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Previous Article | Next Article
Volume 17, Number 24, Issue of December 15, 1997
Activity-Dependent Dendritic Targeting of BDNF and TrkB mRNAs in Hippocampal Neurons
Enrico Tongiorgi, Massimo Righi, and Antonino Cattaneo International School for Advanced Studies (SISSA), Neuroscience Program, 34014 Trieste, Italy
ABSTRACT
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 Na+ channel blocker tetrodotoxin, and involves the activation of glutamate receptors. Extracellular Ca2+, partly flowing through L-type Ca2+ 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.
Key words: neurotrophins; dendritic mRNA; BDNF; TrkB; synaptic plasticity; hippocampal neurons
INTRODUCTION
The neurotrophin BDNF (Leibrock et al., 1989
; Barde, 1990
BDNF and its receptor TrkB are stored in the dendrites (Wetmore et al., 1994
The specific sorting of mRNAs is a possible mechanism to localize proteins to the dendrites (Steward, 1994
-CaMKinase II
TrkB and BDNF mRNAs have been amplified from dendritic growth cones (Crino and Eberwine, 1996
In view of the role of BDNF and TrkB in synaptic plasticity (Thoenen, 1995
MATERIALS AND METHODS
Cell cultures. Primary cell cultures were made from rat hippocampal neurons according to Malgaroli and Tsien (1992)-D-arabinofuranoside from the second day in culture onward.
Electrophysiology and KCl stimulation of cultured hippocampal neurons. Whole-cell recordings were performed at room temperature (rt) (23-25°C) on large pyramidal cells with an EPC 7 patch-clamp amplifier. Patch pipettes were made from thin-wall glass (outside diameter 1.5 µm) with 6-8 M resistance and were filled with 110 mM potassium gluconate, 10 mM NaCl, 5 mM MgCl2, 0.6 mM EGTA, 2 mM Na2-ATP, 49 mM HEPES, pH 7.2. Extracellular oxygenated control solution contained 3.5 mM KCl, 132 mM NaCl, 1 mM MgCl2, 2 mM CaCl2, 20 mM D-glucose, 10 mM HEPES, pH 7.4. Cells were depolarized for 30 min at rt with oxygenated K-medium (10 mM KCl, 1.8 mM CaCl2·2H2O, 0.8 mM MgSO4·7H2O, 101 mM NaCl, 26 mM NaHCO3, 1 mM NaH2PO4·2H2O, 0.7% D-glucose, 15 mM HEPES, pH 7.4, or KK-medium (20 mM KCl, 1.8 mM CaCl2·2H2O, 0.8 mM MgSO4·7H2O, 110 mM NaCl, 26 mM NaHCO3, 1 mM NaH2PO4·2H2O, 0.7% D-glucose, 15 mM HEPES, pH 7.4.
For mRNA or protein localization experiments, cells were depolarized for the indicated times, at 37°C, with the K or the KK high potassium media described above. For pharmacological blockade experiments, cells were incubated in normal culture medium or in K- or KK-medium, supplemented with drugs, for the indicated times at 37°C. Drug concentrations were 1 mM kinurenic acid (Sigma), 1 µM nifedipine (Sigma), 0.5 µM tetrodotoxin (TTX) (Sigma), 5 µg/ml actinomycin-D (Sigma), 1 µM cycloheximide (Sigma), and 1 µg/ml nocodazole (Sigma). When cycloheximide or actinomycin-D were used, preincubation before depolarization was performed as described above for 30 min, whereas in the case of nocodazole, preincubation at 37°C was 6 hr long. Ca2+-free experiments were performed in Ca2+-free control medium containing 5 mM KCl, 1.8 mM MgCl2, 0.8 mM MgSO4·7H2O, 116 mM NaCl, 26 mM NaHCO3, 1 mM NaH2PO4·2H2O, 0.7% D-glucose, 15 mM HEPES, pH 7.4, or in Ca2+-free K-medium containing 10 mM KCl, 1.8 mM MgCl2, 0.8 mM MgSO4·7H2O, 101 mM NaCl, 26 mM NaHCO3, 1 mM NaH2PO4·2H2O, 0.7% D-glucose, 15 mM HEPES, pH 7.4, supplemented with 10 µM EGTA or BAPTA-AM.
