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Upregulation of the immediate early gene arc in the brains of rats exposed to environmental enrichment: implications for molecular plasticity
Molecular Brain Research 91 (2001) 50–56 www.elsevier.com / locate / bres
Research report
Upregulation of the immediate early gene arc in the brains of rats exposed to environmental enrichment: implications for molecular plasticity Raphael Pinaud a , Marsha R. Penner b , Harold A. Robertson b , R. William Currie a , * a
Department of Anatomy and Neurobiology, Laboratory of Molecular Neurobiology, Dalhousie University, Halifax, Nova Scotia, Canada B3 H 4 H7 b Department of Pharmacology, Laboratory of Molecular Neurobiology, Dalhousie University, Halifax, Nova Scotia, Canada B3 H 4 H7 Accepted 3 April 2001
Abstract Exposure to an enriched environment, a procedure that induces plasticity in the cerebral cortex, is associated with pronounced morphological changes, including higher density of dendritic spines, enlargement of synaptic boutons, and other putative correlates of altered neurotransmission. Recently, it has been demonstrated that animals reared in an enriched environment setting for 3 weeks have less neuronal damage as a result of seizures and have decreased rates of spontaneous apoptosis. Even though clear morphological modifications are observed in the cerebral cortex of animals exposed to heightened environmental complexity, the molecular mechanisms that underlie such modifications are yet to be described. In the present work, we investigated the expression of the immediate early gene arc in the cortex of animals exposed to an enriched environment. Animals were exposed daily, for 1 h, to an enriched environment, for a total period of 3 weeks. Brains were processed for in-situ hybridization against arc mRNA. We found a marked upregulation of arc mRNA in the cerebral cortex of animals exposed to the enriched environment, when compared to undisturbed controls, an effect that was most pronounced in cortical layers III and V. Animals in an additional control group that were handled for 5 min daily, displayed intermediate levels of arc mRNA. Furthermore, arc expression was upregulated in the CA1, CA2 and CA3 hippocampal subfields and in the striatum, but to a lesser extent in the dentate gyrus of animals exposed to an enriched environment, as compared to the two control groups. Our results support the association between the upregulation of the immediate early gene arc and plasticity-associated anatomical changes in the cerebral cortex of the adult mammal. 2001 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Neural plasticity Keywords: Arc; Enriched environment; Plasticity; Transcription factor; NGFI-A; Dendrite
1. Introduction In the early 1970s, several research groups demonstrated that exposure of animals to an enriched environment produced marked morphological changes in the sensory cortices of these animals [7,19,23]. For example, it has been demonstrated that exposure of adult rats to increased
*Corresponding author. Tel.: 11-902-494-3343; fax: 11-902-4941212. E-mail address: [email protected] (R.W. Currie).
environmental complexity increases the number of dendritic branches in the visual and somatosensory cortices [19,23]. In addition, other groups have demonstrated morphological modifications in the brains of animals exposed to an enriched environment. These changes included an increase in the number of dendritic spines, enlargement of synapses and an increase in the neuron to glia ratio [7,19]. Behaviorally-induced cellular plasticity reflects the reorganization and re-weighting of cerebral circuits and has been hypothesized to reflect the physical changes that enable learning and memory. More recently, it has been documented that levels of neurotrophic factors, such as the brain-derived neurotrophic factor (BDNF), are
0169-328X / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 01 )00121-8
R. Pinaud et al. / Molecular Brain Research 91 (2001) 50 – 56
increased in the central nervous system (CNS) in response to exposure to a complex environment [5,11,17,22]. Consequentially, exposure to an enriched environment enhances performance on behavioral paradigms of learning, memory, and visual acuity, suggesting that circuits are altered in order to optimize multiple levels of information processing and storage of environmental information [6,15,18]. Exposure to an enriched environment also confers neuroprotection against seizure-related neuronal damage and inhibits spontaneous apoptosis, which may provide mechanisms for longer-term neuroprotection [25]. Several aspects of the mechanisms that guide morphological restructuring, synaptogenesis and synaptic weighting have been characterized. The expression of immediate early genes (IEGs), the focus of the present work, has been postulated as the molecular mechanisms that control plastic changes in the CNS. In 1995, Wallace and collaborators provided strong evidence that the zincfinger IEG nerve growth factor induced gene-A (NGFI-A) is upregulated in the brains of rats exposed to an enriched environment. Expression of this IEG was lower in animals that were simply manipulated or left undisturbed [24]. NGFI-A is a transcriptional regulator that is known to regulate the expression of both synapsin I and II genes, that in turn are involved in the docking of synaptic vesicles, neurite outgrowth and synapse maturation [16,21]. These findings strongly suggest that NGFI-A expression may reflect one of the molecular pathways that mediate neuronal plasticity in the early stages of synaptic reorganization. In the present paper, we characterized the expression of a second candidate-plasticity gene, the activity-regulated cytoskeletal protein (arc), in the brains of animals exposed to the enriched environment setting. Arc is an IEG and growth factor that is rapidly and transiently induced after neural activity [8,13,20]. It has been previously demonstrated that arc expression is activity-regulated and its translation takes place in polyribosomal complexes located in the post-synaptic density [20]. One implication of this finding is that the protein encoded by this IEG might have a localized role in dendritic configuration (i.e., spine shape, dendritic orientation, effective synapse size or efficacy [2,4,9,10,14]. The position of this site of translation is intriguing in light of several hypotheses that suggest that learning induced plasticity results in physical changes in circuitry and that such changes can be demonstrated where neurons are most closely physically associated and fidelity of neurotransmission can be greatest [4,9,10]. Guzowski and collaborators have recently provided additional support for arc in mediating plasticity, as blockade of arc expression with an antisense oligonucleotide impaired the consolidation of long-term memory and the maintenance of long-term potentiation (LTP) in rats [8]. Here we determine the effects of a procedure that induces plasticity (exposure to an enriched environment) in
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the cerebral cortex, on the expression of arc, a gene whose expression may be related to plasticity in dendrites.
