Abstract

Sensory experience alters neuronal circuits, which is believed to form the basis for learning and memory. On a microscopic level, structural changes of the neuronal network are prominently observable as experience-dependent addition and removal of cortical dendritic spines. By environmental enrichment, we here applied broad sensory stimulation to mice and followed the consequences to dendritic spines in the somatosensory cortex utilizing in vivo microscopy. Additionally to apical dendrites of layer V neurons, which are typically analyzed in in vivo imaging experiments, we investigated basal dendrites of layer II/III neurons and describe for the first time experience-dependent alterations on this population of dendrites. On both classes of cortical dendrites, enriched environment-induced substantial changes determined by increases in density and turnover of dendritic spines. Previously established spines were lost after enriched stimulation. A fraction of experience-induced gained spines survived for weeks, which might therefore be functionally integrated into the neuronal network. Furthermore, we observed an increased density of spines that appeared only transiently. Together, we speculate that the cognitive benefits seen in environmental-enriched animals might be a consequence of both, a higher connectivity of the neuronal network due to more established synapses and an enhanced flexibility due to more transient spines.

Introduction

Plastic changes in the neuronal network are believed to form the basis for learning and memory (Bailey and Kandel 1993). These changes occur at synapses either on a molecular level altering the synaptic strength or on a structural level like through the addition and removal of synapses. In the mammalian neocortex, the ability of the network to modulate its connections is most prominent in the developing brain (Hubel and Wiesel 1970; Katz and Shatz 1996), but plasticity was found to remain throughout life (Stepanyants et al. 2002; Chklovskii et al. 2004).

Since Ramon y Cajal dendritic spines have been studied extensively, not least as they can be explored easily with microscopic techniques due to their characteristic morphology. Dendritic spines are small protrusions along the dendrites of pyramidal neurons with a size of 0.5–2 µm and constitute the postsynaptic site of the majority of excitatory synapses (Gray 1959; Nimchinsky et al. 2002). In organotypic slice cultures of hippocampal neurons, a local electrical stimulation, which was sufficient to induce long-term potentiation, lead to the de novo formation of dendritic spines (Engert and Bonhoeffer 1999; Maletic-Savatic et al. 1999; Nagerl et al. 2004, 2007). On the contrary, the retraction of spines was observed after low-frequency stimulation (Nagerl et al. 2004). Time-lapse imaging of dendritic spines in the brain of living mice was enabled by the combined use of multiphoton laser scanning microscopy with transgenic animals expressing fluorescent proteins in neurons (Grutzendler et al. 2002; Trachtenberg et al. 2002). In vivo imaging in the murine cortex revealed 2 classes of dendritic spines, due to their temporal characteristics. Spines that persist for 8 days are in large part stable for much longer time periods and are therefore defined as persistent spines, distinguished from transient spines (Holtmaat et al. 2005). Ultrastructural analysis of in vivo imaged dendritic spines showed that persistent spines have all features of a mature synapse, whereas transient spines lack a presynaptic partner (Knott et al. 2006). Similar results were obtained in cultured hippocampal neurons, confirming that spinogenesis precedes the formation of synapses (Nagerl et al. 2007).

Sensory manipulation was found to strongly affect spine dynamics in vivo in various areas of the cortex of adult mice (reviewed in Fu and Zuo 2011). Most of these investigations used adverse, denervating experimental paradigms like whisker trimming or visual deprivation. Only recently studies report that specific learning tasks like a grabing task, the rotarod, or fear conditioning are associated specifically with either the de novo appearance or, in the latter case, the loss of dendritic spines in corresponding cortical regions (Xu et al. 2009; Yang et al. 2009; Lai et al. 2012). Learning-induced new spines correlate positively with the behavioral outcome and were stable for months. Together, these observations suggest that the experience-dependent formation and the elimination of dendritic spines modulate the cortical neuronal network and might therefore represent a physical basis for life-long memory storage (Xu et al. 2009; Yang et al. 2009).

However, little is known about broad physiological stimulation on the kinetics of cortical dendritic spines. Here, we studied in detail structural plasticity induced by an extensive enriched environment in the somatosensory cortex. Environmental enrichment usually involves a combination of increased sensory, cognitive, and motor stimulation (van Praag et al. 2000). The enrichment stimulation has been shown to improve learning and memory (Meshi et al. 2006), ameliorate neuropathology in models of neurodegenerative diseases like Alzheimer's disease and Parkinsons disease (Bezard et al. 2003; Lazarov et al. 2005), and beneficially influence the consequences of brain injury (Kolb and Gibb 1991). On the structural level, an enriched environment alters brain weight (Bennett et al. 1969) and dendritic branching (Greenough et al. 1973; Mohammed et al. 2002) and increases hippocampal neurogenesis (Kempermann et al. 1997). The spine density of enriched animals was found to be elevated in static ex vivo analysis of rodent brain regions, including the somatosensory cortex (Kolb et al. 2003; Leggio et al. 2005; Gelfo et al. 2009).

The present study aims to expand this current state of knowledge by a temporal description of enriched environment-induced alterations on cortical dendrites, utilizing in vivo microscopy. This allows to quantify changes in spine density at the same dendritic element and to follow the fate of individual spines over time. Additionally, we not only focused on typically in vivo imaged tuft apical dendrites, but also included with basal dendrites of layer II/III neurons a second, poorly characterized population of dendrites.