Riboprobes and oligonucleotides. The 700-bp-long rat end of the oligonucleotides by means of a DIG-oligonucleotide 3
In situ hybridization on cultured hippocampal neurons. For in situ hybridization with riboprobes, cells were fixed for 10 min at room temperature in 4% paraformaldehyde in PBS, washed in PBS, and permeabilized in ethanol for 15 min at 20°C. After rehydration with decreasing ethanol concentrations in PBS at rt, cells were prehybridized at 55°C for 90 min in the hybridization mix containing 20 mM Tris/HCl, pH 7.5, 1 mM EDTA, 1× Denhardt's solution, 300 mM NaCl, 100 mM dithiothreitol, 0.5 mg/ml salmon sperm DNA, 0.5 mg/ml polyadenylic acid, and 50% formamide. In situ hybridization was performed overnight at 55°C in the hybridization mix to which 10% dextrane sulfate and the riboprobes (50-100 ng/ml) were added. High-stringency washes were performed in 0.1% SSC/0.1% Tween-20 at 60°C. Cells hybridized with digoxigenin-labeled riboprobes were incubated overnight at 4°C with anti-DIG Fab fragments coupled to alkaline phosphatase (Boehringer Mannheim), diluted 1:500 in 10% fetal calf serum in PBS + 0.1% Tween 20 (PBST). After a thorough wash in PBST, cells were reacted with 4-nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate in 100 mM Tris/HCl, pH 9.5, 50 mM MgCl2, 100 mM NaCl, and 1 mM Levamisol. Alkaline phosphatase development was performed for 16 hr at 4°C to obtain reproducible results and to avoid saturation of the reaction (Augood et al., 1991
Antibody staining of cultured hippocampal neurons. Double staining of cultured hippocampal neurons was performed with the same in situ hybridization procedure described above, followed by anti-MAP2 immunostaining. Cells were coincubated overnight at 4°C with the anti-MAP2 monoclonal antibodies (Boehringer Mannheim) diluted 1:2000 and the anti DIG-alkaline phosphatase-coupled Fab fragments (Boehringer Mannheim) diluted 1:500 in 10% fetal calf serum in PBST. After washes in PBST, cells were incubated 1 hr at rt with biotinylated anti-mouse IgG antibodies (Vector, Burlingame, CA) diluted 1:100 in 10% fetal calf serum in PBST. Subsequently, cells were washed in PBST and reacted with 4-nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate in 100 mM Tris/HCl, pH 9.5, 50 mM MgCl2, 100 mM NaCl, and 1 mM Levamisol overnight at 4°C. Finally, cells were incubated 20 min at rt in streptavidin-FITC (Sigma) diluted 1:500 in 10 mM HEPES, pH 8.2, 150 mM NaCl buffer, washed in PBS twice, and mounted in Vectashield (Vector). Fluorescence was analyzed with a fluorescein filter under dark field, on a Zeiss Axiophot microscope.
Immunohistochemistry on cultured hippocampal neurons was preceded by the same fixation and permeabilization steps used for in situ hybridization described above. Fixed and permeabilized cells were preincubated for 30 min at rt in 3% BSA in PBS, incubated 3 hr at rt with an antibody recognizing the TrkB full length isoform (Santa Cruz Ab794, made in rabbit, diluted 1:100 in 3% BSA in PBS), or anti-BDNF [(Promega, Madison, WI) made in chicken, diluted 1:100 in 3% BSA in PBS]. After they were washed in PBS, cells were incubated for 1 hr at rt with biotinylated anti-rabbit IgG antibody (Vector) or biotinylated anti-chicken IgG antibody (Promega) diluted 1:200 in 3% BSA in PBS, then washed in PBS, incubated in streptavidin-FITC as described above, and mounted in Vectashield (Vector).
