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Stability of synaptic plasticity in the adult rat visual cortex induced by complex environment exposure
Brain Research 1018 (2004) 130 – 135 www.elsevier.com/locate/brainres
Short communication
Stability of synaptic plasticity in the adult rat visual cortex induced by complex environment exposure Teresita L. Briones a,*, Anna Y. Klintsova b, William T. Greenough c,d,e,f a
Department of Medical-Surgical Nursing, University of Illinois, 845 S. Damen Ave., Rm 707, M/C 802, Chicago, IL 60612, USA b Department of Psychology, Binghamton University, Binghamton, NY, USA c Beckman Institute, University of Illinois, Urbana, IL, USA d Department of Psychology, University of Illinois, Urbana, IL, USA e Department of Psychiatry, University of Illinois, Urbana, IL, USA f Department of Cell and Structural Biology, University of Illinois, Urbana, IL, USA Accepted 4 June 2004 Available online
Abstract Studies have demonstrated the effects of complex environment (EC) housing on brain plasticity both during postnatal development and in adulthood, but it is not clear how long these plastic changes persist nor what happens when environmental exposure is discontinued. Here we examined layer IV in the visual cortex of adult male rats for the: (1) effects of EC housing on synaptic plasticity, and (2) persistence of the synaptic changes after withdrawal from the complex environment. Fifty-eight adult male Long Evans rats were assigned to either: EC, socially paired housing (SC), or individual housing (IC). These rats remained in their assigned environment for 30 days. After 30 days, all rats in SC and some animals from the EC and IC groups were removed and perfused. The remaining animals in EC were then assigned to either remain in EC (ECEC) or be subsequently housed in IC (ECIC) for another 30 days. Similarly, rats in the IC group either remained in IC (ICIC) or were subsequently housed in EC (ICEC) for another 30 days. Electron microscopy results showed that all rats exposed to EC had significantly more synapses/neuron compared to SC, IC, and ICIC animals. Longer exposure to EC (ECEC) did not result in statistically more synapses per neuron; however, decreased neuron volume was seen. EC-induced synaptic changes persisted for an additional 30 days after withdrawal from EC (ECIC) confirming that EC-induced plastic changes occur in the brain regardless of age and indicating that once changes occur they tend to persist. D 2004 Elsevier B.V. All rights reserved. Keywords: Synaptic plasticity; Complex environment; Stereology; Learning; Memory; Enriched environment
1. Introduction Naturally occurring morphological plasticity is an ongoing process that can be evoked by environmental stimulation. Over the past 40 years, there have been numerous reports on the effects of complex environment exposure on brain morphology and function in young animals. Morphological changes in the brain in response to experience have been described in the nervous system of a wide array of species including monkeys, cats, rats, birds, honeybees, and marine snails (reviewed in Refs. [11,22]). Although most of the findings reported in the studies on experience-induced
* Corresponding author. Tel.: +1-312-355-3142; fax: +1-312-996-4979. E-mail address: [email protected] (T.L. Briones). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.06.001
morphological plasticity were demonstrated in young animals, similar changes were seen in older animals [1,7,10,12– 14], suggesting a fundamental role of the external environment in promoting neuronal plasticity regardless of age. Indeed, it has been recently reported that environmental stimulation resulted in increased granule cell proliferation and survival in the dentate gyrus of adult mice and rats [15 – 17,20]. The overall effects of complex environment exposure seem to be beneficial; however, it is not clear how long these effects persist with continuing exposure to the environment, nor what happens when environmental stimulation is discontinued. This lack of clarity regarding the persistence of EC-induced plasticity may be due to: (1) conflicting reports on the stability of changes seen in young and old animals, and (2) the wide range of measures used to assess
T.L. Briones et al. / Brain Research 1018 (2004) 130–135
plasticity, from gross to ultrastructural measurement. Thus, the aim of the present study was to determine whether synaptic changes brought by environmental stimulation were stable both over longer periods than the typical 30 days and in the absence of continued complex environment exposure. Specifically, we measured synaptic addition, in terms of the number of synapses per neuron arising from complex environment exposure in layer IV of the visual cortex using 4-month-old adult rats. In this study, we examined: (1) the effects of 30 and 60 days of complex environment exposure on synaptic plasticity, and (2) the stability and/or reversibility of these synaptic changes.