2. Material and methods
2.1. Animals and experimental groups We used a total of 18 adult male Sprague Dawley rats (Charles River, Canada), weighing between 200 and 225 g. All protocols utilized were in accordance with institutional regulations and the Guide to the Care and Use of Experimental Animals from the Canadian Council on Animal Care. The protocol for handling the animals within the enriched environment experimental paradigm is based on previous work by Wallace and colleagues [24]. All rats were housed in a room under a 12 / 12 h light–dark cycle. Rats were divided into 3 experimental groups: (1) Animals exposed to an enriched environment (EE) for 1 h per day (n56). Animals belonging to this group were housed in a standard laboratory home cage with a larger variety of toys and objects. Each afternoon, all EE animals were transferred together to a ‘play area’ of 3 m31.5 m31 m, where they remained for 1 h. This larger enriched environment also contained a variety of toys, objects, short and long plastic tubes, plastic houses and a pan of water. In addition, cereal flakes were spread throughout the play area every 2 days. After this period, rats were returned to their home cages (3 animals per cage) (Fig. 1). (2) Handledonly animals (HO) (n56) were manipulated for a period of 5 min and returned to their home cages. This group was included in the present experiment to control for stress during cage transfer manipulation that was experienced by EE animals, as well as a possible ‘expectation-related’ gene expression. The manipulation procedure consisted of briefly, but repeatedly, lifting these animals. At the end of the handling period, animals were returned to their standard home cages (Fig. 1). (3) Undisturbed controls (UD) (n56) were left untouched in their home cages throughout the experiment (Fig. 1). The procedures described above were conducted for a total period of 3 weeks. To control for possible circadian influences on gene expression, exposure to the enriched environment and handling protocols were conducted between 3 and 4 pm. Behavior was fully monitored for all experimental groups.
2.2. Perfusion and histology In the last day of the experiment, all animals were anesthetized with sodium pentobarbital (70 mg / kg, i.p.) and perfused transcardially with a solution of 0.1 M phosphate buffered saline (PBS). Brains were rapidly dissected out from skulls, cerebellums were removed and brains were placed inside sterile petri-dishes and fast-
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R. Pinaud et al. / Molecular Brain Research 91 (2001) 50 – 56
Fig. 1. Outline of experimental protocol. EE animals were exposed daily, for a total of 3 weeks, to 1 h inside the enriched play area. HO animals were manipulated for 5 min, while UD controls were left untouched for the total period of the experiment. In the last experimental day, EE animals were exposed to the play area for 1 h and then immediately killed. HO animals were handled for 5 min and then killed with minimal time interval.
frozen in a 2708 freezer. Once frozen, brains were transferred to the cryostat and 20 mm sections were made under RNAse-free conditions. From the 6 animals per group, 4 brains were sectioned in the coronal plane, whereas 2 brains were sectioned parasagittally. Sections were directly mounted onto Fisherbrand Superfrost slides and returned to the 2708C freezer.
2.3. In-situ hybridization Slides were removed from the freezer, briefly fixed in 4% paraformaldehyde solution for 5 min and washed in 150 mM sodium chloride / 15 mM sodium citrate (13 SSC). An oligonucleotide probe, specific for the arc mRNA was commercially synthesized (Genosys, Canada) with the following sequence: 59-ATACAGTGTCTGGTACAGGTCCCGCTTACG-39. All sections were subsequently incubated with the arc cDNA oligonucleotide probe, that was 39 labeled with [ 33 P]dATP. Our arc probe was diluted in hybridization buffer (5310 6 cpm / ml), consisting of 50% deionized formamide, 50% 23hybridization buffer. The following day, sections were washed, at 558C, 4315 min in 23SSC, 4315 min in 13SSC, 4315 min in 0.53SSC, 2320 min in 0.253SSC at room temperature and dipped in MilliQ water. Slides were dried overnight at room temperature, a
procedure that was followed by the exposure to Kodak BioMax MR film (Interscience, Markham, ON) for 1 to 3 days.
2.4. Imaging Autoradiograms were scanned with a desktop scanner (Duoscan T1200, Agfa Inc.) and images were digitalized into an IBM-PC computer. Adobe Photoshop software (Adobe, Pantone Inc.) was used to assemble plates.
2.5. Densitometric and statistical analysis Densitometric analysis was performed using NIH Image software. Optical density (OD) was measured across cortical layers for the four levels presented in Fig. 2 and for the whole extent of the hippocampal formation in Fig. 3. Background values obtained from white matter were subtracted from values collected for cortical regions and hippocampi. Data was collected from 4 animals per experimental group, in which OD was averaged across hemispheres in each animal. OD is represented in a range from 0 (white) to 1 (black). Significant treatment effects were analyzed using one-way analysis of variance (ANOVA). The Tukey HSD-test was used for post-hoc comparisons with the criterion level set at P,0.05.
R. Pinaud et al. / Molecular Brain Research 91 (2001) 50 – 56
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Fig. 2. Autoradiograms depicting arc mRNA hybridization at comparable stereotaxic levels in brains of EE (top panel), HO (middle panel) and UD (bottom panel) animals. Sections are presented from rostral to caudal, left to right. Note the marked upregulation in the striatum, sensory cortices and hippocampus of sections of the EE group. Using bregma as reference, approximate stereotaxic coordinates for the four levels presented in this figure are: Level 1 (AP 2.20), Level 2 (AP 0.70), Level 3 (AP24.16) and Level 4 (AP26.30). Scale bar5500 mm.