Quantitative imaging analysis and statistics. Nonradioactive in situ hybridization was analyzed by viewing stained cultures under bright-field illumination with an Olympus microscope with DIC (Differential Interference Contrast) equipped lens (40× magnification). Stained neurons were acquired with a Hitachi CCD camera and digitized with the image analysis program Imageplus (Microsoft). The function "Trace" was used to measure, starting from the base of the dendrites, the maximal distance of dendritic labeling (MDDL). Dendrites were traced, in a conservative manner, up to the point at which the in situ labeling was clearly distinguishable from the background. The background level obtained in sister cell cultures hybridized with the sense probes was used as a reference to distinguish the actual labeling obtained with the antisense probes from the background. The number of dendrites measured is indicated in Table 1. The individual preparations were coded and analyzed in a blind manner. The data of the MDDL were normalized by dividing each single measurement obtained in the different experimental conditions by the mean of the controls, and they were statistically analyzed with unpaired Student's t tests.
Table 1. Maximal distance of dendritic staining
Mean (µm) SE p n BDNF Control 31.5 ±0.7 403 30 30.9 ±0.8 0.00001a
310 3h K 55.1 ±1.0 310 30 33.6 ±0.8 386 3h KK 46.4 ±1.0 350 3h K kinu 41.7 ±1.0 276 3h K nife 43.4 ±0.9 389 3h KK kinu 39.6 ±0.9 324 3h KK nife 32.3 ±0.9 273 3h K TTX 35.7 ±1.1 221 C act.D 23.6 ±1.5 74 3h K act.D 46.6 ±1.3 146 TrkB Control 24.5 ±0.5 451 30 34.7 ±0.8 306 3h K 52.5 ±1.2 301 30 33.6 ±0.8 313 3h KK 49.8 ±1.3 318 3h K kinu 30.3 ±0.8 321 3h K nife 41.1 ±1.0 305 3h KK kinu 36.5 ±1.2 176 3h KK nife 28.8 ±0.7 304 3h K TTX 31.6 ±1.3 72 C act.D 18.8 ±1.2 86 3h K act.D 35.6 ±1.2 147 SE, Standard error; n, number of dendrites measured. a Respect to 3h K; t test. b Respect to control; t test. c Respect to 3h KK; t test.
Fluorescent immunocytochemistry was analyzed by confocal microscopy with a Molecular Dynamics MultiProbe 2001 (Sunnyvale, CA) confocal device mounted on a Nikon microscope. The individual preparations were coded and analyzed in a blind manner. Individual neurons whose entire dendritic domains were distinguishable from those of neighboring neurons were selected. For each neuron, five optical sections 0.7 µm thick were acquired and then were integrated in a single projection with the function "look-through" to ensure the visualization of the entire width of the dendrites on the z axis. This function gives the same results as the function "maximum intensity" used in similar studies (Blöchl and Thoenen, 1996
Differential extraction of tubulin. Soluble and polymerized tubulin was extracted from control cell cultures or treated for 6 hr with 1 µg/ml nocodazole, essentially as described (Fasulo et al., 1996
RESULTS
BDNF and TrkB mRNAs are localized in the dendrites
The subcellular localization of the mRNA for BDNF and TrkB was studied by nonradioactive in situ hybridization in rat hippocampal neurons in culture. An increasing number of laboratories have published studies using nonradioactive probes for the analysis of the subcellular localization of the mRNAs (Ainger, 1993; Hannan et al., 1995
Almost all of the neurons in the culture are labeled with the BDNF and the TrkB riboprobes, with a prominent staining of the cell body and proximal dendrites (Figs. 1A, 2B for BDNF; 1E, 3A for TrkB). Interestingly, several cells show a strong labeling corresponding to branching points, and a subpopulation of 3% of the cells exhibit labeling corresponding to dendritic varicosities (Fig. 1A,D,H, arrowheads). In double-labeling experiments, dendritic processes were identified with a monoclonal antibody against the microtubule-associated protein MAP2, a specific marker for the dendritic compartment (Caceres et al., 1984 
Fig. 1. Subcellular distribution of BDNF and TrkB mRNAs in cultured hippocampal neurons. Staining by nonradioactive in situ hybridization with digoxigenin-labeled riboprobes and oligonucleotides. Fields are viewed with Nomarski optics, except immunofluorescence in B and F. A, The BDNF riboprobe labels the cell soma and a process identified as a dendrite in B by a double-labeling with an anti-MAP2 monoclonal antibody. C, No staining is observed with a BDNF sense riboprobe. D, The BDNF antisense oligonucleotide probe shows somatodendritic labeling. E, The TrkB riboprobe stains the cell soma and a dendritic process identified as a dendrite, in F, by the anti-MAP2 antibody. G, No labeling is detected with the TrkB sense riboprobe. H, The TrkB antisense oligonucleotide probe labels the cell soma and a dendrite. Arrowheads indicate labeling at dendritic branchings or varicosities. Scale bar (shown in H): 20 µm for A-H.