2. Materials and methods 2.1. Experiment 1 In this experiment, we examined the effects of complex environment housing for 30 days on the number of synapses per neuron. Twenty-five male littermate sets of Long Evans hooded rats were included in the study. All rats were housed in small groups until the age of 4 months (120 days) when littermates were assigned to one of three experimental conditions: complex environment (n = 9), inactive (n = 8), and social (n = 8). In the complex environment condition (EC), rats were housed together in a wire mesh cage (0.85 0.85 0.85 m) filled with a variety of objects such as swing, wooden blocks, plastic cars and trucks, plastic tunnels, and mirrors. In addition, these rats were placed each day in an open field (1.2 1.2 1.2 m) with a novel arrangement of toys and objects and allowed to explore for 30 min while the objects in the home cage were being changed. Objects in the home cage and open field were changed daily to maintain novelty. Rats in both inactive (IC) and social (SC) conditions were housed in standard laboratory cages measuring 0.5 0.27 0.36 m with no other objects in the cage; these animals had very minimal opportunity for learning or exercise. In the inactive condition, animals were housed individually whereas in the social condition, rats were housed in pairs; and standard laboratory cages were used for both of these groups. After 30 days of environmental exposure, rats were perfused and the brains were prepared for electron microscopy as described under Section 2.3. 2.2. Experiment two In this experiment, we examined the effects of complex environment housing for 60 days on the number of synapses per neuron and the stability and/or reversibility of these synaptic changes. Twenty-nine male littermate sets of Long Evans hooded rats were included in the study. Again, all rats were housed in small groups until the age of 4 months then littermates were randomized to one of two experimental conditions: EC and IC. Following 30 days of environmental
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exposure, the EC rats were then randomly assigned to either remain in EC, the ECEC group (n = 8), or transfer to IC, the ECIC group (n = 7). Similarly, half of the IC rats remained in IC, the ICIC group (n = 7), and the other half was placed in EC, the ICEC group (n = 7). These animals remained in their respective environments for another 30 days and were then perfused. 2.3. Tissue preparation At the end of the experiment, the rats were anesthetized with an intraperitoneal (i.p.) injection of pentobarbital (100 mg/kg) and transcardially perfused with 100 ml of Hank’s solution followed by the fixative, 2% paraformaldehyde and 2% glutaraldehyde in 0.14 M sodium cacodylate (pH 7.3). Brains were processed for electron microscopy as previously described (see Ref. [19]). Briefly, after overnight post fixation, the occipital region was sectioned coronally at 250 Am and blocks of visual cortex (Oc1M and Oc1B) [21] extending from pia to white matter were removed under a dissecting microscope. These tissue blocks were then washed in 0.14 M sodium cacodylate (pH 7.3), osmicated (2% osmium), stained en bloc with aqueous 0.5% uranyl acetate for 30 min, dehydrated through graded alcohol, transferred to prophylene oxide, and embedded gradually in LX-112 epoxy resin (Ted Pella, Redding, CA). Blocks were coded to conceal experimental conditions during analysis. 2.4. Microscopy 2.4.1. Neuron density Two blocks from each animal were randomly chosen for sectioning and sixty 1-Am serial sections were obtained from each block for a total of 120 sections. The sections were mounted in chrom alum gelatin-coated slides and stained with toluidine blue and layer IV of the visual cortex was identified within Oc1M and Oc1B. The visual cortex was used in this study because the majority of earlier reports on synaptic plasticity were done in this brain region and it is an area most responsive to behavioral experience since it is directly stimulated by variables present in the complex environment [5]. Layer IV of the primary visual cortex was chosen given that it is the major input area of all sensory information that comes from the lateral geniculate. Using a computer-assisted microscope and a sterelogy software package (StereoInvestogator, MicroBrightfield), the physical disector method (see Ref. [19]) was employed to obtain a measure of neuron density. Cells with characteristics of glia were not included in the analyses (i.e., cells with clumping of chromatin centrally or adjacent to the nuclear membrane and, typically, small nuclei and sparse or irregularly shaped surrounding perikarya). 2.4.2. Synapse density After the 1-Am sectioning, a small pyramid was trimmed that included layer IV of the tissue blocks of visual cortex
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using the toluidine blue-stained sections as a guide. From the pyramid, 20 silver/gray serial sections (approximately 60 – 65 nm thick) were taken using an ultamicrotome (Reichert Ultracut S). The serial sections were collected on Formvar-coated, slotted copper grids (care was taken to obtain sections of uniform color/thickness) and stained with lead citrate. Negatives were printed at a final magnification of 24,000 . The number of synapses per neuron in layer IV of the visual cortex was then obtained by dividing the density of synapses per cubic millimeter by the density of neurons per cubic millimeter. Because measurements of neuronal and synaptic density were obtained from the same resin-embedded samples, tissue shrinkage effects contributed equally to synaptic density and neuronal density data and, therefore were unlikely to bias synapse number per neuron data. Statistical analysis was done using the SAS general linear model analysis of variance (ANOVA). Synaptic plasticity was analyzed using two-way ANOVA for the effects of housing condition by time (30 or 60 days housing). All other data were analyzed using one-way ANOVA for the effects of housing condition and the Scheffe´ test was used for post hoc comparisons.