3. Results In-situ hybridization was used to localize the expression patterns of the immediate early gene arc in the brain. Both coronal and parasagittal sections from all experimental groups were exposed to the same histological conditions in order to minimize reaction variables. In EE animals, arc upregulation was found in several cortical regions, with the most pronounced upregulation observed in the sensory cortices, striatum and hippocampal formation (see Tables 1 and 2). In HO and UD controls, arc mRNA was found at apparent basal levels. Dense arc labeling was found in both the primary and
secondary visual areas of EE animals (Fig. 2). For the primary visual cortex of EE animals, regions that correspond to binocular and monocular vision displayed high levels of arc mRNA expression that were approximately equal in intensity. In these regions, labeling was most pronounced in cortical layers III and V, whereas basal levels of arc expression were found in all other layers. In contrast to EE animals, HO and UD controls displayed basal levels of arc expression in these cortical regions. No clear layering was detectable for either control group (Fig. 2). In the somatosensory cortex, the highest arc expression was found in EE animals, followed by HO and UD
Fig. 3. Autoradiograms depicting the pattern of arc mRNA hybridization in the hippocampus and dentate gyrus of all experimental groups. Arc is expressed in all hippocampal subfields in EE animals, while UD animals displayed minimal arc mRNA levels. Arc signal in HO animals was detected at intermediate levels. In addition, arc expression was detected in the dentate gyrus of both EE and HO animal, while in UD controls mRNA signal was almost undetectable. Scale bar5200 mm.
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Table 1 Effects of environmental enrichment on arc mRNA expression in the cortex a Groups
EE HO UD
Level I
Level II
Level III
Level IV
Mean (6S.E.M.)
Treatment F(2, 37)
Mean (6S.E.M.)
Treatment F(2, 43)
Mean (6S.E.M.)
Treatment F(2, 49)
Mean (6S.E.M.)
Treatment F(2, 41)
0.162060.0009a 0.002060.0002b 0.001560.0007b
171.14*
0.184260.0009a 0.002860.0003b 0.000960.0002b
186.22*
0.184260.0005a 0.004660.0018b 0.003860.0005b
71.01*
0.167560.0008a 0.002660.0004b 0.003660.0004b
174.81*
a
Data are expressed as mean optical density6S.E.M. (n54 per group). *All F values significant P,0.001. a,b5groups with different letters differ significantly (Tukey HSD test P,0.05).
controls (Fig. 2). In EE animals, heightened arc labeling was confined to cortical layers III and V, while no apparent stratification was detected for either control group. The most pronounced difference across experimental groups was observed in the hippocampus. Animals exposed to the enriched environment displayed a significant upregulation of arc mRNA in all subfields of the hippocampal formation. Arc expression was almost undetectable in the hippocampus of UD controls, while HO controls displayed intermediate levels of arc mRNA (Figs. 2 and 3; Table 2). However, arc was expressed in a low level in the dentate gyrus of both control groups, while EE animals had a moderate upregulate of arc expression in this region. (Fig. 3; Table 2). A pronounced upregulation of arc mRNA was also found in the striatum. EE animals had a significant upregulation of arc expression in this region, as compared to either control group. In the remaining cortical regions, arc expression also followed the same layering pattern. While most cortical neurons displayed arc mRNA signal to some degree, the strongest labeling was detected in the EE group for cortical layers III and V (Fig. 4). In addition, HO and UD animals displayed basal levels of arc expression, with the same apparent intensity across all cortical layers.
4. Discussion The major finding of the present work is that the immediate early gene arc is significantly upregulated, in
Table 2 Effects of environmental enrichment on arc mRNA expression in the hippocampus a Groups
Mean (6S.E.M.)
Treatment F(2, 15)
EE HO UD
0.116760.0009a 0.002160.0004b 0.000660.0012b
95.59*
a
Data are expressed as mean optical density6S.E.M. (n54 per group). *All F values significant P,0.001. a, b5groups with different letters differ significantly (Tukey HSD test P,0.05).
several sensory systems, in response to environmental complexity. In addition, we have demonstrated that this upregulation is more pronounced in the hippocampus. Constitutive arc expression was found in the brains of two control groups, therefore suggesting to us that the upregulation of this gene in the EE group may reflect processing of greater complexity in the sensory environment, rather than altered rates of neuronal activation, as an explanation that has been forwarded to account for enhanced NGFI-A and c-fos expression [3]. Even though arc expression has been demonstrated to be activity dependent, neuronal depolarization alone is not able to upregulate this gene. For example, blockade of the N-methyl-D-aspartate (NMDA) type of glutamatergic receptors suppressed arc expression [12]. In the present experiment, rats in the HO group were stimulated by visual, olfactory and somatosensory cues that should have resulted in an enhanced expression of the arc gene, according to our hypothesis described above. However, these animals failed to demonstrate such an increase in arc expression and elevated mRNA levels were restricted to the EE animals. Our findings suggest that another process must be involved in regulating arc expression, in conjunction with increased activity, in a given sensory pathway. Because arc mRNA was increased as a result to exposure to an enriched environment, we postulate that arc is invoked as a means of increasing neuronal plasticity and may be related to information or sensory overload. It has been previously demonstrated that arc is rapidly induced, a characteristic feature of immediate early genes, and that the mRNA synthesized from this gene is also rapidly transported to the dendrites [13,20]. In addition, it has been postulated that translation of the protein encoded by the arc gene takes place in polyribosomal complexes located at postsynaptic sites on dendrites [20]. Given that plasticity in the cerebral cortex involves, in some cases, the physical rearrangement or synthesis of cortical circuits, the Arc protein would be, at least, spatially well located to mediate many aspects of reorganization. More specifically, Arc is a growth factor that could regulate neurite sprouting in response to complex levels of sensory information [13]. In 1998, Steward and colleagues demonstrated that stimulation of specific sites of the perforant path increased arc mRNA accumulation in corresponding sites of the
R. Pinaud et al. / Molecular Brain Research 91 (2001) 50 – 56
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Fig. 4. Pictures from an autoradiogram exposed to in-situ hybridization signal depicting the laminar profile of arc expression in a coronal (A) and parasagittal section (B) of an EE animal. The superficial band in either profile corresponds to cortical layer III and the deep band to cortical layer V. Threshold images from the coronal (C) and parasagittal (D) sections depict clearly this laminar profile. Scale bar5500 mm.
