Neuroscience Letters, 138 (1992) 153-156 153 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

NSL 08547

Increased expression of brain-derived neurotrophic factor mRNA in rat hippocampus is associated with improved spatial memory and enriched

environment

Torkel Fa lkenberg a, Abdu l K. M o h a m m e d b, Bengt Henr iksson b, H~.kan Persson c, Bengt Winblad b and Nils Lindefors a

"Department of Pharmacology, Karolinska Institutet, Stoekholm (Sweden), hDepartment of Geriatric Medicine, Karolinska Institutet, Huddinge University Hospital, Huddinge (Sweden) andCDepartment of Medical Chemistry, Laboratory of Molecular Neurobiology, Karolinska lnstitutet,

Stockholm (Sweden)

(Received 8 November 1991; Revised version received 20 January 1992; Accepted 22 January 1992)

Key words: Enriched environment; Spatial memory; Hippocampus; Brain-derived neurotrophic factor; In situ hybridization

Enriched environment has been shown to enhance learning and memory and to induce morphological changes in the hippocampus. We report that rats housed in an enriched environment showed improved performance in the Morris water maze and decreased spontaneous motor activity. Exposure to behavioural tests increased expression of the mRNA that encodes brain-derived neurotrophie factor in the hippocampus. This was not seen when rats subjected to impoverished housing were tested suggesting that environmental history of the animal is of importance to induce expression of brain-derived neurotrophic factor in the hippocampus that may promote neuronal changes related to learning and memory.

Housing rats in an enriched environment (EE) in con- trast to an impoverished has been shown to facilitate ac- quisition in the Morris water maze [13, 20, 21]. Envi- ronmental enrichment also induces morphological changes in the hippocampus, such as an increased number of synapses per neuron and dendritic branching [9, 10]. The biochemical changes underlying morphologi- cal alterations related to plasticity of function in the hip- pocampus are poorly understood, though consolidation of long-term memory is assumed to involve changes in gene expression [4]. Here we have examined whether en- vironmental influence and cognitive functioning are im- portant factors in determining levels of brain-derived neurotrophic factor (BDNF) mRNA in the hip- pocampus using in situ hybridization. The cognitive functioning was measured by the activity in a novel en- vironment [13] and by the escape response in the Morris water maze [14] after previous prolonged exposure to either enriched or impoverished environment.

Adult Sprague-Dawley rats (n=24; 50 days old; mean b.wt. 220 g; Alab, Sollentuna, Sweden) were housed en- riched or impoverished for 34 days. Twelve rats were

Correspondence: N. Lindefors, Department of Pharmacology, Karo- linska Institutet, Box 60400, S-104 01 Stockholm, Sweden.

housed (6 per cage) in large wire mesh cages (100×60×35 cm) with inbuilt ladders, shelves and tunnels. Various toys were placed in the cages and changed daily (enriched environment). The remaining twelve rats were housed (in the same room) individually in plexiglass cages measur- ing 22.5× 16.5× 13.5 cm (impoverished environment).

After 30 days of housing, 6 rats from each environ- ment were tested for 1 day in open-field boxes (70×70x45 cm) where spontaneous motor activity and rearing was registered followed by testing in the Morris water maze [14] twelve times distributed over 3 days in which rats were trained to learn the spatial location of a submerged transparent platform in a circular pool (140 cm diameter) of water (25_+ 1 °C). The swimming test was performed as described earlier [12]. Performance was monitored by a computer linked video camera registering escape response and the distance swum.

Four hours after the last test session all groups of animals were sacrificed by decapitation, their brains re- moved, frozen on dry ice and stored at -70°C. Coronal brain tissue sections including the dorsal hippocampus were hybridized to an oligonucleotide probe specific for rat BDNF mRNA as previously described [6, 11]. The hybridization specificity was assessed by showing a spe- cific inhibition of the labelling in presence of an excess

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(100x) of unlabelled BDNF probe, while an excess (100x) of an unlabelled NGF probe had no effect on the BDNF mRNA labelling. Probe specificity for the group difference obtained was investigated by hybridization with an oligonucleotide probe specific for rat neurofila- ment-light mRNA [15], however, no group difference was revealed.

