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Environmental enrichment promotes fiber sprouting after deafferentation of the superior colliculus in the adult rat brain
Experimental Neurology 216 (2009) 515–519
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Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r
Short Communication
Environmental enrichment promotes fiber sprouting after deafferentation of the superior colliculus in the adult rat brain Matteo Caleo a,⁎, Daniela Tropea b,1, Chiara Rossi a, Laura Gianfranceschi b, Lamberto Maffei a,b a b
Istituto di Neuroscienze, Consiglio Nazionale delle Ricerche, via G. Moruzzi 1, 56100 Pisa, Italy Scuola Normale Superiore, via G. Moruzzi 1, 56100 Pisa, Italy
a r t i c l e
i n f o
Article history: Received 18 July 2008 Revised 18 December 2008 Accepted 22 December 2008 Available online 6 January 2009 Keywords: Brain injury Retinal ganglion cells Optic tectum Synaptic reorganization
a b s t r a c t Exposure to an enriched environment has proven to be beneficial in the recovery of function after brain lesions, but the underlying mechanisms remain only partly understood. One possibility is that environmental enrichment stimulates the reorganization of areas and fiber tracts that have been spared by the injury. Here we evaluate the effects of enriched environment on the sprouting of undamaged retinal afferents into the deafferented superior colliculus (SC) after a partial retinal lesion in adult rats. Anterograde tracing of retinal axons demonstrated a significant increase in fiber sprouting in the denervated SC of animals reared in enriched environment compared to animals reared in standard conditions. Environmental enrichment also promoted a substantial recovery of synaptic sites within the deafferented SC as shown by both synapsin I and vesicular glutamate transporter 2 immunostaining. These data provide evidence that environmental enrichment stimulates axonal plasticity and synaptic reorganization following brain injury. © 2009 Elsevier Inc. All rights reserved.
The adult mammalian central nervous system exhibits very poor recovery after injury. Finding strategies that enhance the functional reorganization of brain circuitry after a lesion is critical to improve the conditions of patients affected by brain trauma. Environmental enrichment is a housing condition that allows enhanced sensory, cognitive and motor stimulation relative to standard laboratory conditions (Rosenzweig and Bennett, 1996; van Praag et al., 2000). In the enriched environmental condition, animals are housed in large cages containing a variety of objects that are changed daily. There are increased opportunities for social interaction, and running wheels are available for voluntary physical exercise (Nithianantharajah and Hannan, 2006; van Praag et al., 2000; Will et al., 2004). Several studies have shown that living in an enriched environment produces dramatic effects on both the developing and adult brain (Cancedda et al., 2004; Rampon et al., 2000; Rossi et al., 2006; Sale et al., 2007). In particular, exposure to an enriched environment is known to produce functional recovery after various types of brain lesions (Biernaskie and Corbett, 2001; Koopmans et al., 2006; Pizzorusso et al., 2007; Will et al., 2004). For example, enriched housing has beneficial effects on locomotor recovery in rats with spinal cord contusion (Lankhorst et al., 2001) and improves cognitive and motor function following traumatic brain injury (Passineau et al., 2001; Smith et al., 2007). In mouse models of central nervous system
⁎ Corresponding author. Fax: +39 050 3153220. E-mail address: [email protected] (M. Caleo). 1 Present address: Department of Brain and Cognitive Sciences and Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. 0014-4886/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2008.12.016
(CNS) disorders, rearing in an enriched environment ameliorates functional parameters and reduces neurodegeneration (Nithianantharajah and Hannan, 2006; van Dellen et al., 2000). The mechanisms by which environmental enrichment produces these effects are only partly understood. One possibility is that enrichment stimulates the plastic reorganization of unlesioned areas and fiber tracts surrounding the site of injury (Will et al., 2004). Indeed, enriched experience activates molecular and cellular cascades, such as the induction of BDNF (brain-derived neurotrophic factor) and GAP43 (growth-associated protein of 43 kDa) that act to enhance and support brain plasticity (Ickes et al., 2000; Molteni et al., 2004; Pham et al., 1999). Here we investigate the effects of an exposure to an enriched environment on axonal plasticity following injury. Two to 3 months old Long–Evans rats were used in this study. All experiments were carried out in accordance with the European Community Council Directive of 24 November 1986 (86/609/EEC). Animals were either housed in standard laboratory cages (n = 12) or in an enriched environment (n = 12). The enriched environmental condition has been described previously (Bartoletti et al., 2004; Sale et al., 2007). It consisted of a large cage (95 × 75 × 80 cm) containing several food hoppers, two running wheels and differently shaped objects (tunnels, shelters, stairs) that were repositioned once per day and completely substituted with others once per week (Sale et al., 2007). Partial retinal lesions were performed as described previously (Tropea et al., 2003). The animals were anesthetized with avertin and placed in a stereotaxic apparatus. The sclera was exposed and touched with a heat microcauterizer, resulting in the complete disruption of retinal cells and the optic nerve fiber in the heated area. In all cases the