3. Results 3.1. Effects of differential housing for 30 days on synaptic plasticity To examine effects of differential environment housing for 30 days on visual cortex plasticity, we compared neuron and synapse density in the EC, SC, and IC groups. Neuron and synapse density in layer IV of the visual cortex were not significantly different between the groups. However, a significant increase in the number of synapses per neuron was seen in animals exposed to EC as seen in Fig. 1 ( F2,22 = 6.93, p < 0.05). After 30 days of environmental
Fig. 1. The number of synapses per neuron in layer IV of the visual cortex. Error bars show standard error of the means (S.E.M.). *p < 0.05— significantly different from the IC group.
Fig. 2. (A) The number of neurons/mm3 in layer IV of the visual cortex. (B) Number of synapses per neuron in layer IV of the occipital cortex. Data are 106 synapses per neuron (synaptic density/neuronal density). Error bars show standard error of the means (S.E.M.). *p < 0.05—significantly different from the ICIC group.
exposure, EC animals had 21% more synaptic contacts per neuron than SC animals and 27% more than IC animals. These results indicate that experience can induce ultrastructural changes in the visual cortex even in adult rats. 3.2. Effects of differential housing for 60 days on synaptic plasticity To examine the effects of a more prolonged duration of differential environment housing on visual cortex plasticity, a comparison of all groups kept for 60 days was performed. A significant group effect was evident ( F 3,25 = 4.19, p < 0.05) as shown in Fig. 2. That is, post hoc comparisons showed that rats housed in EC for a prolonged period of time have significantly fewer neurons per unit volume of brain tissue within layer IV of the visual cortex ( F3,25 = 3.68, p < 0.05) compared to rats housed in IC for 60 days (ICIC); however, no significant difference was seen in neuron density between the three EC groups (ECEC, ECIC, and ICEC). The lower neuronal density in the ECEC group presumably reflects added neuropil spreading apart the neurons. Additionally, all animals exposed to EC had a significantly higher synapse per neuron ratio ( F3,25 = 4.25, p < 0.05) compared to animals housed in IC only (IC and
T.L. Briones et al. / Brain Research 1018 (2004) 130–135
Fig. 3. Stability of EC-induced synaptic plasticity. Data are 106 synapses per neuron (synaptic density/neuronal density). Error bars show standard error of the means (S.E.M.). *p < 0.05—significantly different from the ICIC group.
ICIC groups). Rats exposed to EC for 60 days (ECEC) had 21% more synapses per neuron compared to animals housed in IC alone (ICIC). Changing housing condition after 30 days also increased synapse number with ICEC and ECIC rats having 15% and 16% more synapses per neuron, respectively, compared to the ICIC animals. These results suggest that prolonged exposure to complex environment can induce changes both in neuron density and synaptic plasticity. 3.3. Stability of synaptic changes induced by complex environment exposure To assess the relative permanence of changes resulting from exposure to a complex environment, the rats housed in EC for the first 30 days and subsequently transferred to IC (ECIC), rats housed in EC for 30 and 60 days, and rats housed in IC for 60 days were examined. No significant differences in neuron and synapse density were seen among the three groups exposed to EC. However, a significant increase in the number of synapses per neuron ( F2,20 = 3.19, p < 0.05) was seen among the group of rats housed in EC compared to rats housed in IC as shown in Fig. 3. Post hoc comparisons showed that the ratio of synapses per neuron among the groups of rats that received environmental stimulation was not significantly different. These results indicate that the gain in synapses seen after 30 days of exposure to a complex environment persisted even after reversing the housing condition from EC to IC.