Materials and Methods

Animals and Housing

Female mice expressing yellow fluorescent protein (YFP) under control of Thy-1 promoter were used in all experiments (YFP-H line) (Feng et al. 2000). Control mice were singly housed in standard cages (30 × 15 × 20 cm). For environmental enrichment, a large cage (80 × 50 × 40 cm) was used with a maximum of 10 animals. The cage was equipped with various nesting materials, platforms, tunnels, huts, and toys of various materials and textures. Furthermore, at least 2 running wheels were provided at a time to allow voluntary physical activity. The content of the cage was relocated and substituted for every 2–3 days, and the location of food and drinking facilities was changed as well. For the infant-start experiment, pregnant mice were transferred to the enriched cage, and after birth the rearranging of the cage was halted for 10 days. At an age of 3 weeks, mothers and male littermates were removed from enrichment, matching the time when control mice were separated from their mother. After surgery or imaging, enriched mice were kept isolated until they fully recovered from anesthesia. Supplementary Figure S1 summarizes in detail the procedure of the infant-start and the adult-start experiment.

All procedures were in accordance with an animal protocol approved by the University of Munich and the government of upper Bavaria.

As a general side note, it shall be mentioned that the broad sensory stimulation provided by the enriched environment rather resembles the natural condition of rodents. Standard caging as it is used by us and others as the control situation might therefore represent an impoverished condition, which might impact the conclusions and the values of such experiments.

Cranial Window Surgery

For in vivo imaging, a chronic cranial window was prepared as described previously (Fuhrmann et al. 2007; Holtmaat et al. 2009). The mice were anesthetized with an intraperitoneal injection of ketamine/xylazine (0.14/0.01 mg/g body weight; WDT/Bayer Health Care). Additionally, dexamethasone (6 µg/g body weight; Sigma) was intraperitoneally administered immediately before surgery. Utilizing the open-skull preparation, a cranial window was placed above the somatosensory cortex. For repositioning during repetitive imaging a small metal bar, containing a hole for a screw, was glued next to the window. After surgery, mice received subcutaneously analgesic treatment with carprophen (7.5 µg/g body weight; Pfizer) and antibiotic treatment with cefotaxim (0.25 mg/g body weight; Pharmore).

Two-Photon In Vivo Imaging

Imaging began after a 3–4-week recovery period post surgery utilizing a LSM510 setup (Zeiss) being equipped with a MaiTai laser (Spectra Physics). For in vivo imaging, mice were anesthetized by an intraperitoneal injection of ketamine/xylazine. Imaging sessions lasted for no longer than 60 min, and laser power was kept below 50 mW to avoid phototoxic effects. Two-photon excitation of YFP was performed at 880 nm, and a ×40 0.8 N water-immersion objective (Zeiss) was used. Stereological coordinates were used to locate the somatosensory cortex. Overview images were taken at low resolution (≈0.4 µm/pixel per image frame in 3 µm steps) up to a depth of 350 µm. These overviews included all cell bodies of YFP-labeled layer II/III neurons, typically at depth of 200–300 µm (Supplementary Fig. S2). Consequently, apical tuft dendrites protruding below the depth of 350 µm arise from layer V pyramidal neurons. Dendritic elements were imaged at high resolution (≈0.1 µm/pixel per frame with 1 µm z-resolution). Care was taken to ensure similar fluorescence levels in space and time.

Image Processing and Data Analysis

Fluorescence images were deconvolved (AutoQuantX2, Media Cybernetics) and filtered with an edge-preserving algorithm, followed by a local contrast change (Imaris 5.0.1, Bitplane). Image processing was performed to enhance contrast, to reduce noise, and to sharpen details, without introducing artifacts (Supplementary Fig. S2). For illustration of in vivo imaged dendrites, distracting neighboring dendritic elements were removed using Adobe Photoshop. Dendritic spines were counted manually and blinded to the experimental condition. In time-series images, a dendritic spine was identified as the same if its location did not change within a range of 1 µm along the dendrite. Because of resolution limitations in z-dimension, only protrusions emanating laterally from the dendritic shaft were analyzed.

Statistical testing was carried out using the GraphPad Prisms software. For column comparisons, student's t-test was conducted to control for statistical significance. To test time-series experiments, 2-way analysis of variance was performed (the significance level is indicated behind the corresponding graph), followed by Bonferroni post-testing for the single time-point comparison (the significance level is indicated above the corresponding data point). All results are reported as mean ± standard error of the mean (SEM), and differences were considered to be significant at P < 0.05.

Results

Infant-Start Enrichment on Spines of Apical Tuft Dendrites of Layer V Neurons

In a first set of experiments, we studied mice that were kept in the enriched environment from birth to elucidate the maximal effect enrichment can have on the neuronal network. Therefore, pregnant mice were placed in the enriched cage that was designed as complex as possible, containing various objects and toys of different materials and textures (Fig. 1A). After weaning, only the female offspring remained in the enrichment. Cranial window implantation was performed at an age of 3 months (week 13) and at 4 months (week 17) 2-photon in vivo microscopy started (Fig. 1B, Supplementary Fig. S1). Parallel-treated single-caged mice at the same age served as controls.

Figure 1.