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lesions were made in the ventro-temporal portion of the retina, at a distance of 2 mm from the optic nerve head. To visualize retinal ganglion cell axons in the superior colliculus, Alexa 594-conjugated cholera toxin subunit B (CTB; Invitrogen, Carlsbad, CA) was administered into the vitreous. Rats were injected with 3 μl of 1% CTB in water 2 days before perfusion of the animals. Three weeks after the lesion, rats were deeply anaesthetized with chloral hydrate (10.5% solution; 0.4 ml/100 g) and perfused transcardially with PBS followed by 4% paraformaldehyde in 0.1 M phosphate buffer. In order to visualize the retinal lesion, eyes were removed and small crystals of the lipophilic tracer DiI (Invitrogen) were applied on the stump of the optic nerve. For the retrograde staining of retinal ganglion cells (RGCs), the eyes were left for 4 weeks at 37 °C in 4% paraformaldehyde. Measurement of the areas affected by the lesion both at the retinal and at the collicular level was performed with Neurolucida (Microbrightfield, Colchester, VT), as described previously (Tropea et al., 2003). Brain coronal sections (40 μm thick) were cut on a freezing microtome and collected in PBS. Synapsin I immunostaining was performed using the mouse monoclonal antibody 355 (Chemicon, Temecula, CA) at a concentration of 2 μg/ml. A rabbit polyclonal antibody (Synaptic Systems, Gottingen, Germany; 1:500) was used for the detection of vesicular glutamate transporter 2 (vGlut-2) immunoreactivity. Bound primary antibodies were revealed with either Alexa 488-conjugated goat anti-mouse or Alexa 488-conjugated goat anti-rabbit antibodies (1:400; Invitrogen, Carlsbad, CA) for 2 h at room temperature. The quantitative analysis of CTB-, synapsin I- and vGlut-2-positive profiles was performed blind to experimental treatment as described previously (Tropea et al., 2003). The number of animals used for the different analyses was as follows: CTB, n = 9; vGlut-2, n = 6; synapsin I, n = 6 per group. Serial sections (one out of three) through the SC were acquired with an Olympus Optical (Tokyo, Japan) confocal microscope. An initial analysis on stained sections from the different groups was performed to establish settings for laser intensity, gain, offset and pinhole size. Care was taken to avoid saturation at either end of the pixel intensity range (0–255). Confocal settings were then held constant through the study. Examination of animals from the various treatment groups was interdigitated to avoid bias caused by slow shifts in laser power. For each coronal section, representative fields (120 × 80 μm) were acquired using the same settings in the undeprived portion of the SC (control region), in a border region (positioned at a distance of 100 μm from the boundary of the lesion; region 2 of Fig. 1B) and in the center of the lesion (region 3 of Fig. 1B). On average, center fields were positioned at 350 μm from the edge of the lesion. All fields were located in the stratum griseum superficiale. For each field, we measured the density of CTB-labelled fibers in confocal images that consisted of the projection of five confocal sections taken at 3 μm intervals. In synapsin I and vGlut-2immunostained samples, a stack of five optical sections separated by 1 μm was collected at the top face of the tissue section. The image within each stack with the highest average pixel intensity was selected for the quantitative analysis of synapsin I/vGlut-2 immunoreactivity (Caleo et al., 2007; Tropea et al., 2003). All image analysis was performed using MCID/M4 software (Imaging Research, St. Catharines, Ontario, Canada). Retinal fiber density was calculated for each confocal micrograph by creating a binary image with a threshold value chosen to include all in-focus axons and arbors and exclude background fluorescence, and calculating the percentage of positive pixels in the field (Tropea et al., 2003). For each section, the threshold was set as the gray scale value obtained by multiplying by four the average background signal. To compensate for tracing efficiency in individual animals, the percentage of positive pixels within the lesion was normalized to axon density in the undeprived portion of the SC (region 1 of Fig. 1B) (Tropea et al., 2003).
Similarly, the area occupied by synapsin I/vGlut-2-positive pixels in the neuropil was calculated for each image by applying a threshold (mean background signal in the cell somas multiplied by four) and masking all blood vessels and cells bodies (Caleo et al., 2007; Silver and Stryker, 2000; Smith et al., 2000; Tropea et al., 2003). vGlut-2 and synapsin I immunoreactivity have been used previously to measure density of presynaptic boutons, and results from this method are in close agreement with those obtained by electron microscopy (Fujiyama et al., 2003; Lund and Lund, 1971; Silver and Stryker, 2000; Smith et al., 2000; Tropea et al., 2003). To compensate for possible differences in the quality of the immunostaining from animal to animal and section to section, the control field outside the lesion served as a within-section reference. All synaptic density data obtained within the lesion were therefore normalized to the values derived from the adjacent undeafferented SC. The statistical analysis was performed with Sigma-Stat. Normality of distributions was assessed with Kolmogorov–Smirnov test. As our samples were found to be drawn from normally distributed populations with the same variances, we used a t-test for all comparisons between standard and enriched animals. All data in the text and figures are presented as mean ± S.E.M. We evaluated sprouting of undamaged retinal afferents into the denervated SC following a partial retinal lesion in standard and enriched animals. Unilateral, focal retinal lesions were performed in adult rats. These lesions result in axotomy of the RGCs located in