dendritic arborization of dentate granule cells. Interestingly, the greatest levels of arc mRNA accumulation were observed in the locations where perforant fibers innervated dendritic branches of dentate granule cells, suggesting that arc expression was directly correlated to the load of information delivered to a given cellular site [20]. Moreover, the stimulation paradigm used in their study has the attributes that has been previously used to induce longterm potentiation in the dentate gyrus [1], a finding that also supports the argument that arc expression may be involved as one of the first genetic mediators of LTPdependent plasticity in the CNS. Unlike the immunoreactive profile that results with markers for c-fos or NGFI-A, which reveal cell nuclei, arc labeling is dendritic. This feature may become important for identifying the locations of plasticity induced changes. For example, arc labeling would enable the identification of reorganization in cells where the sites of plasticity are remote from their cell bodies, such as the reorganization of deep thalamic and brainstem nuclei following deafferentation, in addition to changes demonstrated in the cerebral cortex. Identifying the contributions of these subcortical CNS levels to the reorganizational process has been
limited to functional studies based on electrophysiology, activity markers such as GAD and cytochrome oxidase and tracer studies in control versus reorganized systems. The identification of IEGs that mediate plastic changes provide great opportunities for pinpointing the site and extent of reorganization throughout the ascending sensory pathway, thus, increasing our understanding of the overall process and possibly indicating locations where therapeutic interventions would provide the maximal benefit.
Acknowledgements We would like to thank Dr. Liisa Tremere for carefully reading this manuscript and for useful comments and Ms. Kay Murphy for excellent technical support. Dr. E. Denovan-Wright helped with the design of the arc oligonucleotide probe. Felipe Hess helped with the initial parts of this work. This work was supported by the Canadian Institutes of Health Research, the Canadian Stroke Network, the Heart and Stroke Foundation of New Brunswick, and by a Killam Scholarship to R.P.
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References [1] T.V.P. Bliss, M.A. Lynch, Long-term potentiation of synaptic transmission in the hippocampus: properties and mechanisms, in: P.W. Landfield, S.A. Deadwyler (Eds.), Long-Term Potentiation: From Biophysics to Behavior, Alan R. Liss, New York, 1988, pp. 3–72. [2] A. Caceres, O. Steward, Dendritic reorganization in the denervated dentate gyrus of the rat following entorhinal cortical lesions: a Golgi and electron microscopic analysis, J. Comp. Neurol. 214 (1983) 387–403. [3] A. Chaudhuri, Neural activity mapping with inducible transcription factors, Neuroreport 8 (1997) v–ix. [4] J.C. Eccles, Possible ways in which synaptic mechanisms participate in learning, remembering and forgetting, in: D.P. Kimble (Ed.), The Anatomy of Memory, Science and Behaviour Books, Palo Alto, 1965, p. 97. [5] T. Falkenberg, A.K. Mohammed, B. Henriksson, H. Persson, B. Winblad, N. Lindefors, Increased expression of brain-derived neurotrophic factor mRNA in rat hippocampus is associated with improved spatial memory and enriched environment, Neurosci. Lett. 138 (1992) 153–156. [6] A. Fernandez-Teruel, R.M. Escorihuela, B. Castellano, B. Gonzalez, A. Tobena, Neonatal handling and environmental enrichment effects on emotionality, novelty / reward seeking, and age-related cognitive and hippocampal impairments: focus on the Roman rat lines, Behav. Genet. 27 (1997) 513–526. [7] A. Globus, M.R. Rosenzweig, E.L. Bennett, M.C. Diamond, Effects of differential experience on dendritic spine counts in rat cerebral cortex, J. Comp. Physiol. Psychol. 82 (1973) 175–181. [8] J.F. Guzowski, G.L. Lyford, G.D. Stevenson, F.P. Houston, J.L. McGaugh, P.F. Worley, C.A. Barnes, Inhibition of activity-dependent arc protein expression in the rat hippocampus impairs the maintenance of long-term potentiation and the consolidation of long-term memory, J. Neurosci. 20 (2000) 3993–4001. [9] D.O. Hebb, The Organisation of Behaviour, Wiley, New York, 1949. [10] T. Hosokawa, D.A. Rusakov, T.V.P. Bliss, A. Fine, Repeated confocal imaging of individual dendritic spines in the living hippocampal slice: evidence for changes in length and orientation associated with chemically induced LTP, J. Neurosci. 15 (1995) 5560–5573. [11] B.R. Ickes, T.M. Pham, L.A. Sanders, D.S. Albeck, A.H. Mohammed, A.C. Granholm, Long-term environmental enrichment leads to regional increases in neurotrophin levels in rat brain, Exp. Neurol. 164 (2000) 45–52. [12] H. Kunizuka, H. Kinouchi, S. Arai, K. Izaki, S. Mikawa, H. Kamii, T. Sugawara, A. Suzuki, K. Mizoi, T. Yoshimoto, Activation of arc gene, a dendritic immediate early gene, by middle cerebral artery occlusion in rat brain, Neuroreport 10 (1999) 1717–1722.