Infant-start–enriched environment induces structural plasticity on apical tuft dendrites of layer V neurons. (A) Schematic drawing of an enriched environment next to a standard cage. (B) Transcranial in vivo 2-photon imaging. (C) Lateral view of fluorescently labeled cortical neurons in YFP-H mice. Apical tuft dendrites of layer V neurons were imaged at depth of about 20–70 µm. The basal dendrites of layer II/III neurons are more difficult to image, which was performed typically at depth of about 150–250 µm (scale bar 100 µm). (D) Time series over 30 days of representative in vivo imaged dendrites. Black triangles mark exemplarily dendritic spines that are stable over the whole imaging period. Black and empty arrows mark all gained and lost spines, respectively (scale bar 5 µm). (E) Spine density is elevated in enriched mice and stable over the whole period observed (enriched group: 41 dendrites/6 mice/a total of 1667 µm dendrite analyzed; controls: 37 dendrites/5 mice/a total of 1907 µm dendrite analyzed). (F) Gained and (G) lost spine densities are increase to a similar amount in the enriched group. (H–K) The fate of gained spines, formed within the first 3 imaging time points, was analyzed. (H) The dendritic spines that were newly formed within the first 3 imaging sessions were followed over the consecutive imaging sessions. (J) More newly gained spines in the enriched mice survived a minimum of 22 days. (K) More newly gained spines in the enriched mice disappeared within 8 days after formation and are therefore classified as transient spines (error bars indicate ±SEM; ***P > 0.001, **P > 0.01).

Figure 1.

Infant-start–enriched environment induces structural plasticity on apical tuft dendrites of layer V neurons. (A) Schematic drawing of an enriched environment next to a standard cage. (B) Transcranial in vivo 2-photon imaging. (C) Lateral view of fluorescently labeled cortical neurons in YFP-H mice. Apical tuft dendrites of layer V neurons were imaged at depth of about 20–70 µm. The basal dendrites of layer II/III neurons are more difficult to image, which was performed typically at depth of about 150–250 µm (scale bar 100 µm). (D) Time series over 30 days of representative in vivo imaged dendrites. Black triangles mark exemplarily dendritic spines that are stable over the whole imaging period. Black and empty arrows mark all gained and lost spines, respectively (scale bar 5 µm). (E) Spine density is elevated in enriched mice and stable over the whole period observed (enriched group: 41 dendrites/6 mice/a total of 1667 µm dendrite analyzed; controls: 37 dendrites/5 mice/a total of 1907 µm dendrite analyzed). (F) Gained and (G) lost spine densities are increase to a similar amount in the enriched group. (H–K) The fate of gained spines, formed within the first 3 imaging time points, was analyzed. (H) The dendritic spines that were newly formed within the first 3 imaging sessions were followed over the consecutive imaging sessions. (J) More newly gained spines in the enriched mice survived a minimum of 22 days. (K) More newly gained spines in the enriched mice disappeared within 8 days after formation and are therefore classified as transient spines (error bars indicate ±SEM; ***P > 0.001, **P > 0.01).

The density of dendritic spines of apical tuft dendrites of layer V neurons (Fig. 1C) in the somatosensory cortex was elevated by a third in the enriched group (mean ± SEM; 0.46 ± 0.02 vs. 0.33 ± 0.02/µm dendrite, P > 0.001) and stayed stable over an imaging period of 30 days (Fig. 1D,E). Whereas the majority of dendritic spines were stable under both conditions, the turnover of spines in the enriched group was more than doubled. Specifically, the density of spines that were gained (0.034 ± 0.004 vs. 0.016 ± 0.002/µm dendrite, P > 0.001) and lost (0.035 ± 0.002 vs. 0.014 ± 0.002/µm dendrite, P > 0.001) between 2 imaging sessions was found to be dramatically increased in the enriched mice (Fig. 1D,F,G). The rates for gained and lost spines within the experimental groups are comparable, confirming the stable net spine density.

The fate of newly gained dendritic spines, which were formed within the time span of the first 3 imaging time points, was followed (Fig. 1H). A higher number of these gained spines persisted for at least 22 days in the enriched mice (0.032 ± 0.004 vs. 0.015 ± 0.003/µm dendrite, P = 0.0057) and are therefore believed to be functionally integrated into the neuronal network (Knott et al. 2006; Fig. 1H,J). Nevertheless, more of these new spines were only transiently present in the enriched mice (0.044 ± 0.005 vs. 0.013 ± 0.003/µm dendrite, P > 0.001; Fig. 1K).

Infant-Start Enrichment on Spines of Basal Dendrites of Layer II/III Neurons

In vivo microscopy of experience-dependent changes on basal dendrites of layer II/III neurons has not been reported so far, as these dendrites are located typically at depth of about 150–300 µm and are therefore relatively difficult to image (Supplementary Fig. S2). Under control conditions, the spine density of basal dendrites of layer II/III neurons (Fig. 1C) is higher when compared with apical tuft dendrites of layer V neurons (0.67 ± 0.02/µm dendrite). Infant-start enriched caging leads to an increased spine density of 0.84 ± 0.02/µm dendrite (P > 0.001; Fig. 2A,B). Spine turnover was found to be elevated as well, evidenced by enhanced gain of spines (0.047 ± 0.005 vs. 0.018 ± 0.002/µm dendrite, P > 0.001) and loss of spines (0.043 ± 0.004 vs. 0.020 ± 0.002/µm dendrite; Fig. 2C,D). Similar to the results for apical dendrites of layer V neurons, a higher number of newly gained spines persisted over 3 weeks (0.035 ± 0.006 vs. 0.020 ± 0.004/µm dendrite, P = 0.035), and the density of transient spines increased (0.043 ± 0.007 vs. 0.012 ± 0.003/µm dendrite, P > 0.001; Fig. 2EH).

Figure 2.

Effect of infant-start environmental enrichment on spines of basal dendrites of layer II/III neurons. (A) Representative images of dendrites with gained (black arrow) and lost (empty arrow) spines (scale bar 5 µm). (B) The enriched mice display a higher spine density with (C) more gained spines and (D) more lost spines (enriched group: 34 dendrites/4 mice/a total of 1152 µm dendrite analyzed; controls: 34 dendrites/4 mice/a total of 1627 µm dendrite analyzed). (E) The newly gained spines, formed within the first 8 days of imaging, were followed for at least 22 days. The enriched mice display (F) an enhanced density of spines surviving at least 22 days after formation and (G) an enhanced density of transient spines (error bars indicate ±SEM; ***P > 0.001, **P > 0.05).

