visual cortex turned over more than those in auditory cortex (p < 0.05). Also, rates of terminal loss and gain were well matched in all cortices during the 3 week imaging interval.

To understand whether changes in cortical circuitry occur by coordinated turnover of both presynaptic and postsynaptic elements, we compared dendritic spine and axon terminal turnover rates in our images. On average, at these ages, axon terminals were more stable than dendritic spines (p < 0.001) (Fig. 6A,B). This was true for spines in visual, auditory, and somatosensory cortices. In all areas, the increased turnover of dendritic spines was primarily attributable to an increased rate of spine loss compared with that of axon terminals (Fig. 6A,C).

Rewiring visual input into the auditory cortex does not affect synaptic persistence

We investigated whether changing the type of sensory input to a cortical area would affect spine and axon terminal turnover by rewiring visual projections into the auditory system and investigating spine and terminal persistence in auditory cortex. This manipulation did not affect basal spine turnover rates in auditory cortex in either the 1 or 3 week imaging period (rewired cortex at 1 week, 87.3 ± 7.6%, n = 577 in 4 animals; 3 weeks, 87.9 ± 3.5%, n = 253 in 3 animals) (Fig. 3B). Spines were more likely to disappear (1 week, 6.8 ± 1.8%; 3 weeks, 12.1 ± 3.5%) than to appear (1 week, 5.1 ± 2.1%; 3 weeks, 6.5 ± 2.8%). Axon terminals were equally stable in rewired and nonrewired auditory cortex and turned over less than those in visual cortex (p < 0.05). Similar to the other sensory regions studied, terminals were more stable than dendritic spines (rewired cortex at 1 week, 93.8 ± 3.8%, n = 415 in 4 animals; 3 weeks, 95.3 ± 3.7%, n = 219 in 3 animals) (Fig. 5B).

Dendritic spine stability is likely to be a general feature of cortical circuitry

Our results so far have been limited to synapses occurring on the apical tuft of layer 5 neurons in sensory cortices in young adult animals. To determine whether persistence of dendritic spines is limited to this subset of synapses or whether it is a more general feature of cortical synapses, we imaged dendritic spines of layer 5 neurons in the motor cortex of one P40 animal. After 3 weeks, ∼80% of dendritic spines were stable in this animal, suggesting that dendritic spine stability might not be an exclusive feature of sensory cortex (stable spines, 87.5%; new spines, 12.5%; lost spines, 18.9%; n = 71 spines).

Additionally, in some cases, layer 2/3 neurons in somatosensory cortex were also labeled. We imaged dendritic spines on the proximal apical dendrite of these neurons up to 300 μm below the surface of the pia. Over a 1 week period, 90% of spines were stable with more spines being lost than being formed de novo (stable spines, 90.6 ± 3.7%; new spines, 3.3 ± 3.4%; lost spines, 9.4 ± 3.7%; n = 187 spines from 2 animals) (Fig. 7). Additionally, axon terminals were also very stable (stable, 94.5 ± 2.6%; new, 2.8 ± 3.3%; lost, 5.5 ± 2.6%; n = 137 from 2 animals). These rates of spine formation and disassembly are comparable with those seen on the apical tuft of layer 5 neurons in visual, somatosensory, and auditory cortices (see above). The data from layer 2/3 and from layer 5 neurons in motor cortex suggest that it is likely that stability of presynaptic and postsynaptic structures in adult animals is a general property of cortical pyramidal neurons.

Activity dependence of spine dynamics

Morphological changes in spines may be involved in the formation of new connections during synaptogenesis and network remodeling (Bonhoeffer and Yuste, 2002). Our experiments suggest that, in young animals, spines turn over infrequently but undergo morphological changes. Thin spines are the most motile spines at P28 and are the most likely to turn over and change morphology at P40. This supports the view that thin spines carry weak synapses (Matsuzaki et al., 2001) and, when potentiated, grow to become mushroom spines (Matsuzaki et al., 2004; Okamoto et al., 2004). Thin spines have been implicated previously as preferential sites of synaptic remodeling (Holtmaat et al., 2005), whereas large mushroom spines may be resistant to both potentiation and additional increases in size (Matsuzaki et al., 2004). In our study, we did not see preferential transitions between spine classes, suggesting that neck length, neck diameter, and head diameter can be regulated independently. These parameters may be fine tuned by activity allowing spines to regulate synaptic calcium increases (Majewska et al., 2000a; Noguchi et al., 2005; Oertner and Matus, 2005).

Differences in spine dynamics between sensory cortical areas

Differences in basal spine motility between sensory areas suggest that, in vivo, spine motility is driven by sensory input. Our rewiring experiments, however, suggest that this is unlikely. In rewired animals, the auditory cortex experiences patterns of activity driven by visual input commencing shortly after birth. Synaptic dynamics in these animals, however, are not affected by this different activity pattern. There are several explanations compatible with this result. First, activity may be important during the critical period, which closes at approximately P28 in auditory cortex (Zhang et al., 2001). Similarly, activity-dependent effects on spine motility in somatosensory (Lendvai et al., 2000) and visual (Majewska and Sur, 2003; Oray et al., 2004) cortices decline by P14 and P28, respectively. Second, effects on spine motility were observed after gross manipulation of activity in vitro (Fischer et al., 2000; Korkotian and Segal, 2001) or deprivation paradigms in vivo (Lendvai et al., 2000; Majewska and Sur, 2003; Oray et al., 2004), suggesting that overall levels of activity may be more important than its fine structure. Third, rewiring may affect too few synapses to be detected with our imaging paradigm. This is unlikely because retinal projections to the auditory thalamus are extensive, spanning the entire ventral division of the MGN (Ellsworth et al., 2005). Furthermore, rewiring elicits functional changes in the network architecture of auditory cortex and can drive visual responses and behavior (Sharma et al., 2000; von Melchner et al., 2000; Newton et al., 2004). We favor the fourth possibility, that cues intrinsic to the cortical area regulate synaptic dynamics.