the ventro-temporal quadrant of the retina (see Fig. 1A) (Tropea et al., 2003). There were no significant differences in the extent of the retinal lesion between standard and enriched animals. The lesion occupied 10.6 ± 1.6% and 13.4 ± 1.8% of the retinal area in control and enriched rats, respectively (t-test, p = 0.39). The collicular lesion was invariably located in the rostro-medial portion of the SC and was clearly identifiable in coronal sections through the tectum (see Fig. 1B). Retinal afferents were labeled with fluorescently tagged CTB and fiber sprouting was measured by quantitative laser scanning confocal microscopy. The analysis included a border region (region 2 of Fig. 1B, positioned at 100 μm from the edge of the lesion) and a center region (region 3 of Fig. 1B; see also Tropea et al., 2003). In all coronal sections used for the analysis, the boundary of the lesion was sharp and clearly identifiable, as shown in the representative example of Fig. 1B. We have made sure that we have compared fields taken at correspondent positions within the denervated SC in standard and enriched animals, based on the following observations. First, the fraction of the SC that was deafferented by the lesion was superimposable between the two groups (t-test, p = 0.19). Second, the medio-lateral extent of the lesion in the single coronal sections used for the quantitative analysis did not differ between control and enriched rats (t-test, p = 0.34). The quantitative analysis indicated that environmental enrichment was remarkably effective in promoting the sprouting of RGC axon terminals at the edge of the lesion (Figs. 1C–E). On average, retinal fiber density was increased more than fourfold in enriched vs. standard animals (t-test, p = 0.002; Fig. 1E, left). There was no significant correlation between extent of retinal damage and fiber sprouting at the border of the lesion in both control and enriched rats (Pearson correlation, p N 0.63; Suppl. Fig. 1). Enriched rats tended to have a greater density of RGC fibers also in the center of the lesion, but this effect only approached statistical significance (t-test, p = 0.12; Fig. 1E, right). Analysis of the intensity of retinal fiber labelling in uninjured areas of the SC (region 1; Fig. 1C, top) revealed no significant differences between standard and enriched rats (t-test, p = 0.39). An important issue is whether sprouting RGC terminals establish synaptic contacts within the denervated tectum. As retinotectal terminals selectively employ vGlut-2 (Fujiyama et al., 2003), we used vGlut-2 immunoreactivity to determine the density of retinal synapses in the SC. The results showed a very strong decrease of
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Fig. 1. (A) Neurolucida drawing of a whole-mounted lesioned retina. The extent of the retinal damage is shown in gray. V, ventral; T, temporal. (B) Confocal image of a coronal section through the SC of an animal with partial retinal lesion. The lesioned eye was injected with Alexa594-conjugated CTB 2 days before killing. Note deafferentation of the medial part of the SC (right). The three boxes indicate the location of the fields used for the quantitation of retinal fiber density: region 1 within the undeprived SC and regions 2 and 3 at the edge and in the center of the lesion, respectively. Scale bar = 200 μm. D, dorsal; M, medial. (C) Confocal images of representative fields showing the density of RGC fibers in the SC of a control (standard-reared, left) and an enriched rat (right). Retinal afferents are labeled with Alexa594-conjugated CTB. From top to bottom, images are taken from the undeprived SC (region 1) and from the border and center of the lesion (regions 2 and 3, respectively). Scale bar = 20 μm. (D) Higher magnifications of the images shown in (C) showing sprouting at the border of the lesion. Scale bar = 10 μm. (E) Quantification of the effects of environmental enrichment on retinal fiber density at the border (left) and in the center (right) of the denervated SC. Sprouting is significantly increased at the edge of the lesion in enriched rats (region 2; ⁎⁎, t-test, p b 0.01 vs. control). Enrichment fails to reach statistical significance in the center (region 3; p = 0.12).
vGlut-2 immunoreactivity within the deafferented tectum of standard-reared animals (Figs. 2A, B). Environmental enrichment produced a robust and consistent recovery of the immunostaining at the border (Figs. 2A, B; t-test, p = 0.01), but not in the center of the lesion (t-test, p = 0.3). Finally, we measured total synaptic density at the border and in the center of the lesion by quantifying immunohistochemical staining for synapsin I (Figs. 2C, D). We found in control animals that synapse density was reduced to about 60% of the control value both at the edge and in the center of the lesion (Figs. 2C, D). Notably, environmental enrichment restored a normal cross-sectional area of synapsin I immunolabelling. A significant effect of enrichment was detectable both at the border and in the center of the lesion (p = 0.005 and p = 0.02, respectively; Figs. 2C, D). This study provides evidence that a period of environmental enrichment promotes sprouting of undamaged RGC fibers after retinal lesions in adult rats. To our knowledge, this is the first demonstration that environmental stimulation enhances plasticity of central axons following injury. One major component of environmental enrichment is the increased motor stimulation (Nithianantharajah and Hannan, 2006;