Figure 2.

Effect of infant-start environmental enrichment on spines of basal dendrites of layer II/III neurons. (A) Representative images of dendrites with gained (black arrow) and lost (empty arrow) spines (scale bar 5 µm). (B) The enriched mice display a higher spine density with (C) more gained spines and (D) more lost spines (enriched group: 34 dendrites/4 mice/a total of 1152 µm dendrite analyzed; controls: 34 dendrites/4 mice/a total of 1627 µm dendrite analyzed). (E) The newly gained spines, formed within the first 8 days of imaging, were followed for at least 22 days. The enriched mice display (F) an enhanced density of spines surviving at least 22 days after formation and (G) an enhanced density of transient spines (error bars indicate ±SEM; ***P > 0.001, **P > 0.05).

Adult-Start Enrichment Triggers Structural Plasticity of Apical Tuft Dendrites of Layer V Neurons

For the second part of our study, we were interested to elucidate how the mature neuronal network of single-caged mice reacts toward the broad stimulation of an enriched environment. For this purpose, we implanted a cranial window in adult female mice at an age of 4–5 months (17–21 weeks) that were caged under control conditions. Imaging started at 5–6 months of age (20–25 weeks) for 2 time points to record the baseline status of cortical dendrites. One day before the third imaging session, the mice were transferred to the enriched environment, and the stimulation-induced changes were followed at the previously imaged dendrites (Fig. 3A, Supplementary Fig. S2). Parallel-treated single-caged mice at a comparable age served as controls.

Figure 3.

Adult-start–enriched environment induces changes in structural plasticity on apical tuft dendrites of layer V neurons. (A) Representative time-series images of a mouse that was first kept under control conditions (d0 and d7) before it was placed into the enriched environment. Black and empty arrows indicate gained and lost spines, respectively (scale bar 5 µm). (B) Dendritic spine density increases as a consequence of environmental enrichment stimulation (enriched group: 45 dendrites/8 mice/a total of 2391 µm dendrite analyzed; controls: 31 dendrites/4 mice/a total of 1500 µm dendrite analyzed). (C) The density of newly formed spines significantly rises at the first imaging time point after enrichment, but declines after 14 days. (D) The rise in the density of lost spines is delayed and less strong than the gained spines. (E) Plotted is the mean of the density of gained spines within the first 4 weeks of enrichment (x-axis) versus the mean of the density of lost spines (y-axis) for each single dendrite. The even distribution of the spots of the enriched group indicates no cluster of affected dendrites. (F) A higher number of established spines (stable over both base line imaging time points) is lost after the initiation of enrichment. (G) The fate of newly formed at the first 2 imaging time points in enrichment was followed the consecutive imaging sessions and compared with newly gained spines of controls. (H) In the enriched group, more of these newly gained spines are stable over 28 days. (J) More transient spines are present in enriched mice (error bars indicate ±SEM; ***P > 0.001, **P > 0.01, *P > 0.05).

Figure 3.

Adult-start–enriched environment induces changes in structural plasticity on apical tuft dendrites of layer V neurons. (A) Representative time-series images of a mouse that was first kept under control conditions (d0 and d7) before it was placed into the enriched environment. Black and empty arrows indicate gained and lost spines, respectively (scale bar 5 µm). (B) Dendritic spine density increases as a consequence of environmental enrichment stimulation (enriched group: 45 dendrites/8 mice/a total of 2391 µm dendrite analyzed; controls: 31 dendrites/4 mice/a total of 1500 µm dendrite analyzed). (C) The density of newly formed spines significantly rises at the first imaging time point after enrichment, but declines after 14 days. (D) The rise in the density of lost spines is delayed and less strong than the gained spines. (E) Plotted is the mean of the density of gained spines within the first 4 weeks of enrichment (x-axis) versus the mean of the density of lost spines (y-axis) for each single dendrite. The even distribution of the spots of the enriched group indicates no cluster of affected dendrites. (F) A higher number of established spines (stable over both base line imaging time points) is lost after the initiation of enrichment. (G) The fate of newly formed at the first 2 imaging time points in enrichment was followed the consecutive imaging sessions and compared with newly gained spines of controls. (H) In the enriched group, more of these newly gained spines are stable over 28 days. (J) More transient spines are present in enriched mice (error bars indicate ±SEM; ***P > 0.001, **P > 0.01, *P > 0.05).

On apical tuft dendrites of layer V neurons, spine density increases after the mice were placed in the enriched cage (Fig. 3B). Significant differences were observed after 16 days, when spine density was elevated by about 20%. There was a dramatic increase in the formation of new spines at the first imaging session after enrichment stimulation (Fig. 3C). The enhanced density of gained spines remained at a high order for 2 weeks before declining to a level that was still above the baseline (Fig. 3C). Interestingly, the increase in lost spines started later and did not reach the range of gained spines (Fig. 3D). Questioning if changes in spine turnover were restricted to a cluster of specific dendrites, we plotted the gained and lost spine densities for every imaged dendrite (Fig. 3E). This analysis revealed an equal distribution of turnover per dendrite favoring a rather broad effect regarding all dendrites. Furthermore, this illustration highlights that the equilibrium of gain and loss of spines in control mice is shifted toward gained spines after environmental enrichment.