[13] G.L. Lyford, K. Yamagata, W.E. Kaufmann, C.A. Barnes, L.K. Sanders, N.G. Copeland, D.J. Gilbert, N.A. Jenkins, A.A. Lanahan, P.F. Worley, Arc, a growth factor and activity-regulated gene, encodes a novel cytoskeleton-associated protein that is enriched in neuronal dendrites, Neuron 14 (1995) 433–445. [14] J.G. Parnavelas, G. Lynch, N. Brecha, C.W.W. Cotman, A. Globus, Spine loss and regrowth in hippocampus following deafferentation, Nature 248 (1974) 71–73. [15] R. Paylor, S.K. Morrison, J.W. Rudy, L.T. Waltrip, J.M. Wehner, Brief exposure to an enriched environment improves performance on the Morris water task and increases hippocampal cytosolic protein kinase C activity in young rats, Behav. Brain Res. 52 (1992) 49–59. [16] D. Petersohn, S. Schoch, D.R. Brinkmann, G. Thiel, The human synapsin II gene promoter. Possible role for the transcription factor zif268 / egr-1, polyoma enhancer activator 3, and AP2, J. Biol. Chem. 270 (1995) 24361–24369. [17] T.M. Pham, B. Ickes, D. Albeck, S. Soderstrom, A.C. Granholm, A.H. Mohammed, Changes in brain nerve growth factor levels and nerve growth factor receptors in rats exposed to environmental enrichment for one year, Neuroscience 94 (1999) 279–286. [18] G.T. Prusky, C. Reidel, R.M. Douglas, Environmental enrichment from birth enhances visual acuity but not place learning in mice, Behav. Brain Res. 114 (2000) 11–15. [19] M.R. Rozenzweig, E.L. Bennett, M.C. Diamond, Brain changes in relation to experience, Sci. Am. 226 (1972) 22–29. [20] O. Steward, C.S. Wallace, G.L. Lyford, P.F. Worley, Synaptic activation causes the mRNA for the IEG Arc to localize selectively near activated postsynaptic sites on dendrites, Neuron 21 (1998) 741–751. [21] G. Thiel, S. Schoch, D. Petersohn, Regulation of synapsin I gene expression by the zinc finger transcription factor zif268 / egr-1, J. Biol. Chem. 269 (1994) 15294–15301. [22] M. Torasdotter, M. Metsis, B.G. Henriksson, B. Winblad, A.H. Mohammed, Environmental enrichment results in higher levels of nerve growth factor mRNA in the rat visual cortex and hippocampus, Behav. Brain Res. 93 (1998) 83–90. [23] F.R. Volkmar, W.T. Greenough, Rearing complexity affects branching of dendrites in the visual cortex of the rat, Science 176 (1972) 1445–1447. [24] C.S. Wallace, G.S. Withers, I.J. Weiler, J.M. George, D.F. Clayton, W.T. Greenough, Correspondence between sites of NGFI-A induction and sites of morphological plasticity following exposure to environmental complexity, Mol. Brain Res. 32 (1995) 211–220. [25] D. Young, P.A. Lawlor, P. Leone, M. Dragunow, M.J. During, Environmental enrichment inhibits spontaneous apoptosis, prevents seizures and is neuroprotective, Nature Med. 5 (1999) 448–453.
Research report
Upregulation of the immediate early gene arc in the brains of rats exposed to environmental enrichment: implications for molecular plasticity Raphael Pinaud a , Marsha R. Penner b , Harold A. Robertson b , R. William Currie a , * a
Department of Anatomy and Neurobiology, Laboratory of Molecular Neurobiology, Dalhousie University, Halifax, Nova Scotia, Canada B3 H 4 H7 b Department of Pharmacology, Laboratory of Molecular Neurobiology, Dalhousie University, Halifax, Nova Scotia, Canada B3 H 4 H7 Accepted 3 April 2001
Abstract Exposure to an enriched environment, a procedure that induces plasticity in the cerebral cortex, is associated with pronounced morphological changes, including higher density of dendritic spines, enlargement of synaptic boutons, and other putative correlates of altered neurotransmission. Recently, it has been demonstrated that animals reared in an enriched environment setting for 3 weeks have less neuronal damage as a result of seizures and have decreased rates of spontaneous apoptosis. Even though clear morphological modifications are observed in the cerebral cortex of animals exposed to heightened environmental complexity, the molecular mechanisms that underlie such modifications are yet to be described. In the present work, we investigated the expression of the immediate early gene arc in the cortex of animals exposed to an enriched environment. Animals were exposed daily, for 1 h, to an enriched environment, for a total period of 3 weeks. Brains were processed for in-situ hybridization against arc mRNA. We found a marked upregulation of arc mRNA in the cerebral cortex of animals exposed to the enriched environment, when compared to undisturbed controls, an effect that was most pronounced in cortical layers III and V. Animals in an additional control group that were handled for 5 min daily, displayed intermediate levels of arc mRNA. Furthermore, arc expression was upregulated in the CA1, CA2 and CA3 hippocampal subfields and in the striatum, but to a lesser extent in the dentate gyrus of animals exposed to an enriched environment, as compared to the two control groups. Our results support the association between the upregulation of the immediate early gene arc and plasticity-associated anatomical changes in the cerebral cortex of the adult mammal. 2001 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Neural plasticity Keywords: Arc; Enriched environment; Plasticity; Transcription factor; NGFI-A; Dendrite
1. Introduction In the early 1970s, several research groups demonstrated that exposure of animals to an enriched environment produced marked morphological changes in the sensory cortices of these animals [7,19,23]. For example, it has been demonstrated that exposure of adult rats to increased
*Corresponding author. Tel.: 11-902-494-3343; fax: 11-902-4941212. E-mail address: [email protected] (R.W. Currie).