Will et al., 2004). Substantial evidence indicates that physical activity alone can account for some of the beneficial effects of enrichment (van Praag et al., 2000). In this context, it has been reported that dorsal root ganglion neurons from exercised animals show enhanced neurite extension and regeneration as compared to sedentary animals (Molteni et al., 2004). It is likely that a neurotrophin-mediated mechanism is involved in the effects of enrichment reported here. Indeed, enrichment boosts BDNF expression in the visual system (Cancedda et al., 2004; Landi et al., 2007), and antisense oligonucleotides to BDNF blocks the effects of enrichment at the retinal level (Landi et al., 2007). In keeping with an involvement of BDNF, delivery of this neurotrophin enhances RGC sprouting to the same extent as environmental enrichment does (Tropea et al., 2003). In addition to promoting sprouting of RGC axons, we found that environmental enrichment increased the density of vGlut-2-positive puncta within the lesion. As vGlut-2 is the glutamate transporter used by retinal terminals (Fujiyama et al., 2003), these data suggest synapse formation by the sprouting RGC axons. Environmental enrichment also enabled a complete recovery of synapsin I labelling within the deafferented SC. We found that while 40% fewer synapses
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Fig. 2. (A) Confocal images of vGlut-2 immunoreactivity in a control (left) and in an enriched animal (right). From top to bottom, images are taken from the undeprived SC (region 1) and from the border and center of the lesion (regions 2 and 3, respectively). Note stronger vGlut-2 staining within the lesion in the enriched rat. (B) Quantification of vGlut-2 immunoreactivity. There is a significant recovery of vGlut-2-positive puncta at the border of the lesion in rats reared in environmental enrichment (⁎⁎, t-test, p = 0.01). (C) Confocal images of synapsin I immunoreactivity in a control (left) and in an enriched animal (right). From top to bottom, images are taken from the undeprived SC (region 1) and from the border and center of the lesion (regions 2 and 3, respectively). Note complete recovery of synapsin I immunoreactivity within the lesion of the enriched rat compared to the control animal. Scale bar = 20 μm. (D) Quantification of the effects of enriched environment on total synaptic density. In standard lesioned rats synaptic density is about 60% of the control value. Environmental enrichment restores normal synaptic density values both at the border (left; ⁎⁎, t-test, p b 0.01) and in the center of the lesion (right; ⁎, t-test, p b 0.05).
are detectable in the denervated tectum of standard lesioned animals, synaptic density was completely recovered in enriched rats. It is interesting to note that a restoration of normal synaptic density was found both at the edge and in the center of the lesion, while the effects of enrichment on retinal fiber sprouting were significant only at the border. This discrepancy may be explained by postulating an effect of enrichment on the sprouting of other afferents to the denervated tectum. For example, a major input to the SC is represented by cortical afferents arising from layer V pyramidal neurons, and this pathway might undergo some remodelling following retinal lesions and environmental enrichment (Garcia del Cano et al., 2002). Another important issue is whether the effects reported here are specifically due to a “visual” enrichment or rather reflect the increased opportunities for movement, exercise and socialization. Previous data from our laboratory support the view that vision is not required for the effects of enrichment. Indeed, enrichment drives maturation of the
developing visual pathway even in the total absence of light (Bartoletti et al., 2004). Other studies have shown that environmental enrichment affects the visual system via the upregulation of trophic factors such as BDNF and insulin-like growth factor-1 (IGF-1) (Ciucci et al., 2007; Landi et al., 2007; Sale et al., 2007). The physiological consequences of the enrichment-mediated sprouting also remain to be determined. In particular, it will be important in future studies to evaluate whether the ectopic sprouting in the SC (with ventrotemporal retinal projections being replaced by axons from other areas) is beneficial or detrimental for visual behavior. In conclusion, this study indicates for the first time that environmental factors can induce axon remodelling after a CNS insult. As collateral sprouting of unlesioned fiber tracts mediates recovery of function after CNS trauma (Bareyre et al., 2004; Bradbury et al., 2002; Chen et al., 2002; Massey et al., 2006; Smith et al., 2007; Thallmair et al., 1998), this finding can have therapeutic implications in the repair of the diseased brain.
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Acknowledgments This work was supported by a grant (P83) from the International Institute for Research in Paraplegia, Zurich, Switzerland. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.expneurol.2008.12.016. References Bareyre, F.M., Kerschensteiner, M., Raineteau, O., Mettenleiter, T.C., Weinmann, O., Schwab, M.E., 2004. The injured spinal cord spontaneously forms a new intraspinal circuit in adult rats. Nat. Neurosci. 7, 269–277. Bartoletti, A., Medini, P., Berardi, N., Maffei, L., 2004. Environmental enrichment prevents effects of dark-rearing in the rat visual cortex. Nat. Neurosci. 7, 215–216. Biernaskie, J., Corbett, D., 2001. Enriched rehabilitative training promotes improved forelimb motor function and enhanced dendritic growth after focal ischemic injury. J. Neurosci. 21, 5272–5280. Bradbury, E.J., Moon, L.D., Popat, R.J., King, V.R., Bennett, G.S., Patel, P.N., Fawcett, J.W., McMahon, S.B., 2002. Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature 416, 636–640. Caleo, M., Restani, L., Gianfranceschi, L., Costantin, L., Rossi, C., Rossetto, O., Montecucco, C., Maffei, L., 2007. Transient synaptic silencing of developing striate cortex has persistent effects on visual function and plasticity. J. Neurosci. 