Next, we ask how established synapses react on the enrichment-associated stimulation. The fate of dendritic spines, which were stable over the 2 time points of baseline imaging and therefore considered as persistent spines, was followed over the whole period of enrichment. Whereas, in control animals, 86.8 ± 1.2% of established spines survived, enriched mice had a survival of only 80.4 ± 1.4% persistent spines (P > 0.001; Fig. 3F). This indicates a significant reorganization of the neuronal network after enriched environment.

Furthermore, we analyzed spines that were newly formed within the first 2 imaging sessions after enriched environment. A much higher number of gained spines remained stable over the following 4 weeks compared with control levels (0.044 ± 0.004 vs. 0.013 ± 0.003/µm dendrite, P > 0.001; Fig. 3H). The density of transient spines was elevated as well in the enriched group (0.025 ± 0.003 vs. 0.008 ± 0.002/µm dendrite, P > 0.001; Fig. 3J). Interestingly, comparing the survival of gained spines between the infant-start and the adult-start–enriched animals at a similar time point reveals a higher fraction of newly stable integrated spines in the latter group (infant start: 37% survival; adult start: 60% survival; Figs 1H and 3G).

Adult-Start Enrichment Triggered Structural Plasticity of Basal Dendrites of Layer II/III Neurons

The enrichment-induced changes of dendritic spines of basal dendrites of layer II/III recapitulate in a similar way the findings described above for apical tuft dendrites of layer V neurons. Spine density increases by more than 20% due to an instant rise in gained spines which is accompanied by a delayed and less prominent increase in lost spines (Fig. 4AD). The observed changes in lost and gained spines are distributed evenly over the imaged dendritic branches (Fig. 4E). More persistent spines, which have existed under control conditions, get lost after acute enrichment stimulation over the period observed when compared with single-caged animals (12.7 ± 1.1% vs. 8.2 ± 1.2%, P < 0.001; Fig. 4F). Likewise, it turned out that enrichment-induced newly gained spines are to a higher proportion stable until the end of the imaging period (0.060 ± 0.006 vs. 0.016 ± 0.004/µm dendrite, P < 0.001; Fig. 4H). The density of transient spines is enhanced in the enriched group as well (0.021 ± 0.004 vs. 0.010 ± 0.003/µm dendrite, P < 0.05; Fig. 4J).

Figure 4.

Basal dendrites of layer II/III neurons are in a similar way affected by the adult-start–enriched environment as apical tuft dendrites of layer V neuron. (A) Representative time-series images of a mouse that was first kept under control conditions (d0 and d7) before it was placed into the enriched environment. Black and empty arrows indicate gained and lost spines, respectively (scale bar 5 µm). (B) Dendritic spine density increases after enrichment (enriched group: 41 dendrites/5 mice/a total of 1246 µm dendrite analyzed; controls: 18 dendrites/4 mice/a total of 785 µm dendrite analyzed). (C) The density of newly formed spines significantly rises at the first imaging time point after enrichment. (D) The rise in the density of lost spines is delayed and less strong than the gained spines. (E) Plotted is the mean of the density of gained spines within the first 4 weeks of enrichment (x-axis) versus the mean of the density of lost spines (y-axis) for each single dendrite. The even distribution of the spots of the enriched group indicates no cluster of affected dendrites. (F) A higher number of established spines (stable over both base line imaging time points) is lost after the initiation of enrichment. (G) The fate of newly gained spines, formed at the first 2 imaging time points after enriched stimulation, was followed the consecutive imaging sessions and compared with the gained spines of controls. (H) In the enriched group, more of these newly gained spines are stable over 28 days. (J) More transient spines are present in enriched mice (error bars indicate ±SEM; ***P > 0.001, **P > 0.01, *P > 0.05).

Figure 4.

Basal dendrites of layer II/III neurons are in a similar way affected by the adult-start–enriched environment as apical tuft dendrites of layer V neuron. (A) Representative time-series images of a mouse that was first kept under control conditions (d0 and d7) before it was placed into the enriched environment. Black and empty arrows indicate gained and lost spines, respectively (scale bar 5 µm). (B) Dendritic spine density increases after enrichment (enriched group: 41 dendrites/5 mice/a total of 1246 µm dendrite analyzed; controls: 18 dendrites/4 mice/a total of 785 µm dendrite analyzed). (C) The density of newly formed spines significantly rises at the first imaging time point after enrichment. (D) The rise in the density of lost spines is delayed and less strong than the gained spines. (E) Plotted is the mean of the density of gained spines within the first 4 weeks of enrichment (x-axis) versus the mean of the density of lost spines (y-axis) for each single dendrite. The even distribution of the spots of the enriched group indicates no cluster of affected dendrites. (F) A higher number of established spines (stable over both base line imaging time points) is lost after the initiation of enrichment. (G) The fate of newly gained spines, formed at the first 2 imaging time points after enriched stimulation, was followed the consecutive imaging sessions and compared with the gained spines of controls. (H) In the enriched group, more of these newly gained spines are stable over 28 days. (J) More transient spines are present in enriched mice (error bars indicate ±SEM; ***P > 0.001, **P > 0.01, *P > 0.05).

Discussion

In the present study, we show that enriched environment strongly influences dendritic spines in the somatosensory cortex in both of our experimental in vivo paradigms, the infant-start and the adult-start enrichment. At 2 populations of dendrites, apical tuft dendrites of layer V neurons and basal dendrites of layer II/III neurons, the spine density is elevated, and the formation and elimination of spines are enhanced. More new spines are stabilized, and the sudden enrichment also leads to the elimination of established spines. This indicates that substantial and lasting changes of the neuronal network are induced by the enriched environment.