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Fig. 2. High potassium increases the dendritic localization of BDNF mRNA in cultured hippocampal neurons. A, Whole-cell patch-clamp recordings, under current-clamp conditions, after isotonic depolarization with 10 mM (top trace) and 20 mM KCl (bottom trace). The arrows mark the onset of the depolarization. The arrowheads mark the last 3 min of a 30 min recording time. Calibration: 45 mV, 1 min. B, In situ hybridization on cultured hippocampal neurons with BDNF riboprobe, viewed with Nomarski optics. Small arrows mark the maximal distance at which a dendritic labeling was scored (MDDL). In the dendrites, staining appears granular (large arrows). C, D, Depolarization with 10 mM KCl for 3 hr increases the dendritic localization of BDNF mRNA. E, At higher magnification, after 3 hr depolarization in 10 mM KCl, intensely labeled granules are detected within varicosities at great distance from the cell body (large arrows). Scale bar (shown in E): 20 µm for B, C, D; 14 µm for E.
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KCl depolarization extends BDNF and TrkB mRNA dendritic labeling
BDNF mRNA expression is upregulated by physiological stimuli (Castrén et al., 1992
The depolarization of hippocampal cells with KCl affects the subcellular localization of both BDNF and TrkB mRNAs. The in situ staining for BDNF mRNA is found at much greater distances from the cell body than in control cells after a 3 hr incubation with 10 mM KCl (compare Fig. 2, B, control, and C, D, and E, 10 mM KCl) and 20 mM KCl (not shown). The increase in the dendritic localization of BDNF mRNA was observed in cells of different morphologies (compare Fig. 2, C and D). The mRNA for BDNF is found in both large and thin caliber dendrites (Fig. 2E). In the majority of the dendrites, regardless of their caliber, the staining appears to be discontinuous, in the form of granules localized, in some cases, to varicosities (Fig. 2B,E, large arrows). These granules may correspond to the RNA-containing granules described in living neurons (Knowles et al., 1996
Similar results were obtained with hippocampal cultures labeled with the probe for the full length isoform of TrkB. Control cells show labeling of the cell body and of proximal dendrites (Fig. 3A). Similar to what is observed for BDNF, a strong increase is also observed for TrkB in the extent of mRNA dendritic staining on depolarization for 3 hr in 10 mM KCl (compare Fig. 3, A and B) and 20 mM KCl (not shown). In Figure 3B, one of the two neurons shown displays TrkB mRNA labeling in the distal regions of the dendrites, whereas the other does not. These experiments demonstrate that neuronal depolarization is able to increase the extent of dendritic localization of both BDNF and TrkB mRNAs. 