27, 4530–4540. Cancedda, L., Putignano, E., Sale, A., Viegi, A., Berardi, N., Maffei, L., 2004. Acceleration of visual system development by environmental enrichment. J. Neurosci. 24, 4840–4848. Chen, P., Goldberg, D.E., Kolb, B., Lanser, M., Benowitz, L.I., 2002. Inosine induces axonal rewiring and improves behavioral outcome after stroke. Proc. Natl. Acad. Sci. U. S. A. 99, 9031–9036. Ciucci, F., Putignano, E., Baroncelli, L., Landi, S., Berardi, N., Maffei, L., 2007. Insulin-like growth factor 1 (IGF-1) mediates the effects of enriched environment (EE) on visual cortical development. PLoS ONE 2, e475. Fujiyama, F., Hioki, H., Tomioka, R., Taki, K., Tamamaki, N., Nomura, S., Okamoto, K., Kaneko, T., 2003. Changes of immunocytochemical localization of vesicular glutamate transporters in the rat visual system after the retinofugal denervation. J. Comp. Neurol. 465, 234–249. Garcia del Cano, G., Gerrikagoitia, I., Martinez-Millan, L., 2002. Plastic reaction of the rat visual corticocollicular connection after contralateral retinal deafferentiation at the neonatal or adult stage: axonal growth versus reactive synaptogenesis. J. Comp. Neurol. 446, 166–178. Ickes, B.R., Pham, T.M., Sanders, L.A., Albeck, D.S., Mohammed, A.H., Granholm, A.C., 2000. Long-term environmental enrichment leads to regional increases in neurotrophin levels in rat brain. Exp. Neurol. 164, 45–52. Koopmans, G.C., Brans, M., Gomez-Pinilla, F., Duis, S., Gispen, W.H., Torres-Aleman, I., Joosten, E.A., Hamers, F.P., 2006. Circulating insulin-like growth factor I and functional recovery from spinal cord injury under enriched housing conditions. Eur. J. Neurosci. 23, 1035–1046. Landi, S., Sale, A., Berardi, N., Viegi, A., Maffei, L., Cenni, M.C., 2007. Retinal functional development is sensitive to environmental enrichment: a role for BDNF. FASEB J. 21, 130–139.
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Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r
Short Communication
Environmental enrichment promotes fiber sprouting after deafferentation of the superior colliculus in the adult rat brain Matteo Caleo a,⁎, Daniela Tropea b,1, Chiara Rossi a, Laura Gianfranceschi b, Lamberto Maffei a,b a b
Istituto di Neuroscienze, Consiglio Nazionale delle Ricerche, via G. Moruzzi 1, 56100 Pisa, Italy Scuola Normale Superiore, via G. Moruzzi 1, 56100 Pisa, Italy
a r t i c l e
i n f o
Article history: Received 18 July 2008 Revised 18 December 2008 Accepted 22 December 2008 Available online 6 January 2009 Keywords: Brain injury Retinal ganglion cells Optic tectum Synaptic reorganization
a b s t r a c t Exposure to an enriched environment has proven to be beneficial in the recovery of function after brain lesions, but the underlying mechanisms remain only partly understood. One possibility is that environmental enrichment stimulates the reorganization of areas and fiber tracts that have been spared by the injury. Here we evaluate the effects of enriched environment on the sprouting of undamaged retinal afferents into the deafferented superior colliculus (SC) after a partial retinal lesion in adult rats. Anterograde tracing of retinal axons demonstrated a significant increase in fiber sprouting in the denervated SC of animals reared in enriched environment compared to animals reared in standard conditions. Environmental enrichment also promoted a substantial recovery of synaptic sites within the deafferented SC as shown by both synapsin I and vesicular glutamate transporter 2 immunostaining. These data provide evidence that environmental enrichment stimulates axonal plasticity and synaptic reorganization following brain injury. © 2009 Elsevier Inc. All rights reserved.
The adult mammalian central nervous system exhibits very poor recovery after injury. Finding strategies that enhance the functional reorganization of brain circuitry after a lesion is critical to improve the conditions of patients affected by brain trauma. Environmental enrichment is a housing condition that allows enhanced sensory, cognitive and motor stimulation relative to standard laboratory conditions (Rosenzweig and Bennett, 1996; van Praag et al., 2000). In the enriched environmental condition, animals are housed in large cages containing a variety of objects that are changed daily. There are increased opportunities for social interaction, and running wheels are available for voluntary physical exercise (Nithianantharajah and Hannan, 2006; van Praag et al., 2000; Will et al., 2004). Several studies have shown that living in an enriched environment produces dramatic effects on both the developing and adult brain (Cancedda et al., 2004; Rampon et al., 2000; Rossi et al., 2006; Sale et al., 2007). In particular, exposure to an enriched environment is known to produce functional recovery after various types of brain lesions (Biernaskie and Corbett, 2001; Koopmans et al., 2006; Pizzorusso et al., 2007; Will et al., 2004). For example, enriched housing has beneficial effects on locomotor recovery in rats with spinal cord contusion (Lankhorst et al., 2001) and improves cognitive and motor function following traumatic brain injury (Passineau et al., 2001; Smith et al., 2007). In mouse models of central nervous system
⁎ Corresponding author. Fax: +39 050 3153220. E-mail address: [email protected] (M. Caleo). 1 Present address: Department of Brain and Cognitive Sciences and Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. 0014-4886/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2008.12.016