environmental complexity increases the number of dendritic branches in the visual and somatosensory cortices [19,23]. In addition, other groups have demonstrated morphological modifications in the brains of animals exposed to an enriched environment. These changes included an increase in the number of dendritic spines, enlargement of synapses and an increase in the neuron to glia ratio [7,19]. Behaviorally-induced cellular plasticity reflects the reorganization and re-weighting of cerebral circuits and has been hypothesized to reflect the physical changes that enable learning and memory. More recently, it has been documented that levels of neurotrophic factors, such as the brain-derived neurotrophic factor (BDNF), are
0169-328X / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 01 )00121-8
R. Pinaud et al. / Molecular Brain Research 91 (2001) 50 – 56
increased in the central nervous system (CNS) in response to exposure to a complex environment [5,11,17,22]. Consequentially, exposure to an enriched environment enhances performance on behavioral paradigms of learning, memory, and visual acuity, suggesting that circuits are altered in order to optimize multiple levels of information processing and storage of environmental information [6,15,18]. Exposure to an enriched environment also confers neuroprotection against seizure-related neuronal damage and inhibits spontaneous apoptosis, which may provide mechanisms for longer-term neuroprotection [25]. Several aspects of the mechanisms that guide morphological restructuring, synaptogenesis and synaptic weighting have been characterized. The expression of immediate early genes (IEGs), the focus of the present work, has been postulated as the molecular mechanisms that control plastic changes in the CNS. In 1995, Wallace and collaborators provided strong evidence that the zincfinger IEG nerve growth factor induced gene-A (NGFI-A) is upregulated in the brains of rats exposed to an enriched environment. Expression of this IEG was lower in animals that were simply manipulated or left undisturbed [24]. NGFI-A is a transcriptional regulator that is known to regulate the expression of both synapsin I and II genes, that in turn are involved in the docking of synaptic vesicles, neurite outgrowth and synapse maturation [16,21]. These findings strongly suggest that NGFI-A expression may reflect one of the molecular pathways that mediate neuronal plasticity in the early stages of synaptic reorganization. In the present paper, we characterized the expression of a second candidate-plasticity gene, the activity-regulated cytoskeletal protein (arc), in the brains of animals exposed to the enriched environment setting. Arc is an IEG and growth factor that is rapidly and transiently induced after neural activity [8,13,20]. It has been previously demonstrated that arc expression is activity-regulated and its translation takes place in polyribosomal complexes located in the post-synaptic density [20]. One implication of this finding is that the protein encoded by this IEG might have a localized role in dendritic configuration (i.e., spine shape, dendritic orientation, effective synapse size or efficacy [2,4,9,10,14]. The position of this site of translation is intriguing in light of several hypotheses that suggest that learning induced plasticity results in physical changes in circuitry and that such changes can be demonstrated where neurons are most closely physically associated and fidelity of neurotransmission can be greatest [4,9,10]. Guzowski and collaborators have recently provided additional support for arc in mediating plasticity, as blockade of arc expression with an antisense oligonucleotide impaired the consolidation of long-term memory and the maintenance of long-term potentiation (LTP) in rats [8]. Here we determine the effects of a procedure that induces plasticity (exposure to an enriched environment) in
51
the cerebral cortex, on the expression of arc, a gene whose expression may be related to plasticity in dendrites.
2. Material and methods
2.1. Animals and experimental groups We used a total of 18 adult male Sprague Dawley rats (Charles River, Canada), weighing between 200 and 225 g. All protocols utilized were in accordance with institutional regulations and the Guide to the Care and Use of Experimental Animals from the Canadian Council on Animal Care. The protocol for handling the animals within the enriched environment experimental paradigm is based on previous work by Wallace and colleagues [24]. All rats were housed in a room under a 12 / 12 h light–dark cycle. Rats were divided into 3 experimental groups: (1) Animals exposed to an enriched environment (EE) for 1 h per day (n56). Animals belonging to this group were housed in a standard laboratory home cage with a larger variety of toys and objects. Each afternoon, all EE animals were transferred together to a ‘play area’ of 3 m31.5 m31 m, where they remained for 1 h. This larger enriched environment also contained a variety of toys, objects, short and long plastic tubes, plastic houses and a pan of water. In addition, cereal flakes were spread throughout the play area every 2 days. After this period, rats were returned to their home cages (3 animals per cage) (Fig. 1). (2) Handledonly animals (HO) (n56) were manipulated for a period of 5 min and returned to their home cages. This group was included in the present experiment to control for stress during cage transfer manipulation that was experienced by EE animals, as well as a possible ‘expectation-related’ gene expression. The manipulation procedure consisted of briefly, but repeatedly, lifting these animals. At the end of the handling period, animals were returned to their standard home cages (Fig. 1). (3) Undisturbed controls (UD) (n56) were left untouched in their home cages throughout the experiment (Fig. 1). The procedures described above were conducted for a total period of 3 weeks. To control for possible circadian influences on gene expression, exposure to the enriched environment and handling protocols were conducted between 3 and 4 pm. Behavior was fully monitored for all experimental groups.