In postmortem tissue of rodents, studies have revealed an enrichment-induced increase in dendritic spine density in the hippocampus (Rampon et al. 2000) and striatum (Comery et al. 1995) as well as in various areas of the neocortex (Kolb et al. 2003; Leggio et al. 2005; Gelfo et al. 2009). In the cortex, this was consistent for apical and basal dendritic segments, although one study found no effect specifically on apical dendrites of layer V neurons in the parietal cortex (Gelfo et al. 2009). With the help of in vivo imaging, we confirmed that the enhanced cortical spine density induced by the enriched environment in an unbiased manner, as we were able to compare the same dendritic elements before and during enriched stimulation.

Beyond that, the in vivo approach certainly provides important information on spine turnover. The experience-dependent increase in the formation and elimination of dendritic spines has been shown mainly with adverse sensory manipulations, and only recently physiological challenges have also been used for stimulation (Fu and Zuo 2011). Consistent with our findings, Yang et al. (2009) also found a rapid formation of new spines and an increased elimination of existing spines in the somatosensory cortex, when whiskers were permanently stimulated. In an enrichment similar to ours, Fu et al. (2012) observed a doubled spine formation in the motor cortex within 4 days. All of these studies exclusively focused on apical tuft dendrites and, therefore, we additionally describe here for the first time experience-dependent changes on the basal dendrites of layer II/III neurons in vivo. Although showing under standard conditions a higher spine density, basal dendrites of layer II/III neurons are influenced by the enriched environment in the same way as apical dendrites in every parameter recorded here.

The mammalian cerebral cortex exhibits highest plasticity in developmental stages, when synapses are initially overproduced, but substantially decrease to form the mature, more stable neuronal network (Rakic et al. 1986; Hua and Smith 2004; Petanjek et al. 2011). Similarly, in the murine cortex the density of dendritic spines was found to decline in postnatal development reaching a balance of continuous gain and loss of spines at an age of about 4 months (Holtmaat et al. 2005; Zuo et al. 2005). We here observed a more pronounced elevation of spine density in infant-start–enriched mice, which was not reached by the adult-start group. This difference might therefore result from experiencing the enriched environment during the postnatal neuronal system development. Although the adult neuronal circuits retain the capability of structural changes, the young network features a higher flexibility. Interestingly, in aged rats, the enrichment-induced increase in spine density did not attain a significant difference in the cortex, whereas in the hippocampus of the same mice, the elevation in spine density was still prominent (Darmopil et al. 2009; Rasin et al. 2011). Hence, development but also aging influences the ability of structural spine plasticity in a brain region-dependant manner.

In the adult-start experiment, the switch from control to enriched housing was accompanied by a substantial rearrangement of the neuronal network. Importantly, the changes of the neuronal system started with the formation and stabilization of new spines, which was counteracted at later time points by a loss of spines. Xu et al. (2009) describe for the grabbing task a similar kinetic with a rapid gain of spines, which is later on followed by the elimination of pre-existing spines. For this specific task, the loss of spines leads to a balancing of synapse number resulting in an unaltered spine density. The broad stimulation of enriched housing might therefore cover a different quality of structural changes, leading to a net gain in spine density.

But how and to what degree do these structural changes in the neuronal network, induced by enriched environment, reflect specific experiences? The fact that environmental enrichment influences brain structure at various stages, ranging from increased brain weight, increased gliogenesis and hippocampal neurogenesis, and enhanced synaptogenesis (Bennett et al. 1969; Kolb and Gibb 1991; Kempermann et al. 1997; Soffie et al. 1999) argues for a more general influence. Furthermore in several brain regions, neurotrophic factors like brain-derived neurotrophic factor and nerve growth factor (NGF) are increased by enriched housing (Ickes et al. 2000). In line with this is our finding that modifications in dendritic spine turnover are evenly distributed over the imaged dendritic branches. Additionally, the observation that apical tuft dendrites of layer V neurons and basal dendrites of layer II/III neurons are in a similar way affected by enriched stimulation further speaks for general impact of the enriched environment on the neuronal network.

On the other hand, our results on structural plasticity parallel what has been shown for specific learning tasks, namely a grabbing task and motor learning on the rotarod (Xu et al. 2009; Yang et al. 2009). In all studies, established spines get lost after the new stimulus. Additionally, out of a large pool of newly gained spines only few get stabilized over long time, which might therefore represent a physical correlate of new memorizable experiences. Hence, one could imagine a combination of both, one more general aspect and one specific aspect of structural changes of dendritic spines in the enriched mice. Interestingly, the 2 classes of spines, persistent spines and transient spines, both are significant elevated under enriched conditions. Therefore, we speculate that the cognitive benefits seen in environmental-enriched animals might result from 1) a higher connectivity of the neuronal network, due to an increased number of established synapses and 2) an enhanced flexibility, due to more transient spines that, if needed, could be transformed into functional synapses.

Supplementary Material

Supplementary material can be found at: http://www.cercor.oxfordjournals.org/.

Funding

This work was supported by the Deutsche Forschungsgemeinschaft (SFB 596) and the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung, 13N11130).

Notes

Specifically, we thank Sonja Steinbach for her dedication and excellent technical support. We further thank M. Biel, S. Michalakis, K. Keppler, M. Fuhrmann, S. Burgold, M. Dorostkar, and A. Gupta for helpful advice in planning the experiments and preparing the manuscript. Conflict of Interest: None declared.

