Transgenic mice overexpressing the full-length neurotrophin receptor trkB exhibit increased activation of the trkB–PLCγ pathway, reduced anxiety, and facilitated learning
Introduction
Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, has emerged as a potent molecular mediator of synaptic plasticity and behavior (reviewed by Poo, 2001, Segal, 2003, Tyler et al., 2002). BDNF together with its cognate receptor trkB is widely and similarly distributed in the brain and is especially rich in the hippocampus, an important area for memory and plasticity Fryer et al., 1996, Hofer et al., 1990, Kokaia et al., 1993, Maisonpierre et al., 1990. Neurotrophin binding to trk is followed by receptor dimerization and kinase activation. Subsequently, the phosphorylated cytoplasmic tyrosines provide docking sites for adaptor proteins that initiate intracellular signaling events including Ras–Erk, PI3K–Akt, and PLCγ-1 pathways (reviews by Kaplan and Miller, 2000, Patapoutian and Reichardt, 2001). PLCγ-1 activation leads to release of Ca2+ stores, followed by activation of enzymes controlled by cytoplasmic Ca2+ such as CaM kinases and protein kinase C isoforms. Shc recruitment results in activation of the Ras–Erk pathway regulating neuronal differentiation and survival. The survival promoting PI3K–Akt pathway is activated either in a Ras-dependent manner or by Shc–Grb-2–Gab-1 adaptor proteins Huang and Reichardt, 2003, Patapoutian and Reichardt, 2001.
Previous studies have provided evidence to support the involvement of BDNF in activity-induced long-term potentiation (LTP) (recently reviewed by Schinder and Poo, 2000, Tyler et al., 2002). In normal hippocampal slices, LTP impairment occurs if endogenous BDNF is sequestered Chen et al., 1999, Figurov et al., 1996, Kang et al., 1997 and intrahippocampal application of BDNF induces late-phase LTP Messaoudi et al., 2002, Ying et al., 2002. Furthermore, BDNF knockout mice show LTP impairment Bartoletti et al., 2002, Korte et al., 1995, Patterson et al., 1996, Pozzo-Miller et al., 1999; however, the effect is rescued by hippocampal re-expression of BDNF Korte et al., 1996, Patterson et al., 1996. Mice lacking the trkB receptor gene show reduced CA1-LTP Minichiello et al., 1999, Xu et al., 2000a. Moreover, mutations in trkB docking sites suggest that hippocampal plasticity mediated by trkB happens via PLCγ pathway He et al., 2002, Minichiello et al., 1998, Minichiello et al., 2002, whereas the Shc-binding site is not required for hippocampal LTP (Korte et al., 2000).
Several lines of evidence suggest a role for the BDNF–trkB system in memory acquisition and consolidation. First, spatial learning is shown to induce BDNF and trkB expression in activated brain areas Falkenberg et al., 1992, Gomez-Pinilla et al., 2001, Kesslak et al., 1998, Mizuno et al., 2000. Second, BDNF withdrawal by antisense oligonucleotides or anti-BDNF antibodies severely impairs spatial learning Ma et al., 1998, Mu et al., 1999. Third, mutant mice with reduced BDNF levels show impairments in learning and memory (Linnarsson et al., 1997, but see Montkowski and Holsboer, 1997). Also, alterations in the trkB receptor function lead to learning deficits. Forebrain-specific trkB null mice show a dramatic deficit in complex learning paradigms (Minichiello et al., 1999) and behavioral flexibility (Vyssotski et al., 2002). Furthermore, mice overexpressing the dominant-negative form of trkB receptor, trkB.T1, display a mild learning deficit (Saarelainen et al., 2000a). Surprisingly, artificially increased BDNF levels have not revealed improvements of the cognitive functions. On the contrary, mice overexpressing BDNF develop a passive avoidance deficit that is dependent on BDNF overexpression levels (Croll et al., 1999).
In this study, we have used an in vivo paradigm of overexpressing the full-length trkB receptor in adult neuronal cells. We have characterized the overall pattern of trkB.TK+ overexpression and investigated the consequences on receptor signaling. We subjected the mice overexpressing trkB.TK+ to extensive behavioral analysis and provide data on the effects of enhanced BDNF signaling on learning, memory, and LTP.
Section snippets
General characterization of transgenic trkB.TK+ mice
We have produced a transgenic mouse line overexpressing the full-length neurotrophin receptor trkB (trkB.TK+) in postnatal neurons under the Thy-1 promoter control. Heterozygous mice overexpressing trkB.TK+ were viable, fertile, and possessed normal life span. They were indistinguishable from wild-type littermates by general appearance, weight, and development. Histological analysis by Nissl staining did not reveal any major changes in lamination and morphology of the brain (unpublished
Increased trkB receptor activation in transgenic trkB.TK+ mice
To assess the expression and functions of transgenic trkB.TK+ protein in these mice, biochemical analysis was performed on cortical and hippocampal tissue extracts. Western blot results showed dramatically elevated levels of the full-length trkB receptor protein (p145TrkB) in transgenic mice (Fig. 2A, bottom). Analysis using the trkBout antibody showed a 17-fold increase in the full-length trkB protein in cortex (P < 0.001) and a 18-fold increase in hippocampus (P < 0.001) of transgenic mice
Discussion
In this study, we have examined the role of the full-length neurotrophin receptor trkB in vivo by creating a transgenic mouse overexpressing the trkB.TK+ receptor in postnatal CNS neurons. The existing evidence on the importance of the BDNF–trkB system on plasticity and behavior was supported by the following findings. First, the overexpression of trkB.TK+ in the adult CNS led to a continuous dramatic increase in receptor activity without any major defects in the general well being of the mice.
Animal preparation
The production of a trkB.TK+ overexpressing mouse line and all animal experiments were done according the guidelines of The Society for Neuroscience and were approved by the Experimental Animal Ethics Committee of the National Laboratory Animal Center, University of Kuopio, Finland. Animals were housed in metal cages under standard animal room conditions (12:12 h light cycle, ambient temperature of 23°C, and free access to food and water).
The expression cassette used for the generation of
Acknowledgements
This work was supported by grants from The Academy of Finland, TEKES and Sigrid Juselius Foundation for E.C, and Finnish Cultural Foundation for E.K. We thank Dr. D. Kaplan (University of Toronto, Canada) for the trkB antibody and Dr. H. Nawa (Niigata University, Japan) for the BDNF antibodies, Drs. G. Wong, A. Haapasalo, and MSc S. Lähteinen for their comments on the manuscript, and A-M Haapaniemi for assistance. E.K is a graduate student at the A.I. Virtanen Institute Graduate School,
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