4. Discussion This study demonstrates that housing adult rats in a complex environment can induce synaptogenesis. We demonstrated that adult animals exposed to a complex environ-
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ment for 30 or 60 days had a significantly higher synapse per neuron ratio in layer IV of the visual cortex. This suggests that the synaptic plasticity seen in our adult animals exposed to environmental complexity is comparable directionally if not in absolute magnitude to that seen in young animals housed in EC. The absolute values from an earlier study that used weanling rats [25] cannot be directly compared with the present values due to the differences in stereological techniques used in the two studies. Nonetheless, the magnitudes of the differences among the 30-day EC, SC, and IC groups are very comparable across the two studies. Assuming that neuron number does not change dramatically, the decreased neuron density seen here after prolonged EC exposure probably reflects the increase in volume of tissue elements such as dendrites, axons, glia, and capillaries, pushing apart the neurons [1,8,23,24]. Thus, the increased volume of tissue seen in the visual cortex of adult animals exposed to a complex environment may be explained in part by the appearance of larger and more complex dendritic fields such as increased dendritic branchings and longer terminal branches in the dendrites [3,4,6,9]. However, this type of neuropil expansion resulting from complex environment exposure is predominantly seen during early development and declines with age. Given that adult rats were used in this study, true differences in synapse to neuron ratio between the ECIC, ECEC, and ICEC animals may have been washed out. It is possible that the mechanisms whereby synaptogenesis resulting from earlier (ECEC and ECIC) and later exposure to EC (ICEC) may be different; in that, increased synapses per neuron seen in earlier and prolonged exposure to EC may be a result of a de novo formation of multiple synaptic contacts with the same neuron. In contrast, synaptogenesis resulting from later exposure to EC (ICEC) may be due to an addition of synaptic contacts onto a pre-existing synaptic bouton. In either case, the net synapse to neuron ratio would increase, thus the manner and direction of change would be similar but the difference in mechanisms in the EC-induced synaptogenesis between the groups will be hard to identify. The persistence of the changes in the adult brain caused by exposure to a complex environment is an understudied problem. Some studies have demonstrated that in young animals the increases in cortical thickness in the visual cortex outlast the experience [14]. The stability in the increase in cortical thickness seen in the young animals reported in these studies as a result of experience depends on the: (1) length of the initial EC exposure and (2) duration of subsequent IC housing. Others have shown that experience-induced changes in neurochemistry and ‘total brain weight’ in older animals dissipated fairly rapidly upon withdrawal from EC. In contrast, dendritic alterations in the visual cortex persistent in young animals exposed to EC for 30 days followed by a subsequent period of individual housing. The difference in relative stability of EC-induced changes reported in these studies suggests the possibility that gross measures of plasticity, such as cortical
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thickness, ‘total brain weight’, and ‘total DNA and RNA content’, may reflect the sum of underlying changes in other tissue components such as glial and vascular elements; whereas specific neuronal measures of synaptic connectivity, such as dendritic branchings, dendritic spine density, and ratio of synapses per neuron, may accurately reflect relatively permanent plastic changes. Although the plasticity of synapses and dendrites without a doubt contributes to the increase in cortical size, other tissue components do as well, making gross measurements less specific. In the present study, we showed that in adult rats, the synaptic alterations resulting from exposure to EC persisted essentially undiminished 1 month after subsequent IC housing. These results are indeed compatible with findings from another model of experience-induced plasticity, complex motor skill learning, in which a training-dependent increase in synaptic number in cerebellar cortex lasted for at least 28 days [18]. Our findings indicate that the adult brain remains dynamic and adapts to the demands of the external environment while emphasizing the stability of experience-induced synaptic numerical change. Complex environment housing provides the animals with opportunity for increased physical activity, social interaction, and environmental novelty. Of all these factors, it is possible that environmental novelty plays a significant role in influencing the changes in synapse to neuron ratio observed in the EC rats compared to the individually or socially paired housed animals. This line of reasoning is supported by our findings that synapse to neuron ratio in the SC and IC rats were not significantly different compared to the EC animals, suggesting that social interaction alone was not sufficient to result in significant visual cortical synaptogenesis. Furthermore, others have reported that although physical activity alone can result in increased angiogenesis, no changes were seen in neuron volume and synapses per neuron [2]. The EC-induced synaptogenesis seen in this study may be due to the ‘learning’ aspects of the behavioral experience because complex environment provides not just novelty but a continuing opportunity for exploration, play, and spatial learning. However, it is not known at this time whether the benefits of complex environment housing have a ceiling effect beyond which continued exposure will not be useful. Therefore, more controlled studies that will examine EC effects throughout the lifespan on brain morphology are merited.
Acknowledgements We are grateful to Kathy Bates for editorial assistance. We also thank Pinky Shah for animal handling, Brian Belt and Grace Manipon for tissue sectioning. This work was supported by the National Institutes of Health grant nos. MH35321 and MH10422-W.T.G., training grant T32 NR07075-T.L.B, and AA09838-A.Y.K.
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T.L. Briones et al. / Brain Research 1018 (2004) 130–135 postnatal binge alcohol exposure: preliminary report, Alcoholism, Clinical and Experimental Research 21 (1997) 1257 – 1263. [20] M. Nillsson, E. Perfilieva, U. Johansson, O. Orwar, P.S. Eriksson, Enriched environment increases neurogenesis in the adult rat dentate gyrus and improves spatial memory, Journal of Neurobiology 39 (1999) 569 – 578. [21] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, 3rd edn., Academic Press, London, 1997. [22] M.R. Rosenzweig, E.L. Bennett, Experimental influences on brain anatomy and brain chemistry in rodents, in: G. Gottlieb (Ed.), Studies on the Development of Behavior and the Nervous System, vol. 4, Academic Press, New York, 1978, pp. 429 – 532.
