Enrico Tongiorgi, Massimo Righi, and Antonino Cattaneo International School for Advanced Studies (SISSA), Neuroscience Program, 34014 Trieste, Italy.
The mechanisms underlying the subcellular localization of neurotrophins and their receptors are poorly understood. We show that in cultured hippocampal neurons, the mRNAs for BDNF and TrkB have a somatodendritic localization, and we quantify the extent of their dendritic mRNA localization. In the dendrites the labeling covers on average the proximal 30% of the total dendritic length. On high potassium depolarization, the labeling of BDNF and TrkB mRNA extends on average to 68% of the dendritic length. This increase does not depend on new RNA synthesis, is inhibited by the Na1 channel blocker tetrodotoxin, and involves the activation of glutamate receptors. Extracellular Ca21, partly flowing through L-type Ca21 channels, is absolutely required for this process to occur. At the protein level, a brief stimulation of hippocampal neurons with 10 mM KCl leads to a marked increase of BDNF and TrkB immunofluorescence density in the distal portion of dendrites, which also occurs, even if at lower levels, when transport is inhibited by nocodazole. The protein synthesis inhibitor cycloheximide abolishes this increase. The activity-dependent modulation of mRNA targeting and protein accumulation in the dendrites may provide a mechanism for achieving a selective local regulation of the activity of neurotrophins and their receptors, close to their sites of action. The neurotrophin BDNF (Leibrock et al., 1989; Barde, 1990) has been involved in modulating synaptic plasticity (Thoenen, 1995; Bonhoeffer, 1996). In particular, BDNF can increase synaptic transmission (Lohof et al., 1993; Knipper et al., 1994a,b; Leßmann et al., 1994; Kang and Schumann, 1995; Levine et al., 1995; Carmignoto et al., 1997) and has been implicated in hippocampal LTP (Korte et al., 1995, 1996; Patterson et al., 1996). Furthermore, BDNF enhances synaptic transmission in the rat hippocampus requiring local dendritic synthesis of proteins whose identity was not determined (Kang and Schumann, 1996). BDNF and its receptor TrkB are stored in the dendrites (Wetmore et al., 1994; Dugich-Djordjevic et al., 1995; Cabelli et al., 1996; Cellerino et al., 1996; Goodman et al., 1996), but the mechanisms of targeting of these two proteins are largely unknown. The specific sorting of mRNAs is a possible mechanism to localize proteins to the dendrites (Steward, 1994, 1997; Johnston, 1995). An increasing number of dendritic mRNAs have been identified, including those encoding MAP2, ARC/arg 3.1, a- CaMKinase II a-subunit, IP3-receptor, NMDAR1 subunit, glycine receptor a-subunit, vasopressin, and dendrin (Garner et al., 1988; Burgin et al., 1990; Furuichi et al., 1993; Link et al., 1995; Lyford et al., 1995; Gazzaley et al., 1997; Herb et al., 1997; Prakash et al., 1997; Racca et al., 1997). Recent studies demonstrated the existence of a protein synthesis machinery in dendrites, including ribosomes, tRNA, translation factors (Steward and Levy, 1982; Tiedge and Brosius, 1996), endoplasmic reticulum, and Golgi-like apparatus (Torre and Steward, 1996; Spacek and Harris, 1997). Moreover, the local protein synthesis in fully developed dendrites (Torre and Steward, 1992) and in isolated growth cones (Crino and Eberwine, 1996) has been demonstrated unequivocally. TrkB and BDNF mRNAs have been amplified from dendritic growth cones (Crino and Eberwine, 1996); however, the localization of these two mRNAs in the dendrites of mature neurons is controversial. Most studies did not describe a BDNF mRNA dendritic localization (Ernfors et al., 1990, 1992; Isackson et al., 1991; Kokaia et al., 1993; Miranda et al., 1993; Ringstedt et al., 1993; Tinmusk et al., 1993; Castre´n et al., 1995; Lauterborn et al., 1996; Conner et al., 1997), whereas a few others were suggestive of a very proximal dendritic localization (Wetmore et al., 1990, 1994; Dugich-Djordjevich et al., 1992; Schmidt-Kastner et al., 1996a,b). Recently, the localization of TrkB mRNA in the dendritic compartment of retinal neurons at the end of their development was shown (Ugolini et al., 1995). However, other studies did not report a similar localization (Ringstedt et al., 1993; Schmidt-Kastner et al., 1996a,b). In view of the role of BDNF and TrkB in synaptic plasticity (Thoenen, 1995; Bonhoeffer, 1996), the clarification of this issue is crucial. This study demonstrates that BDNF and TrkB mRNAs are localized in the dendrites of hippocampal neurons in culture, that depolarization extends these mRNAs to the distal dendrites, and that a brief depolarization after blockade of dendritic trans-port increases the levels of BDNF and TrkB proteins in the distal dendritic compartment.
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