Fig. 3. High potassium increases the dendritic localization of TrkB, but not of
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To determine whether the effect of KCl reflects an overall change in the subcellular distribution of the mRNAs, rather than a specific effect on the BDNF and TrkB mRNAs, we examined the distribution of the
After this first qualitative evaluation, a quantitative statistical analysis was performed. The maximum distance from the cell body at which labeled BDNF and TrkB mRNA could be detected in individual dendrites (MDDL) was determined. An example of this procedure is marked by the small arrows in Figure 2B,C. For these measurements, cells with a well identified dendritic tree were randomly chosen within the cell population. In control cultures, the average MDDL for BDNF mRNA is 31.5 µm (Table 1). Incubation with 10 mM or 20 mM KCl for 3 hr leads to a 1.75- and 1.47-fold increase of the MDDL, respectively (Fig. 4A, Table 1). After shorter incubation times in 10 or 20 mM KCl (30 min), no significant variation could be observed. The quantitative analysis also confirmed that incubation for 3 hr in depolarizing media leads to a great increase of the extent of TrkB mRNA dendritic labeling (Fig. 4B). In control cultures the average MDDL for TrkB mRNA was 24.5 µm (Table 1). After 3 hr stimulation in 10 or 20 mM KCl the MDDL increases by approximately twofold (Fig. 4B, Table 1). At variance with BDNF, incubation in high potassium for shorter times (30 min) leads to a small but statistically significant (p 
Fig. 4. Quantitative analysis of dendritic localization of BDNF and TrkB mRNAs after depolarization of hippocampal neurons in culture. Bars in A and B indicate the fold increase, with respect to controls, of the mean maximal distance at which the in situ labeling in dendrites was detectable (MDDL). A, BDNF mRNA. Depolarization with either 10 (K) or 20 mM KCl (KK) increases the mean maximal distance of dendritic labeling (MDDL) at 3 hr (3 h K and 3 h KK) but not at 30 min (30, Significantly different with respect to the 3 h K-stimulated;
, significantly different with respect to the 3 h KK stimulated; and *, significantly different with respect to the control (no stimulation). Error bars represent SE. The corresponding numerical values, number of dendrites measured, and significance values are shown in Table 1. C, D, E, F, Correlation plots of the MDDL versus dendritic length. Each point refers to one MDDL determination, together with the length of the corresponding dendrite, under the experimental conditions indicated. The scatter plots were fitted by linear regression lines through the origin. C, Slope = 0.30, correlation coefficient r = 0.57; D, slope = 0.68; r = 0.80; E, slope = 0.26; r = 0.31; F, slope = 0.67; r = 0.72.
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The previous results represent the average MDDL calculated on the total population of dendrites, including dendrites with very different length. To put the extent of the dendritic labeling into relationship with the length of the dendrites analyzed, each individual MDDL measurement was plotted as a function of the length of the corresponding dendrite. For this set of measurements we selected the most isolated cells in the culture, in which individual processes could be followed for their entire length. The results of the correlation plots for BDNF and TrkB are reported in Figure 4C-F. In control conditions, the correlation between the MDDL and the length of the dendrites is 0.57 for BDNF and 0.31 for TrkB. The regression lines have slopes of 0.30 and 0.26 for BDNF and TrkB, respectively. This slope corresponds to the actual average value of dendritic "filling" with BDNF and TrkB mRNA, ~30 and 26% of the entire dendritic length, respectively. Depolarization of the cultures with 10 mM KCl for 3 hr induces an increase in the slope as well as in the correlation coefficient of the regression lines, for both BDNF (slope = 0.68; r = 0.80) and TrkB (slope = 0.67; r = 0.72). On average, BDNF and TrkB mRNAs label 68 and 67% of the total dendritic length, respectively. It is worth noting here that under depolarizing conditions several dendrites are stained for 100% of their length (Fig. 4D,F). In general, under depolarizing conditions staining extends to secondary and tertiary branchings, especially of large-caliber dendrites, but similar to MAP2 mRNA, was rarely seen in the very fine, distal dendritic processes (Kleiman et al., 1990 The effects of KCl depolarization do not depend on new mRNA synthesis
Previous studies have shown that depolarization of hippocampal neurons in culture with high potassium concentrations (50 mM) results in an increase of the total amount of BDNF mRNA that peaks after 6 hr (Zafra et al., 1990
Fig. 5. The effects of KCl depolarization do not depend on new mRNA synthesis. Nonradioactive in situ hybridization on cultured hippocampal neurons viewed with Nomarski optics. In control conditions, cells were kept in the presence of actinomycin-D for 3.5 hr and then stained with (A) BDNF or (C) TrkB riboprobes. After a pretreatment of 30 min with actinomycin-D, cultures were depolarized for 3 hr in 10 mM KCl in the continuous presence of actinomycin-D and stained with (B) BDNF or (D) TrkB riboprobes. Scale bar (shown in D): 20 µm for A-D. E, Quantitative analysis of the MDDL for BDNF and TrkB mRNAs with actinomycin-D. Bars indicate the fold increase, with respect to controls, of the mean maximal distance at which the in situ labeling was detectable (MDDL). Results are similar for both BDNF and TrkB mRNAs: treatment of control cultures with actinomycin-D for 3.5 hr (C act.D) reduces the MDDL with respect to untreated controls (C), whereas a strong increase of MDDL induced by 3 hr in 10 mM KCl also occurs in the presence of actinomycin-D (3h K act.D). Error bars represent SE. *, Significantly different from controls. The corresponding numerical values, the number of dendrites measured, and the significance values are shown in Table 1.