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[23] A.M. Sirevaag, W.T. Greenough, Differential rearing effects on rat visual cortex synapses: III. Neuronal and glial nuclei, boutons, dendrites, and capillaries, Brain Research 424 (1987) 320 – 332. [24] A.M. Sirevaag, W.T. Greenough, Plasticity of GFAP-immunoreactive astrocyte size and number in visual cortex of rats reared in complex environment, Brain Research 540 (1991) 273 – 278. [25] A.M. Turner, W.T. Greenough, Differential rearing effects on rat visual cortex synapses: I. Synaptic and neuronal density and synapses per neuron, Brain Research 329 (1985) 195 – 203.
Short communication
Stability of synaptic plasticity in the adult rat visual cortex induced by complex environment exposure Teresita L. Briones a,*, Anna Y. Klintsova b, William T. Greenough c,d,e,f a
Department of Medical-Surgical Nursing, University of Illinois, 845 S. Damen Ave., Rm 707, M/C 802, Chicago, IL 60612, USA b Department of Psychology, Binghamton University, Binghamton, NY, USA c Beckman Institute, University of Illinois, Urbana, IL, USA d Department of Psychology, University of Illinois, Urbana, IL, USA e Department of Psychiatry, University of Illinois, Urbana, IL, USA f Department of Cell and Structural Biology, University of Illinois, Urbana, IL, USA Accepted 4 June 2004 Available online
Abstract Studies have demonstrated the effects of complex environment (EC) housing on brain plasticity both during postnatal development and in adulthood, but it is not clear how long these plastic changes persist nor what happens when environmental exposure is discontinued. Here we examined layer IV in the visual cortex of adult male rats for the: (1) effects of EC housing on synaptic plasticity, and (2) persistence of the synaptic changes after withdrawal from the complex environment. Fifty-eight adult male Long Evans rats were assigned to either: EC, socially paired housing (SC), or individual housing (IC). These rats remained in their assigned environment for 30 days. After 30 days, all rats in SC and some animals from the EC and IC groups were removed and perfused. The remaining animals in EC were then assigned to either remain in EC (ECEC) or be subsequently housed in IC (ECIC) for another 30 days. Similarly, rats in the IC group either remained in IC (ICIC) or were subsequently housed in EC (ICEC) for another 30 days. Electron microscopy results showed that all rats exposed to EC had significantly more synapses/neuron compared to SC, IC, and ICIC animals. Longer exposure to EC (ECEC) did not result in statistically more synapses per neuron; however, decreased neuron volume was seen. EC-induced synaptic changes persisted for an additional 30 days after withdrawal from EC (ECIC) confirming that EC-induced plastic changes occur in the brain regardless of age and indicating that once changes occur they tend to persist. D 2004 Elsevier B.V. All rights reserved. Keywords: Synaptic plasticity; Complex environment; Stereology; Learning; Memory; Enriched environment
1. Introduction Naturally occurring morphological plasticity is an ongoing process that can be evoked by environmental stimulation. Over the past 40 years, there have been numerous reports on the effects of complex environment exposure on brain morphology and function in young animals. Morphological changes in the brain in response to experience have been described in the nervous system of a wide array of species including monkeys, cats, rats, birds, honeybees, and marine snails (reviewed in Refs. [11,22]). Although most of the findings reported in the studies on experience-induced
* Corresponding author. Tel.: +1-312-355-3142; fax: +1-312-996-4979. E-mail address: [email protected] (T.L. Briones). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.06.001
morphological plasticity were demonstrated in young animals, similar changes were seen in older animals [1,7,10,12– 14], suggesting a fundamental role of the external environment in promoting neuronal plasticity regardless of age. Indeed, it has been recently reported that environmental stimulation resulted in increased granule cell proliferation and survival in the dentate gyrus of adult mice and rats [15 – 17,20]. The overall effects of complex environment exposure seem to be beneficial; however, it is not clear how long these effects persist with continuing exposure to the environment, nor what happens when environmental stimulation is discontinued. This lack of clarity regarding the persistence of EC-induced plasticity may be due to: (1) conflicting reports on the stability of changes seen in young and old animals, and (2) the wide range of measures used to assess
T.L. Briones et al. / Brain Research 1018 (2004) 130–135
plasticity, from gross to ultrastructural measurement. Thus, the aim of the present study was to determine whether synaptic changes brought by environmental stimulation were stable both over longer periods than the typical 30 days and in the absence of continued complex environment exposure. Specifically, we measured synaptic addition, in terms of the number of synapses per neuron arising from complex environment exposure in layer IV of the visual cortex using 4-month-old adult rats. In this study, we examined: (1) the effects of 30 and 60 days of complex environment exposure on synaptic plasticity, and (2) the stability and/or reversibility of these synaptic changes.