(CNS) disorders, rearing in an enriched environment ameliorates functional parameters and reduces neurodegeneration (Nithianantharajah and Hannan, 2006; van Dellen et al., 2000). The mechanisms by which environmental enrichment produces these effects are only partly understood. One possibility is that enrichment stimulates the plastic reorganization of unlesioned areas and fiber tracts surrounding the site of injury (Will et al., 2004). Indeed, enriched experience activates molecular and cellular cascades, such as the induction of BDNF (brain-derived neurotrophic factor) and GAP43 (growth-associated protein of 43 kDa) that act to enhance and support brain plasticity (Ickes et al., 2000; Molteni et al., 2004; Pham et al., 1999). Here we investigate the effects of an exposure to an enriched environment on axonal plasticity following injury. Two to 3 months old Long–Evans rats were used in this study. All experiments were carried out in accordance with the European Community Council Directive of 24 November 1986 (86/609/EEC). Animals were either housed in standard laboratory cages (n = 12) or in an enriched environment (n = 12). The enriched environmental condition has been described previously (Bartoletti et al., 2004; Sale et al., 2007). It consisted of a large cage (95 × 75 × 80 cm) containing several food hoppers, two running wheels and differently shaped objects (tunnels, shelters, stairs) that were repositioned once per day and completely substituted with others once per week (Sale et al., 2007). Partial retinal lesions were performed as described previously (Tropea et al., 2003). The animals were anesthetized with avertin and placed in a stereotaxic apparatus. The sclera was exposed and touched with a heat microcauterizer, resulting in the complete disruption of retinal cells and the optic nerve fiber in the heated area. In all cases the
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lesions were made in the ventro-temporal portion of the retina, at a distance of 2 mm from the optic nerve head. To visualize retinal ganglion cell axons in the superior colliculus, Alexa 594-conjugated cholera toxin subunit B (CTB; Invitrogen, Carlsbad, CA) was administered into the vitreous. Rats were injected with 3 μl of 1% CTB in water 2 days before perfusion of the animals. Three weeks after the lesion, rats were deeply anaesthetized with chloral hydrate (10.5% solution; 0.4 ml/100 g) and perfused transcardially with PBS followed by 4% paraformaldehyde in 0.1 M phosphate buffer. In order to visualize the retinal lesion, eyes were removed and small crystals of the lipophilic tracer DiI (Invitrogen) were applied on the stump of the optic nerve. For the retrograde staining of retinal ganglion cells (RGCs), the eyes were left for 4 weeks at 37 °C in 4% paraformaldehyde. Measurement of the areas affected by the lesion both at the retinal and at the collicular level was performed with Neurolucida (Microbrightfield, Colchester, VT), as described previously (Tropea et al., 2003). Brain coronal sections (40 μm thick) were cut on a freezing microtome and collected in PBS. Synapsin I immunostaining was performed using the mouse monoclonal antibody 355 (Chemicon, Temecula, CA) at a concentration of 2 μg/ml. A rabbit polyclonal antibody (Synaptic Systems, Gottingen, Germany; 1:500) was used for the detection of vesicular glutamate transporter 2 (vGlut-2) immunoreactivity. Bound primary antibodies were revealed with either Alexa 488-conjugated goat anti-mouse or Alexa 488-conjugated goat anti-rabbit antibodies (1:400; Invitrogen, Carlsbad, CA) for 2 h at room temperature. The quantitative analysis of CTB-, synapsin I- and vGlut-2-positive profiles was performed blind to experimental treatment as described previously (Tropea et al., 2003). The number of animals used for the different analyses was as follows: CTB, n = 9; vGlut-2, n = 6; synapsin I, n = 6 per group. Serial sections (one out of three) through the SC were acquired with an Olympus Optical (Tokyo, Japan) confocal microscope. An initial analysis on stained sections from the different groups was performed to establish settings for laser intensity, gain, offset and pinhole size. Care was taken to avoid saturation at either end of the pixel intensity range (0–255). Confocal settings were then held constant through the study. Examination of animals from the various treatment groups was interdigitated to avoid bias caused by slow shifts in laser power. For each coronal section, representative fields (120 × 80 μm) were acquired using the same settings in the undeprived portion of the SC (control region), in a border region (positioned at a distance of 100 μm from the boundary of the lesion; region 2 of Fig. 1B) and in the center of the lesion (region 3 of Fig. 1B). On average, center fields were positioned at 350 μm from the edge of the lesion. All fields were located in the stratum griseum superficiale. For each field, we measured the density of CTB-labelled fibers in confocal images that consisted of the projection of five confocal sections taken at 3 μm intervals. In synapsin I and vGlut-2immunostained samples, a stack of five optical sections separated by 1 μm was collected at the top face of the tissue section. The image within each stack with the highest average pixel intensity was selected for the quantitative analysis of synapsin I/vGlut-2 immunoreactivity (Caleo et al., 2007; Tropea et al., 2003). All image analysis was performed using MCID/M4 software (Imaging Research, St. Catharines, Ontario, Canada). Retinal fiber density was calculated for each confocal micrograph by creating a binary image with a threshold value chosen to include all in-focus axons and arbors and exclude background fluorescence, and calculating the percentage of positive pixels in the field (Tropea et al., 2003). For each section, the threshold was set as the gray scale value obtained by multiplying by four the average background signal. To compensate for tracing efficiency in individual animals, the percentage of positive pixels within the lesion was normalized to axon density in the undeprived portion of the SC (region 1 of Fig. 1B) (Tropea et al., 2003).