References

Bailey
CH
Kandel
ER
Structural changes accompanying memory storage
Annu Rev Physiol
 , 
1993
, vol. 
55
 (pg. 
397
-
426
)
Bennett
EL
Rosenzweig
MR
Diamond
MC
Rat brain: effects of environmental enrichment on wet and dry weights
Science
 , 
1969
, vol. 
163
 (pg. 
825
-
826
)
Bezard
E
Dovero
S
Belin
D
Duconger
S
Jackson-Lewis
V
Przedborski
S
Piazza
PV
Gross
CE
Jaber
M
Enriched environment confers resistance to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and cocaine: involvement of dopamine transporter and trophic factors
J Neurosci
 , 
2003
, vol. 
23
 (pg. 
10999
-
11007
)
Chklovskii
DB
Mel
BW
Svoboda
K
Cortical rewiring and information storage
Nature
 , 
2004
, vol. 
431
 (pg. 
782
-
788
)
Comery
TA
Shah
R
Greenough
WT
Differential rearing alters spine density on medium-sized spiny neurons in the rat corpus striatum: evidence for association of morphological plasticity with early response gene expression
Neurobiol Learn Mem
 , 
1995
, vol. 
63
 (pg. 
217
-
219
)
Darmopil
S
Petanjek
Z
Mohammed
AH
Bogdanovic
N
Environmental enrichment alters dentate granule cell morphology in oldest-old rat
J Cell Mol Med
 , 
2009
, vol. 
13
 (pg. 
1845
-
1856
)
Engert
F
Bonhoeffer
T
Dendritic spine changes associated with hippocampal long-term synaptic plasticity
Nature
 , 
1999
, vol. 
399
 (pg. 
66
-
70
)
Feng
G
Mellor
RH
Bernstein
M
Keller-Peck
C
Nguyen
QT
Wallace
M
Nerbonne
JM
Lichtman
JW
Sanes
JR
Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP
Neuron
 , 
2000
, vol. 
28
 (pg. 
41
-
51
)
Fu
M
Yu
X
Lu
J
Zuo
Y
Repetitive motor learning induces coordinated formation of clustered dendritic spines in vivo
Nature
 , 
2012
, vol. 
483
 (pg. 
92
-
95
)
Fu
M
Zuo
Y
Experience-dependent structural plasticity in the cortex
Trends Neurosci
 , 
2011
, vol. 
34
 (pg. 
177
-
187
)
Fuhrmann
M
Mitteregger
G
Kretzschmar
H
Herms
J
Dendritic pathology in prion disease starts at the synaptic spine
J Neurosci
 , 
2007
, vol. 
27
 (pg. 
6224
-
6233
)
Gelfo
F
De Bartolo
P
Giovine
A
Petrosini
L
Leggio
MG
Layer and regional effects of environmental enrichment on the pyramidal neuron morphology of the rat
Neurobiol Learn Mem
 , 
2009
, vol. 
91
 (pg. 
353
-
365
)
Gray
EG
Electron microscopy of synaptic contacts on dendrite spines of the cerebral cortex
Nature
 , 
1959
, vol. 
183
 (pg. 
1592
-
1593
)
Greenough
WT
Volkmar
FR
Juraska
JM
Effects of rearing complexity on dendritic branching in frontolateral and temporal cortex of the rat
Exp Neurol
 , 
1973
, vol. 
41
 (pg. 
371
-
378
)
Grutzendler
J
Kasthuri
N
Gan
WB
Long-term dendritic spine stability in the adult cortex
Nature
 , 
2002
, vol. 
420
 (pg. 
812
-
816
)
Holtmaat
A
Bonhoeffer
T
Chow
DK
Chuckowree
J
De Paola
V
Hofer
SB
Hubener
M
Keck
T
Knott
G
Lee
WC
, et al.  . 
Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window
Nat Protoc
 , 
2009
, vol. 
4
 (pg. 
1128
-
1144
)
Holtmaat
AJ
Trachtenberg
JT
Wilbrecht
L
Shepherd
GM
Zhang
X
Knott
GW
Svoboda
K
Transient and persistent dendritic spines in the neocortex in vivo
Neuron
 , 
2005
, vol. 
45
 (pg. 
279
-
291
)
Hua
JY
Smith
SJ
Neural activity and the dynamics of central nervous system development
Nat Neurosci
 , 
2004
, vol. 
7
 (pg. 
327
-
332
)
Hubel
DH
Wiesel
TN
The period of susceptibility to the physiological effects of unilateral eye closure in kittens
J Physiol
 , 
1970
, vol. 
206
 (pg. 
419
-
436
)
Ickes
BR
Pham
TM
Sanders
LA
Albeck
DS
Mohammed
AH
Granholm
AC
Long-term environmental enrichment leads to regional increases in neurotrophin levels in rat brain
Exp Neurol
 , 
2000
, vol. 
164
 (pg. 
45
-
52
)
Katz
LC
Shatz
CJ
Synaptic activity and the construction of cortical circuits
Science
 , 
1996
, vol. 
274
 (pg. 
1133
-
1138
)
Kempermann
G
Kuhn
HG
Gage
FH
More hippocampal neurons in adult mice living in an enriched environment
Nature
 , 
1997
, vol. 
386
 (pg. 
493
-
495
)
Knott
GW
Holtmaat
A
Wilbrecht
L
Welker
E
Svoboda
K
Spine growth precedes synapse formation in the adult neocortex in vivo
Nat Neurosci
 , 
2006
, vol. 
9
 (pg. 
1117
-
1124
)
Kolb
B
Gibb
R
Environmental enrichment and cortical injury: behavioral and anatomical consequences of frontal cortex lesions
Cereb Cortex
 , 
1991
, vol. 
1
 (pg. 
189
-
198
)
Kolb
B
Gorny
G
Soderpalm
AH
Robinson
TE
Environmental complexity has different effects on the structure of neurons in the prefrontal cortex versus the parietal cortex or nucleus accumbens
Synapse
 , 
2003
, vol. 
48
 (pg. 
149
-
153
)
Lai
CS
Franke
TF
Gan
WB
Opposite effects of fear conditioning and extinction on dendritic spine remodelling
Nature
 , 
2012
, vol. 
483
 (pg. 
87
-
91
)
Lazarov
O
Robinson
J
Tang
YP
Hairston
IS
Korade-Mirnics
Z
Lee
VM
Hersh
LB
Sapolsky
RM
Mirnics
K
Sisodia
SS
Environmental enrichment reduces Abeta levels and amyloid deposition in transgenic mice
Cell
 , 
2005
, vol. 
120
 (pg. 
701
-
713
)
Leggio
MG
Mandolesi
L
Federico
F
Spirito
F
Ricci
B
Gelfo
F
Petrosini
L
Environmental enrichment promotes improved spatial abilities and enhanced dendritic growth in the rat
Behav Brain Res
 , 
2005
, vol. 
163
 (pg. 
78
-
90
)
Maletic-Savatic
M
Malinow
R
Svoboda
K
Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity
Science
 , 
1999
, vol. 
283
 (pg. 
1923
-
1927
)
Meshi
D
Drew
MR
Saxe
M
Ansorge
MS
David
D
Santarelli
L
Malapani
C
Moore
H
Hen
R
Hippocampal neurogenesis is not required for behavioral effects of environmental enrichment
Nat Neurosci
 , 
2006
, vol. 
9
 (pg. 
729
-
731
)
Mohammed
AH
Zhu
SW
Darmopil
S
Hjerling-Leffler
J
Ernfors
P
Winblad
B
Diamond
MC
Eriksson
PS
Bogdanovic
N
Environmental enrichment and the brain
Prog Brain Res
 , 
2002
, vol. 
138
 (pg. 
109
-
133
)
Nagerl
UV
Eberhorn
N
Cambridge
SB
Bonhoeffer
T
Bidirectional activity-dependent morphological plasticity in hippocampal neurons
Neuron
 , 
2004
, vol. 
44
 (pg. 
759
-
767
)
Nagerl
UV
Kostinger
G
Anderson
JC
Martin
KA
Bonhoeffer
T
Protracted synaptogenesis after activity-dependent spinogenesis in hippocampal neurons
J Neurosci
 , 
2007
, vol. 
27
 (pg. 
8149
-
8156
)
Nimchinsky
EA
Sabatini
BL
Svoboda
K
Structure and function of dendritic spines
Annu Rev Physiol
 , 
2002
, vol. 
64
 (pg. 
313
-
353
)
Petanjek
Z
Judas
M
Simic
G
Rasin
MR
Uylings
HB
Rakic
P
Kostovic
I
Extraordinary neoteny of synaptic spines in the human prefrontal cortex
Proc Natl Acad Sci USA
 , 
2011
, vol. 
108
 (pg. 
13281
-
13286
)
Rakic
P
Bourgeois
JP
Eckenhoff
MF
Zecevic
N
Goldman-Rakic
PS
Concurrent overproduction of synapses in diverse regions of the primate cerebral cortex
Science
 , 
1986
, vol. 
232
 (pg. 
232
-
235
)
Rampon
C
Tang
YP
Goodhouse
J
Shimizu
E
Kyin
M
Tsien
JZ
Enrichment induces structural changes and recovery from nonspatial memory deficits in CA1 NMDAR1-knockout mice
Nat Neurosci
 , 
2000
, vol. 
3
 (pg. 
238
-
244
)
Rasin
MR
Darmopil
S
Petanjek
Z
Tomic-Mahecic
T
Mohammed
AH
Bogdanovic
N
Effect of environmental enrichment on morphology of deep layer III and layer V pyramidal cells of occipital cortex in oldest-old rat—a quantitative golgi cox study
Coll Antropol
 , 
2011
, vol. 
35
 