2. Materials and methods 2.1. Experiment 1 In this experiment, we examined the effects of complex environment housing for 30 days on the number of synapses per neuron. Twenty-five male littermate sets of Long Evans hooded rats were included in the study. All rats were housed in small groups until the age of 4 months (120 days) when littermates were assigned to one of three experimental conditions: complex environment (n = 9), inactive (n = 8), and social (n = 8). In the complex environment condition (EC), rats were housed together in a wire mesh cage (0.85 0.85 0.85 m) filled with a variety of objects such as swing, wooden blocks, plastic cars and trucks, plastic tunnels, and mirrors. In addition, these rats were placed each day in an open field (1.2 1.2 1.2 m) with a novel arrangement of toys and objects and allowed to explore for 30 min while the objects in the home cage were being changed. Objects in the home cage and open field were changed daily to maintain novelty. Rats in both inactive (IC) and social (SC) conditions were housed in standard laboratory cages measuring 0.5 0.27 0.36 m with no other objects in the cage; these animals had very minimal opportunity for learning or exercise. In the inactive condition, animals were housed individually whereas in the social condition, rats were housed in pairs; and standard laboratory cages were used for both of these groups. After 30 days of environmental exposure, rats were perfused and the brains were prepared for electron microscopy as described under Section 2.3. 2.2. Experiment two In this experiment, we examined the effects of complex environment housing for 60 days on the number of synapses per neuron and the stability and/or reversibility of these synaptic changes. Twenty-nine male littermate sets of Long Evans hooded rats were included in the study. Again, all rats were housed in small groups until the age of 4 months then littermates were randomized to one of two experimental conditions: EC and IC. Following 30 days of environmental
131
exposure, the EC rats were then randomly assigned to either remain in EC, the ECEC group (n = 8), or transfer to IC, the ECIC group (n = 7). Similarly, half of the IC rats remained in IC, the ICIC group (n = 7), and the other half was placed in EC, the ICEC group (n = 7). These animals remained in their respective environments for another 30 days and were then perfused. 2.3. Tissue preparation At the end of the experiment, the rats were anesthetized with an intraperitoneal (i.p.) injection of pentobarbital (100 mg/kg) and transcardially perfused with 100 ml of Hank’s solution followed by the fixative, 2% paraformaldehyde and 2% glutaraldehyde in 0.14 M sodium cacodylate (pH 7.3). Brains were processed for electron microscopy as previously described (see Ref. [19]). Briefly, after overnight post fixation, the occipital region was sectioned coronally at 250 Am and blocks of visual cortex (Oc1M and Oc1B) [21] extending from pia to white matter were removed under a dissecting microscope. These tissue blocks were then washed in 0.14 M sodium cacodylate (pH 7.3), osmicated (2% osmium), stained en bloc with aqueous 0.5% uranyl acetate for 30 min, dehydrated through graded alcohol, transferred to prophylene oxide, and embedded gradually in LX-112 epoxy resin (Ted Pella, Redding, CA). Blocks were coded to conceal experimental conditions during analysis. 2.4. Microscopy 2.4.1. Neuron density Two blocks from each animal were randomly chosen for sectioning and sixty 1-Am serial sections were obtained from each block for a total of 120 sections. The sections were mounted in chrom alum gelatin-coated slides and stained with toluidine blue and layer IV of the visual cortex was identified within Oc1M and Oc1B. The visual cortex was used in this study because the majority of earlier reports on synaptic plasticity were done in this brain region and it is an area most responsive to behavioral experience since it is directly stimulated by variables present in the complex environment [5]. Layer IV of the primary visual cortex was chosen given that it is the major input area of all sensory information that comes from the lateral geniculate. Using a computer-assisted microscope and a sterelogy software package (StereoInvestogator, MicroBrightfield), the physical disector method (see Ref. [19]) was employed to obtain a measure of neuron density. Cells with characteristics of glia were not included in the analyses (i.e., cells with clumping of chromatin centrally or adjacent to the nuclear membrane and, typically, small nuclei and sparse or irregularly shaped surrounding perikarya). 2.4.2. Synapse density After the 1-Am sectioning, a small pyramid was trimmed that included layer IV of the tissue blocks of visual cortex
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using the toluidine blue-stained sections as a guide. From the pyramid, 20 silver/gray serial sections (approximately 60 – 65 nm thick) were taken using an ultamicrotome (Reichert Ultracut S). The serial sections were collected on Formvar-coated, slotted copper grids (care was taken to obtain sections of uniform color/thickness) and stained with lead citrate. Negatives were printed at a final magnification of 24,000 . The number of synapses per neuron in layer IV of the visual cortex was then obtained by dividing the density of synapses per cubic millimeter by the density of neurons per cubic millimeter. Because measurements of neuronal and synaptic density were obtained from the same resin-embedded samples, tissue shrinkage effects contributed equally to synaptic density and neuronal density data and, therefore were unlikely to bias synapse number per neuron data. Statistical analysis was done using the SAS general linear model analysis of variance (ANOVA). Synaptic plasticity was analyzed using two-way ANOVA for the effects of housing condition by time (30 or 60 days housing). All other data were analyzed using one-way ANOVA for the effects of housing condition and the Scheffe´ test was used for post hoc comparisons.