Similarly, the area occupied by synapsin I/vGlut-2-positive pixels in the neuropil was calculated for each image by applying a threshold (mean background signal in the cell somas multiplied by four) and masking all blood vessels and cells bodies (Caleo et al., 2007; Silver and Stryker, 2000; Smith et al., 2000; Tropea et al., 2003). vGlut-2 and synapsin I immunoreactivity have been used previously to measure density of presynaptic boutons, and results from this method are in close agreement with those obtained by electron microscopy (Fujiyama et al., 2003; Lund and Lund, 1971; Silver and Stryker, 2000; Smith et al., 2000; Tropea et al., 2003). To compensate for possible differences in the quality of the immunostaining from animal to animal and section to section, the control field outside the lesion served as a within-section reference. All synaptic density data obtained within the lesion were therefore normalized to the values derived from the adjacent undeafferented SC. The statistical analysis was performed with Sigma-Stat. Normality of distributions was assessed with Kolmogorov–Smirnov test. As our samples were found to be drawn from normally distributed populations with the same variances, we used a t-test for all comparisons between standard and enriched animals. All data in the text and figures are presented as mean ± S.E.M. We evaluated sprouting of undamaged retinal afferents into the denervated SC following a partial retinal lesion in standard and enriched animals. Unilateral, focal retinal lesions were performed in adult rats. These lesions result in axotomy of the RGCs located in the ventro-temporal quadrant of the retina (see Fig. 1A) (Tropea et al., 2003). There were no significant differences in the extent of the retinal lesion between standard and enriched animals. The lesion occupied 10.6 ± 1.6% and 13.4 ± 1.8% of the retinal area in control and enriched rats, respectively (t-test, p = 0.39). The collicular lesion was invariably located in the rostro-medial portion of the SC and was clearly identifiable in coronal sections through the tectum (see Fig. 1B). Retinal afferents were labeled with fluorescently tagged CTB and fiber sprouting was measured by quantitative laser scanning confocal microscopy. The analysis included a border region (region 2 of Fig. 1B, positioned at 100 μm from the edge of the lesion) and a center region (region 3 of Fig. 1B; see also Tropea et al., 2003). In all coronal sections used for the analysis, the boundary of the lesion was sharp and clearly identifiable, as shown in the representative example of Fig. 1B. We have made sure that we have compared fields taken at correspondent positions within the denervated SC in standard and enriched animals, based on the following observations. First, the fraction of the SC that was deafferented by the lesion was superimposable between the two groups (t-test, p = 0.19). Second, the medio-lateral extent of the lesion in the single coronal sections used for the quantitative analysis did not differ between control and enriched rats (t-test, p = 0.34). The quantitative analysis indicated that environmental enrichment was remarkably effective in promoting the sprouting of RGC axon terminals at the edge of the lesion (Figs. 1C–E). On average, retinal fiber density was increased more than fourfold in enriched vs. standard animals (t-test, p = 0.002; Fig. 1E, left). There was no significant correlation between extent of retinal damage and fiber sprouting at the border of the lesion in both control and enriched rats (Pearson correlation, p N 0.63; Suppl. Fig. 1). Enriched rats tended to have a greater density of RGC fibers also in the center of the lesion, but this effect only approached statistical significance (t-test, p = 0.12; Fig. 1E, right). Analysis of the intensity of retinal fiber labelling in uninjured areas of the SC (region 1; Fig. 1C, top) revealed no significant differences between standard and enriched rats (t-test, p = 0.39). An important issue is whether sprouting RGC terminals establish synaptic contacts within the denervated tectum. As retinotectal terminals selectively employ vGlut-2 (Fujiyama et al., 2003), we used vGlut-2 immunoreactivity to determine the density of retinal synapses in the SC. The results showed a very strong decrease of
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Fig. 1. (A) Neurolucida drawing of a whole-mounted lesioned retina. The extent of the retinal damage is shown in gray. V, ventral; T, temporal. (B) Confocal image of a coronal section through the SC of an animal with partial retinal lesion. The lesioned eye was injected with Alexa594-conjugated CTB 2 days before killing. Note deafferentation of the medial part of the SC (right). The three boxes indicate the location of the fields used for the quantitation of retinal fiber density: region 1 within the undeprived SC and regions 2 and 3 at the edge and in the center of the lesion, respectively. Scale bar = 200 μm. D, dorsal; M, medial. (C) Confocal images of representative fields showing the density of RGC fibers in the SC of a control (standard-reared, left) and an enriched rat (right). Retinal afferents are labeled with Alexa594-conjugated CTB. From top to bottom, images are taken from the undeprived SC (region 1) and from the border and center of the lesion (regions 2 and 3, respectively). Scale bar = 20 μm. (D) Higher magnifications of the images shown in (C) showing sprouting at the border of the lesion. Scale bar = 10 μm. (E) Quantification of the effects of environmental enrichment on retinal fiber density at the border (left) and in the center (right) of the denervated SC. Sprouting is significantly increased at the edge of the lesion in enriched rats (region 2; ⁎⁎, t-test, p b 0.01 vs. control). Enrichment fails to reach statistical significance in the center (region 3; p = 0.12).