Autoradiograms were analyzed with a Microcomputer Image Device (Imaging Research Inc., Canada) and optical density values were measured bilaterally over a total area of 0.072 mm 2 on three consecutive sections and converted to mRNA levels, in an interval with linear correlation, using external radioactivity standards ex- posed to the same film.

To examine possible differences in stress levels follow- ing the behavioural testing corticosterone levels were measured in two separate groups of animals. Seven en- riched and impoverished animals were decapitated 1 h following the last water maze trial. Plasma corticoster- one levels were measured using a commercial radioim- munoassay kit (ICN Biomedicals, Costa Mesa, CA, USA).

Behavioural data was analyzed with two-way ANOVA and repeated postcomparative tests at each mo- nitored time interval were performed using the Scheffe test. BDNF mRNA levels in each region were analyzed using two-way ANOVA and differences due to environ- ment as well as behavioural testing were examined using postcomparative Bonferroni test.

Housing in different environments resulted in different behavioural responses to the testing situation. Impov- erished animals were hyperactive for a longer duration due to significantly (P<0.05) higher locomotion counts during each measured 10-min interval from 10 to 50 min when placed in a novel activity cage (Fig. 1 a). A signifi-

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cant (F5.5o=10.25, P<0.0001) interaction effect was ob- tained for rearing scores, whereby enriched animals showed significantly (P<0.05) higher rearing scores dur- ing the first 10 rain interval compared to impoverished animals, indicating increased exploration by the enriched animals (Fig. lb). The water maze also discriminated the two groups and a significant (FL10=5.75, P<0.05) en- vironment vs trials interaction was obtained whereby the animals housed in EE performed significantly (P<0.05) better on trials 4 and 5 (Fig. l c).

As previously reported [6, 8, 11, 17], BDNF mRNA expressing cells were seen in the granule layer of the dentate gyrus and throughout the pyramidal layer of the hippocampus with higher levels in the CA3 region than in CA 1. Housing animals for 34 days in the two different environments did not influence the basal levels of BDNF mRNA in different regions of the hippocampus (Table I and Fig. 2). However, the exposure to repeated behav- ioural tests revealed differences in activation of BDNF mRNA expression in the two differentially housed animals. Two-way ANOVA indicated a significant (F3.20=4.48, P<0.05) environment vs behavioural testing interaction in the CA 1 region whereby the spatial learn- ing test was followed by a significant (P<0.05; Bon- ferroni P<0.0125) 48% increase in the level of BDNF mRNA in the pyramidal cells of CA1 (not in the CA3 or dentate gyrus) in the EE animals as compared to the non-tested animals (Table I, Figs. 2 and 3).

The analysis of corticosterone plasma levels did not reveal any difference between enriched and impoverished animals tested (299+97 nM and 325+82 nM, respec- tively; mean+S.E.M.), indicating that the distinction in BDNF mRNA expression cannot be attributed to dif- ferences in the level of stress between the two groups.

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Fig. I. Accumulated locomotion (a) and rearing counts (b) during 10-min intervals, for 1 h, of rats housed in enriched ( ) and impoverished (O--o) environments. Note prolonged increase in locomotion by the impoverished animals and increased initial rearing by the enriched animals. Acquisition of spatial learning in the Morris water maze (c) by rats housed in different environments. Performance is represented as mean latency to find the platform of 12 trials given in groups of four on three consecutive test days. The latency value of 65 s was given for an unsuccessful

trial. Note the significantly improved spatial memory revealed by trials 4 and 5. Values are mean_+S.E.M.