3. Results 3.1. Effects of differential housing for 30 days on synaptic plasticity To examine effects of differential environment housing for 30 days on visual cortex plasticity, we compared neuron and synapse density in the EC, SC, and IC groups. Neuron and synapse density in layer IV of the visual cortex were not significantly different between the groups. However, a significant increase in the number of synapses per neuron was seen in animals exposed to EC as seen in Fig. 1 ( F2,22 = 6.93, p < 0.05). After 30 days of environmental
Fig. 1. The number of synapses per neuron in layer IV of the visual cortex. Error bars show standard error of the means (S.E.M.). *p < 0.05— significantly different from the IC group.
Fig. 2. (A) The number of neurons/mm3 in layer IV of the visual cortex. (B) Number of synapses per neuron in layer IV of the occipital cortex. Data are 106 synapses per neuron (synaptic density/neuronal density). Error bars show standard error of the means (S.E.M.). *p < 0.05—significantly different from the ICIC group.
exposure, EC animals had 21% more synaptic contacts per neuron than SC animals and 27% more than IC animals. These results indicate that experience can induce ultrastructural changes in the visual cortex even in adult rats. 3.2. Effects of differential housing for 60 days on synaptic plasticity To examine the effects of a more prolonged duration of differential environment housing on visual cortex plasticity, a comparison of all groups kept for 60 days was performed. A significant group effect was evident ( F 3,25 = 4.19, p < 0.05) as shown in Fig. 2. That is, post hoc comparisons showed that rats housed in EC for a prolonged period of time have significantly fewer neurons per unit volume of brain tissue within layer IV of the visual cortex ( F3,25 = 3.68, p < 0.05) compared to rats housed in IC for 60 days (ICIC); however, no significant difference was seen in neuron density between the three EC groups (ECEC, ECIC, and ICEC). The lower neuronal density in the ECEC group presumably reflects added neuropil spreading apart the neurons. Additionally, all animals exposed to EC had a significantly higher synapse per neuron ratio ( F3,25 = 4.25, p < 0.05) compared to animals housed in IC only (IC and
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Fig. 3. Stability of EC-induced synaptic plasticity. Data are 106 synapses per neuron (synaptic density/neuronal density). Error bars show standard error of the means (S.E.M.). *p < 0.05—significantly different from the ICIC group.
ICIC groups). Rats exposed to EC for 60 days (ECEC) had 21% more synapses per neuron compared to animals housed in IC alone (ICIC). Changing housing condition after 30 days also increased synapse number with ICEC and ECIC rats having 15% and 16% more synapses per neuron, respectively, compared to the ICIC animals. These results suggest that prolonged exposure to complex environment can induce changes both in neuron density and synaptic plasticity. 3.3. Stability of synaptic changes induced by complex environment exposure To assess the relative permanence of changes resulting from exposure to a complex environment, the rats housed in EC for the first 30 days and subsequently transferred to IC (ECIC), rats housed in EC for 30 and 60 days, and rats housed in IC for 60 days were examined. No significant differences in neuron and synapse density were seen among the three groups exposed to EC. However, a significant increase in the number of synapses per neuron ( F2,20 = 3.19, p < 0.05) was seen among the group of rats housed in EC compared to rats housed in IC as shown in Fig. 3. Post hoc comparisons showed that the ratio of synapses per neuron among the groups of rats that received environmental stimulation was not significantly different. These results indicate that the gain in synapses seen after 30 days of exposure to a complex environment persisted even after reversing the housing condition from EC to IC.