Dendritic spines are persistent in adult cortex

Previous studies yielded contradictory results on dendritic spine turnover in young adult cortex. In somatosensory cortex, 60% of spines remained stable over 8 d (Trachtenberg et al., 2002). In visual cortex, however, 90% of spines persisted over a period of months (Grutzendler et al., 2002). Recent reports agree that, in older animals (>3 months) and longer imaging intervals, spines are equally stable in both cortices (Holtmaat et al., 2005; Zuo et al., 2005). Our data in juvenile animals confirm that spines in somatosensory cortex are more motile than those in visual cortex. Additionally, in developing animals, thin spines and filopodia are more prevalent in somatosensory cortex (Holtmaat et al., 2005). However, these differences are not apparent when examining spine turnover in young adult animals, and, at 6–9 weeks, spine class composition is similar in the two areas. Our data support the view that spines in visual and somatosensory cortices are stable (∼85% over 3 weeks starting at P40). At these ages, we also find higher rates of spine elimination than addition, in agreement with Zuo et al. (2005). Additionally, our data suggest that spine stability may be a general feature of cortical neurons.

Although surgical preparation could be responsible for the difference in turnover in these studies, recent experiments show that comparable images are obtained with the window (Trachtenberg et al., 2002; Holtmaat et al., 2005) and thinned skull (Grutzendler et al., 2002; Zuo et al., 2005) preparations. Our control experiments also showed comparable results in the two preparations (see Materials and Methods). Additionally, differences in spine motility were observed in thinned skull preparations at P28, and thin spines were easily found in somatosensory cortex at these ages. It is possible that other factors affect spine turnover. For instance, the frequency of imaging could cause photodamage or increase the delivery of anesthetics that affect cortical function and spine morphology (Kaech et al., 1999). Additionally, there might be differences caused by environmental and rearing conditions (Cancedda et al., 2004).

Although our data point to spines being more stable than originally reported in somatosensory cortex (Trachtenberg et al., 2002), it is necessary to note that, over 3 weeks, 10–20% of spines are lost and ∼10% are formed. Although some of this turnover is possibly explained by limitations of the imaging technique in identifying functional synapses, it is likely that the adult cortex maintains a level of structural plasticity that is greater than previously expected. It is likely that the remodeling of synapses made onto inhibitory neurons is governed by different rules as dendrites of these cells are structrally more plastic than those on pyramidal neurons (Lee et al., 2006). Thus, although the adult cortex learns and adapts through altering the strength of existing synapses, it likely also remodels through physically disconnecting and wiring up new synapses. Such changes in connectivity must be tightly regulated so as not to affect long-term memory but allow flexibility during learning of new behaviors. The fact that adult synapses are structurally labile may prove important for the treatment of brain trauma and age-related cognitive defects.

Spines are more plastic than axon terminals

Although spine turnover rates have been studied, little is known about the dynamics of axonal structures (but see Portera-Cailliau et al., 2005). Our data suggest that axon terminals are highly stable, confirming previous reports on synaptic motility in the cerebellum (Deng and Dunaevsky, 2005). Because higher actin concentrations are generally associated with motile structures, it is perhaps not surprising that the presynaptic terminal with less actin is more stable than the actin-filled postsynaptic spine (Matus et al., 1982). The difference in turnover rates is primarily mediated by a lower rate of terminal loss in the young adult animal. This suggests that dendritic spines are overproduced during synaptogenesis and that pruning of spurious synapses in the mature animal progresses through the elimination of excess dendritic spines.

At least three technical aspects must be considered in these data. First, boutons are larger than spines and are therefore easier to detect in vivo. It is unlikely, however, that the differences observed are attributable to detection thresholds, because axons, which are much thinner and dimmer than most spines, were always visible in subsequent imaging sessions. Additionally, finger-like terminals were also more persistent than dendritic spines (data not shown). Second, although spine morphology has routinely been used to identify postsynaptic sites, boutons determined morphologically may not correspond to functional presynaptic sites (Shepherd and Harris, 1998). Unless our morphological criteria do not pick up a population of transient presynapses, this would, however, lead to an underestimation of the stability of terminals. Third, we cannot be sure that the bouton population we are studying makes synapses with the spines imaged. Although in some cases we see close apposition of these two elements suggesting that connections between them are possible, determining the connectivity would require an enormous effort and is beyond the scope of this study.

Our results suggest that dendritic spines are persistent in sensory cortices and, possibly, in all of cortex. A small population of spines, however, turns over even in adult cortex, possibly allowing for pruning and rearrangements of synapses. This scenario is appealing in that it allows the setting up of a coarse scaffold of connectivity during development that can be refined through changes in synaptic strength but not easily dismantled or rearranged in the adult animal (Filbin, 2003). In fact, presynaptic elements appear more structurally stable than dendritic spines, suggesting that they act as a scaffold for connectivity, whereas spines carry most of the structural plasticity in adult synapses. Indeed, our data show that, although spines are persistent in the adult animal, they still undergo changes in structure, suggesting that they retain a level of structural plasticity that might correspond to changes in synaptic strength.