2.2. Perfusion and histology In the last day of the experiment, all animals were anesthetized with sodium pentobarbital (70 mg / kg, i.p.) and perfused transcardially with a solution of 0.1 M phosphate buffered saline (PBS). Brains were rapidly dissected out from skulls, cerebellums were removed and brains were placed inside sterile petri-dishes and fast-
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Fig. 1. Outline of experimental protocol. EE animals were exposed daily, for a total of 3 weeks, to 1 h inside the enriched play area. HO animals were manipulated for 5 min, while UD controls were left untouched for the total period of the experiment. In the last experimental day, EE animals were exposed to the play area for 1 h and then immediately killed. HO animals were handled for 5 min and then killed with minimal time interval.
frozen in a 2708 freezer. Once frozen, brains were transferred to the cryostat and 20 mm sections were made under RNAse-free conditions. From the 6 animals per group, 4 brains were sectioned in the coronal plane, whereas 2 brains were sectioned parasagittally. Sections were directly mounted onto Fisherbrand Superfrost slides and returned to the 2708C freezer.
2.3. In-situ hybridization Slides were removed from the freezer, briefly fixed in 4% paraformaldehyde solution for 5 min and washed in 150 mM sodium chloride / 15 mM sodium citrate (13 SSC). An oligonucleotide probe, specific for the arc mRNA was commercially synthesized (Genosys, Canada) with the following sequence: 59-ATACAGTGTCTGGTACAGGTCCCGCTTACG-39. All sections were subsequently incubated with the arc cDNA oligonucleotide probe, that was 39 labeled with [ 33 P]dATP. Our arc probe was diluted in hybridization buffer (5310 6 cpm / ml), consisting of 50% deionized formamide, 50% 23hybridization buffer. The following day, sections were washed, at 558C, 4315 min in 23SSC, 4315 min in 13SSC, 4315 min in 0.53SSC, 2320 min in 0.253SSC at room temperature and dipped in MilliQ water. Slides were dried overnight at room temperature, a
procedure that was followed by the exposure to Kodak BioMax MR film (Interscience, Markham, ON) for 1 to 3 days.
2.4. Imaging Autoradiograms were scanned with a desktop scanner (Duoscan T1200, Agfa Inc.) and images were digitalized into an IBM-PC computer. Adobe Photoshop software (Adobe, Pantone Inc.) was used to assemble plates.
2.5. Densitometric and statistical analysis Densitometric analysis was performed using NIH Image software. Optical density (OD) was measured across cortical layers for the four levels presented in Fig. 2 and for the whole extent of the hippocampal formation in Fig. 3. Background values obtained from white matter were subtracted from values collected for cortical regions and hippocampi. Data was collected from 4 animals per experimental group, in which OD was averaged across hemispheres in each animal. OD is represented in a range from 0 (white) to 1 (black). Significant treatment effects were analyzed using one-way analysis of variance (ANOVA). The Tukey HSD-test was used for post-hoc comparisons with the criterion level set at P,0.05.
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Fig. 2. Autoradiograms depicting arc mRNA hybridization at comparable stereotaxic levels in brains of EE (top panel), HO (middle panel) and UD (bottom panel) animals. Sections are presented from rostral to caudal, left to right. Note the marked upregulation in the striatum, sensory cortices and hippocampus of sections of the EE group. Using bregma as reference, approximate stereotaxic coordinates for the four levels presented in this figure are: Level 1 (AP 2.20), Level 2 (AP 0.70), Level 3 (AP24.16) and Level 4 (AP26.30). Scale bar5500 mm.
3. Results In-situ hybridization was used to localize the expression patterns of the immediate early gene arc in the brain. Both coronal and parasagittal sections from all experimental groups were exposed to the same histological conditions in order to minimize reaction variables. In EE animals, arc upregulation was found in several cortical regions, with the most pronounced upregulation observed in the sensory cortices, striatum and hippocampal formation (see Tables 1 and 2). In HO and UD controls, arc mRNA was found at apparent basal levels. Dense arc labeling was found in both the primary and
secondary visual areas of EE animals (Fig. 2). For the primary visual cortex of EE animals, regions that correspond to binocular and monocular vision displayed high levels of arc mRNA expression that were approximately equal in intensity. In these regions, labeling was most pronounced in cortical layers III and V, whereas basal levels of arc expression were found in all other layers. In contrast to EE animals, HO and UD controls displayed basal levels of arc expression in these cortical regions. No clear layering was detectable for either control group (Fig. 2). In the somatosensory cortex, the highest arc expression was found in EE animals, followed by HO and UD
Fig. 3. Autoradiograms depicting the pattern of arc mRNA hybridization in the hippocampus and dentate gyrus of all experimental groups. Arc is expressed in all hippocampal subfields in EE animals, while UD animals displayed minimal arc mRNA levels. Arc signal in HO animals was detected at intermediate levels. In addition, arc expression was detected in the dentate gyrus of both EE and HO animal, while in UD controls mRNA signal was almost undetectable. Scale bar5200 mm.
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Table 1 Effects of environmental enrichment on arc mRNA expression in the cortex a Groups
EE HO UD
Level I
Level II
Level III
Level IV
Mean (6S.E.M.)
Treatment F(2, 37)
Mean (6S.E.M.)
Treatment F(2, 43)
Mean (6S.E.M.)
Treatment F(2, 49)
Mean (6S.E.M.)