Suppl 1
(pg. 
253
-
258
)
Soffie
M
Hahn
K
Terao
E
Eclancher
F
Behavioural and glial changes in old rats following environmental enrichment
Behav Brain Res
 , 
1999
, vol. 
101
 (pg. 
37
-
49
)
Stepanyants
A
Hof
PR
Chklovskii
DB
Geometry and structural plasticity of synaptic connectivity
Neuron
 , 
2002
, vol. 
34
 (pg. 
275
-
288
)
Trachtenberg
JT
Chen
BE
Knott
GW
Feng
G
Sanes
JR
Welker
E
Svoboda
K
Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex
Nature
 , 
2002
, vol. 
420
 (pg. 
788
-
794
)
van Praag
H
Kempermann
G
Gage
FH
Neural consequences of environmental enrichment
Nat Rev Neurosci
 , 
2000
, vol. 
1
 (pg. 
191
-
198
)
Xu
T
Yu
X
Perlik
AJ
Tobin
WF
Zweig
JA
Tennant
K
Jones
T
Zuo
Y
Rapid formation and selective stabilization of synapses for enduring motor memories
Nature
 , 
2009
, vol. 
462
 (pg. 
915
-
919
)
Yang
G
Pan
F
Gan
WB
Stably maintained dendritic spines are associated with lifelong memories
Nature
 , 
2009
, vol. 
462
 (pg. 
920
-
924
)
Zuo
Y
Lin
A
Chang
P
Gan
WB
Development of long-term dendritic spine stability in diverse regions of cerebral cortex
Neuron
 , 
2005
, vol. 
46
 (pg. 
181
-
189
)