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Fig. 2. Autoradiograms of in situ hybridizations with a [~-35S]dATP-labelled BDNF oligonucleotide probe to coronal sections through the dorsal hippocampus of behaviourally tested or non-tested rats following different housing. The increased BDNF mRNA in the pyramidal cells of CA1 in (a) enriched tested animal can be discerned by comparison with the same region in the (b) enriched non-tested animal. No change in BDNF mRNA levels are seen in (c) isolated tested compared with (d) isolated non-tested animals. Arrows delineate parts of each area where hybridization signals

were measured.

The involvement of the hippocampus in higher cogni- tive functions such as learning and memory is well estab- lished. The neurotrophic factors nerve growth factor (NGF) and BDNF, members of the same neurotrophic factor family [6], are expressed at high levels in the hip- pocampus [6, 8, 17]. NGF is known to exert a target- derived trophic support for basal forebrain cholinergic neurons [5] associated with cognitive functions, and BDNF supports the survival of these neurons in cell cul- tures [1]. Expression of NGF and BDNF mRNAs in the brain appears to be regulated by neuronal activity pos- sibly via release of glutamate followed by activation of non-NMDA receptors [2, 7, 11, 22] and by cholinergic

TABLE I

RELATIVE LEVELS OF BDNF mRNA IN REGIONS OF THE HIPPOCAMPUS OF BEHAVIOURALLY TESTED AND NON- TESTED RATS FOLLOWING DIFFERENT HOUSING

Values are mean _+ S.E.M.; n -- 6; arbitrary scale derived from an ex- ternal radioactivity standard.

Housing Group Pyramidal Pyramidal Granule cells cells of CA1 Cells of CA3 of the denate

gyrus

Enriched Tested 514+40" 1140+95 1083+_70 Enriched Non-tested 347_+15 908+95 778+-91 Impoverished Tested 423_+23 1 0 3 6 + - 9 7 1033_+121 Impoverished Non-tested 373_+28 863_+68 716_+107

*Significant difference (P<0.05) compared to enriched, non-tested animals.

(firnbria fornix afferents) activation of muscarinic recep- tors [11]. Induction of long-term potentiation, probably related to memory formation, changes synaptic density in the CA1 region of the hippocampus [3]. The increased expression of BDNF mRNA in EE animals tested for spatial memory shown here raise the possibility that the trophic support for morphological changes known to be

Fig. 3. High magnification bright-field photomicrographs of in situ hybridizations for BDNF mRNA in the pyramidal layer of CA 1 in the dorsal hippocampus in (a) enriched tested and (b) enriched non-tested

animals, respectively.

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induced by behavioural testing [16] is, at least in part, due to an autocrine or paracrine BDNF activity in the CA1 region.

The finding that behavioural testing is followed by in- creased BDNF mRNA levels in the CA1 region also sug- gests a possible role for hippocampal BDNF in this region in more subtle cognitive processes. In tests of ex- ploratory behaviour enriched animals displayed greater diversity of behaviours in exploring novel objects com- pared to impoverished animals, possibly reflecting dif- ferences in the way animals gather information and solve problems [18, 19]. Our finding of increased BDNF mRNA expression reinforce the difference between en- riched and impoverished animals at the molecular level.

The present data indicate that the improved formation of spatial memory following exposure to an enriched en- vironment is associated with an induction of BDNF mRNA expression in the pyramidal cells of the CA1 sub- field in the hippocampus. Thus the environmental his- tory of an individual appears to be an important factor in superior spatial performance which is coupled to an in- duction of BDNF in the hippocampus. Our results also have implications for future studies on the use of neuro- trophic factors during aging and in senile dementia such as of the Alzheimer type since they suggest that subtle but important differences in the levels of neurotrophic factors in the hippocampus may not be present during basal conditions but may be apparent and important when an individual is cognitively challenged.

This work was supported by grants from The Swedish Medical Research Council (8653, 9705), The Swedish Natural Science Research Council, Ake Wibergs Stiftelse and Greta and Johan Kocks foundation. We thank Dr. Stefan Bren6 for helpful suggestions on the in situ hy- bridization technique.

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