vGlut-2 immunoreactivity within the deafferented tectum of standard-reared animals (Figs. 2A, B). Environmental enrichment produced a robust and consistent recovery of the immunostaining at the border (Figs. 2A, B; t-test, p = 0.01), but not in the center of the lesion (t-test, p = 0.3). Finally, we measured total synaptic density at the border and in the center of the lesion by quantifying immunohistochemical staining for synapsin I (Figs. 2C, D). We found in control animals that synapse density was reduced to about 60% of the control value both at the edge and in the center of the lesion (Figs. 2C, D). Notably, environmental enrichment restored a normal cross-sectional area of synapsin I immunolabelling. A significant effect of enrichment was detectable both at the border and in the center of the lesion (p = 0.005 and p = 0.02, respectively; Figs. 2C, D). This study provides evidence that a period of environmental enrichment promotes sprouting of undamaged RGC fibers after retinal lesions in adult rats. To our knowledge, this is the first demonstration that environmental stimulation enhances plasticity of central axons following injury. One major component of environmental enrichment is the increased motor stimulation (Nithianantharajah and Hannan, 2006;
Will et al., 2004). Substantial evidence indicates that physical activity alone can account for some of the beneficial effects of enrichment (van Praag et al., 2000). In this context, it has been reported that dorsal root ganglion neurons from exercised animals show enhanced neurite extension and regeneration as compared to sedentary animals (Molteni et al., 2004). It is likely that a neurotrophin-mediated mechanism is involved in the effects of enrichment reported here. Indeed, enrichment boosts BDNF expression in the visual system (Cancedda et al., 2004; Landi et al., 2007), and antisense oligonucleotides to BDNF blocks the effects of enrichment at the retinal level (Landi et al., 2007). In keeping with an involvement of BDNF, delivery of this neurotrophin enhances RGC sprouting to the same extent as environmental enrichment does (Tropea et al., 2003). In addition to promoting sprouting of RGC axons, we found that environmental enrichment increased the density of vGlut-2-positive puncta within the lesion. As vGlut-2 is the glutamate transporter used by retinal terminals (Fujiyama et al., 2003), these data suggest synapse formation by the sprouting RGC axons. Environmental enrichment also enabled a complete recovery of synapsin I labelling within the deafferented SC. We found that while 40% fewer synapses
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Fig. 2. (A) Confocal images of vGlut-2 immunoreactivity in a control (left) and in an enriched animal (right). From top to bottom, images are taken from the undeprived SC (region 1) and from the border and center of the lesion (regions 2 and 3, respectively). Note stronger vGlut-2 staining within the lesion in the enriched rat. (B) Quantification of vGlut-2 immunoreactivity. There is a significant recovery of vGlut-2-positive puncta at the border of the lesion in rats reared in environmental enrichment (⁎⁎, t-test, p = 0.01). (C) Confocal images of synapsin I immunoreactivity in a control (left) and in an enriched animal (right). From top to bottom, images are taken from the undeprived SC (region 1) and from the border and center of the lesion (regions 2 and 3, respectively). Note complete recovery of synapsin I immunoreactivity within the lesion of the enriched rat compared to the control animal. Scale bar = 20 μm. (D) Quantification of the effects of enriched environment on total synaptic density. In standard lesioned rats synaptic density is about 60% of the control value. Environmental enrichment restores normal synaptic density values both at the border (left; ⁎⁎, t-test, p b 0.01) and in the center of the lesion (right; ⁎, t-test, p b 0.05).
are detectable in the denervated tectum of standard lesioned animals, synaptic density was completely recovered in enriched rats. It is interesting to note that a restoration of normal synaptic density was found both at the edge and in the center of the lesion, while the effects of enrichment on retinal fiber sprouting were significant only at the border. This discrepancy may be explained by postulating an effect of enrichment on the sprouting of other afferents to the denervated tectum. For example, a major input to the SC is represented by cortical afferents arising from layer V pyramidal neurons, and this pathway might undergo some remodelling following retinal lesions and environmental enrichment (Garcia del Cano et al., 2002). Another important issue is whether the effects reported here are specifically due to a “visual” enrichment or rather reflect the increased opportunities for movement, exercise and socialization. Previous data from our laboratory support the view that vision is not required for the effects of enrichment. Indeed, enrichment drives maturation of the
developing visual pathway even in the total absence of light (Bartoletti et al., 2004). Other studies have shown that environmental enrichment affects the visual system via the upregulation of trophic factors such as BDNF and insulin-like growth factor-1 (IGF-1) (Ciucci et al., 2007; Landi et al., 2007; Sale et al., 2007). The physiological consequences of the enrichment-mediated sprouting also remain to be determined. In particular, it will be important in future studies to evaluate whether the ectopic sprouting in the SC (with ventrotemporal retinal projections being replaced by axons from other areas) is beneficial or detrimental for visual behavior. In conclusion, this study indicates for the first time that environmental factors can induce axon remodelling after a CNS insult. As collateral sprouting of unlesioned fiber tracts mediates recovery of function after CNS trauma (Bareyre et al., 2004; Bradbury et al., 2002; Chen et al., 2002; Massey et al., 2006; Smith et al., 2007; Thallmair et al., 1998), this finding can have therapeutic implications in the repair of the diseased brain.
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