4. Discussion This study demonstrates that housing adult rats in a complex environment can induce synaptogenesis. We demonstrated that adult animals exposed to a complex environ-
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ment for 30 or 60 days had a significantly higher synapse per neuron ratio in layer IV of the visual cortex. This suggests that the synaptic plasticity seen in our adult animals exposed to environmental complexity is comparable directionally if not in absolute magnitude to that seen in young animals housed in EC. The absolute values from an earlier study that used weanling rats [25] cannot be directly compared with the present values due to the differences in stereological techniques used in the two studies. Nonetheless, the magnitudes of the differences among the 30-day EC, SC, and IC groups are very comparable across the two studies. Assuming that neuron number does not change dramatically, the decreased neuron density seen here after prolonged EC exposure probably reflects the increase in volume of tissue elements such as dendrites, axons, glia, and capillaries, pushing apart the neurons [1,8,23,24]. Thus, the increased volume of tissue seen in the visual cortex of adult animals exposed to a complex environment may be explained in part by the appearance of larger and more complex dendritic fields such as increased dendritic branchings and longer terminal branches in the dendrites [3,4,6,9]. However, this type of neuropil expansion resulting from complex environment exposure is predominantly seen during early development and declines with age. Given that adult rats were used in this study, true differences in synapse to neuron ratio between the ECIC, ECEC, and ICEC animals may have been washed out. It is possible that the mechanisms whereby synaptogenesis resulting from earlier (ECEC and ECIC) and later exposure to EC (ICEC) may be different; in that, increased synapses per neuron seen in earlier and prolonged exposure to EC may be a result of a de novo formation of multiple synaptic contacts with the same neuron. In contrast, synaptogenesis resulting from later exposure to EC (ICEC) may be due to an addition of synaptic contacts onto a pre-existing synaptic bouton. In either case, the net synapse to neuron ratio would increase, thus the manner and direction of change would be similar but the difference in mechanisms in the EC-induced synaptogenesis between the groups will be hard to identify. The persistence of the changes in the adult brain caused by exposure to a complex environment is an understudied problem. Some studies have demonstrated that in young animals the increases in cortical thickness in the visual cortex outlast the experience [14]. The stability in the increase in cortical thickness seen in the young animals reported in these studies as a result of experience depends on the: (1) length of the initial EC exposure and (2) duration of subsequent IC housing. Others have shown that experience-induced changes in neurochemistry and ‘total brain weight’ in older animals dissipated fairly rapidly upon withdrawal from EC. In contrast, dendritic alterations in the visual cortex persistent in young animals exposed to EC for 30 days followed by a subsequent period of individual housing. The difference in relative stability of EC-induced changes reported in these studies suggests the possibility that gross measures of plasticity, such as cortical
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thickness, ‘total brain weight’, and ‘total DNA and RNA content’, may reflect the sum of underlying changes in other tissue components such as glial and vascular elements; whereas specific neuronal measures of synaptic connectivity, such as dendritic branchings, dendritic spine density, and ratio of synapses per neuron, may accurately reflect relatively permanent plastic changes. Although the plasticity of synapses and dendrites without a doubt contributes to the increase in cortical size, other tissue components do as well, making gross measurements less specific. In the present study, we showed that in adult rats, the synaptic alterations resulting from exposure to EC persisted essentially undiminished 1 month after subsequent IC housing. These results are indeed compatible with findings from another model of experience-induced plasticity, complex motor skill learning, in which a training-dependent increase in synaptic number in cerebellar cortex lasted for at least 28 days [18]. Our findings indicate that the adult brain remains dynamic and adapts to the demands of the external environment while emphasizing the stability of experience-induced synaptic numerical change. Complex environment housing provides the animals with opportunity for increased physical activity, social interaction, and environmental novelty. Of all these factors, it is possible that environmental novelty plays a significant role in influencing the changes in synapse to neuron ratio observed in the EC rats compared to the individually or socially paired housed animals. This line of reasoning is supported by our findings that synapse to neuron ratio in the SC and IC rats were not significantly different compared to the EC animals, suggesting that social interaction alone was not sufficient to result in significant visual cortical synaptogenesis. Furthermore, others have reported that although physical activity alone can result in increased angiogenesis, no changes were seen in neuron volume and synapses per neuron [2]. The EC-induced synaptogenesis seen in this study may be due to the ‘learning’ aspects of the behavioral experience because complex environment provides not just novelty but a continuing opportunity for exploration, play, and spatial learning. However, it is not known at this time whether the benefits of complex environment housing have a ceiling effect beyond which continued exposure will not be useful. Therefore, more controlled studies that will examine EC effects throughout the lifespan on brain morphology are merited.
Acknowledgements We are grateful to Kathy Bates for editorial assistance. We also thank Pinky Shah for animal handling, Brian Belt and Grace Manipon for tissue sectioning. This work was supported by the National Institutes of Health grant nos. MH35321 and MH10422-W.T.G., training grant T32 NR07075-T.L.B, and AA09838-A.Y.K.
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