Treatment F(2, 41)
0.162060.0009a 0.002060.0002b 0.001560.0007b
171.14*
0.184260.0009a 0.002860.0003b 0.000960.0002b
186.22*
0.184260.0005a 0.004660.0018b 0.003860.0005b
71.01*
0.167560.0008a 0.002660.0004b 0.003660.0004b
174.81*
a
Data are expressed as mean optical density6S.E.M. (n54 per group). *All F values significant P,0.001. a,b5groups with different letters differ significantly (Tukey HSD test P,0.05).
controls (Fig. 2). In EE animals, heightened arc labeling was confined to cortical layers III and V, while no apparent stratification was detected for either control group. The most pronounced difference across experimental groups was observed in the hippocampus. Animals exposed to the enriched environment displayed a significant upregulation of arc mRNA in all subfields of the hippocampal formation. Arc expression was almost undetectable in the hippocampus of UD controls, while HO controls displayed intermediate levels of arc mRNA (Figs. 2 and 3; Table 2). However, arc was expressed in a low level in the dentate gyrus of both control groups, while EE animals had a moderate upregulate of arc expression in this region. (Fig. 3; Table 2). A pronounced upregulation of arc mRNA was also found in the striatum. EE animals had a significant upregulation of arc expression in this region, as compared to either control group. In the remaining cortical regions, arc expression also followed the same layering pattern. While most cortical neurons displayed arc mRNA signal to some degree, the strongest labeling was detected in the EE group for cortical layers III and V (Fig. 4). In addition, HO and UD animals displayed basal levels of arc expression, with the same apparent intensity across all cortical layers.
4. Discussion The major finding of the present work is that the immediate early gene arc is significantly upregulated, in
Table 2 Effects of environmental enrichment on arc mRNA expression in the hippocampus a Groups
Mean (6S.E.M.)
Treatment F(2, 15)
EE HO UD
0.116760.0009a 0.002160.0004b 0.000660.0012b
95.59*
a
Data are expressed as mean optical density6S.E.M. (n54 per group). *All F values significant P,0.001. a, b5groups with different letters differ significantly (Tukey HSD test P,0.05).
several sensory systems, in response to environmental complexity. In addition, we have demonstrated that this upregulation is more pronounced in the hippocampus. Constitutive arc expression was found in the brains of two control groups, therefore suggesting to us that the upregulation of this gene in the EE group may reflect processing of greater complexity in the sensory environment, rather than altered rates of neuronal activation, as an explanation that has been forwarded to account for enhanced NGFI-A and c-fos expression [3]. Even though arc expression has been demonstrated to be activity dependent, neuronal depolarization alone is not able to upregulate this gene. For example, blockade of the N-methyl-D-aspartate (NMDA) type of glutamatergic receptors suppressed arc expression [12]. In the present experiment, rats in the HO group were stimulated by visual, olfactory and somatosensory cues that should have resulted in an enhanced expression of the arc gene, according to our hypothesis described above. However, these animals failed to demonstrate such an increase in arc expression and elevated mRNA levels were restricted to the EE animals. Our findings suggest that another process must be involved in regulating arc expression, in conjunction with increased activity, in a given sensory pathway. Because arc mRNA was increased as a result to exposure to an enriched environment, we postulate that arc is invoked as a means of increasing neuronal plasticity and may be related to information or sensory overload. It has been previously demonstrated that arc is rapidly induced, a characteristic feature of immediate early genes, and that the mRNA synthesized from this gene is also rapidly transported to the dendrites [13,20]. In addition, it has been postulated that translation of the protein encoded by the arc gene takes place in polyribosomal complexes located at postsynaptic sites on dendrites [20]. Given that plasticity in the cerebral cortex involves, in some cases, the physical rearrangement or synthesis of cortical circuits, the Arc protein would be, at least, spatially well located to mediate many aspects of reorganization. More specifically, Arc is a growth factor that could regulate neurite sprouting in response to complex levels of sensory information [13]. In 1998, Steward and colleagues demonstrated that stimulation of specific sites of the perforant path increased arc mRNA accumulation in corresponding sites of the
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Fig. 4. Pictures from an autoradiogram exposed to in-situ hybridization signal depicting the laminar profile of arc expression in a coronal (A) and parasagittal section (B) of an EE animal. The superficial band in either profile corresponds to cortical layer III and the deep band to cortical layer V. Threshold images from the coronal (C) and parasagittal (D) sections depict clearly this laminar profile. Scale bar5500 mm.
dendritic arborization of dentate granule cells. Interestingly, the greatest levels of arc mRNA accumulation were observed in the locations where perforant fibers innervated dendritic branches of dentate granule cells, suggesting that arc expression was directly correlated to the load of information delivered to a given cellular site [20]. Moreover, the stimulation paradigm used in their study has the attributes that has been previously used to induce longterm potentiation in the dentate gyrus [1], a finding that also supports the argument that arc expression may be involved as one of the first genetic mediators of LTPdependent plasticity in the CNS. Unlike the immunoreactive profile that results with markers for c-fos or NGFI-A, which reveal cell nuclei, arc labeling is dendritic. This feature may become important for identifying the locations of plasticity induced changes. For example, arc labeling would enable the identification of reorganization in cells where the sites of plasticity are remote from their cell bodies, such as the reorganization of deep thalamic and brainstem nuclei following deafferentation, in addition to changes demonstrated in the cerebral cortex. Identifying the contributions of these subcortical CNS levels to the reorganizational process has been
limited to functional studies based on electrophysiology, activity markers such as GAD and cytochrome oxidase and tracer studies in control versus reorganized systems. The identification of IEGs that mediate plastic changes provide great opportunities for pinpointing the site and extent of reorganization throughout the ascending sensory pathway, thus, increasing our understanding of the overall process and possibly indicating locations where therapeutic interventions would provide the maximal benefit.
Acknowledgements We would like to thank Dr. Liisa Tremere for carefully reading this manuscript and for useful comments and Ms. Kay Murphy for excellent technical support. Dr. E. Denovan-Wright helped with the design of the arc oligonucleotide probe. Felipe Hess helped with the initial parts of this work. This work was supported by the Canadian Institutes of Health Research, the Canadian Stroke Network, the Heart and Stroke Foundation of New Brunswick, and by a Killam Scholarship to R.P.
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