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Role of Ca2+ in the activity-dependent increase of dendritic localization of BDNF and TrkB mRNAs
Ca2+ is a fundamental second messenger through which electrical activity can influence intracellular processes. To study the Ca2+ dependence of the activity-dependent targeting of BDNF and TrkB mRNAs, cells were incubated with 10 mM KCl in Ca2+-free medium supplemented with EGTA to block the external Ca2+ or with BAPTA-AM to block the internal Ca2+. In these Ca2+-free conditions, the general levels of staining are much reduced in comparison with control cultures, and several neurons appeared flattened and partially swollen (not shown). Measurement of the MDDL in neurons having a normal morphology shows that on depolarization with 10 mM KCl for 3 hr, the MDDL for BDNF mRNA (Fig. 6A) or for TrkB mRNA (Fig. 6B) does not change with respect to controls. Interestingly, no statistically significant difference can be observed when either EGTA or BAPTA-AM are used. These results demonstrate that extracellular Ca2+ is absolutely required for the KCl-induced increase in the dendritic targeting of BDNF and TrkB mRNA.
Fig. 6. Effects of Ca2+-free medium on the KCl-induced increase in dendritic localization of BDNF and TrkB mRNAs. Quantitative analysis of the MDDL for BDNF and TrkB mRNAs in nominally Ca2+-free medium supplemented with 10 µM EGTA or BAPTA-AM. Bars indicate the fold increase, with respect to controls, of the mean maximal distance at which the in situ labeling was detectable (MDDL) for 80-150 dendrites. A, In nominally Ca2+-free medium the MDDL for BDNF mRNA, induced by depolarization with 10 mM KCl for 3 hr, does not change with respect to controls. The effects are identical in the presence of either EGTA or BAPTA-AM. B, MDDL for TrkB mRNA. At the level of p
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TTX, kinurenic acid, and nifedipine counteract the KCl-induced increase in dendritic localization of BDNF and TrkB mRNA
To further characterize the activity-dependent increase in MDDL for the BDNF and TrkB mRNAs, the Na+ channel blocker TTX was used. Depolarization with 10 mM KCl in the presence of 0.5 µM TTX for 3 hr results in a strong inhibition of the KCl-induced increase of MDDL, for both BDNF (81% inhibition) and TrkB mRNA (75% inhibition) (Fig. 7A,C, Table 1). These results indicate that the increase in the dendritic localization of BDNF and TrkB mRNAs observed in 10 mM KCl requires Na+-dependent action potentials to occur.
Fig. 7. Effects of TTX, kinurenic acid, and nifedipine on the KCl-induced increase in dendritic localization of BDNF and TrkB mRNAs. Quantitative analysis of the MDDL for BDNF and TrkB mRNAs in the presence of TTX, kinurenic acid, and nifedipine. Bars indicate the fold increase, with respect to controls, of the mean maximal distance at which the in situ labeling was detectable (MDDL). A, Continuous presence of tetrodotoxin (TTX) inhibits the increase in MDDL for BDNF mRNA, induced by depolarization with 10 mM KCl for 3 hr (3h K TTX). The glutamate receptor antagonist kinurenic acid partially counteracts the KCl-induced increase by either 10 (3h K kinu) or 20 mM KCl (3h KK kinu). B, The L-type Ca2+ channel blocker nifedipine has distinct effects at different KCl concentrations, with a partial inhibition at 10 mM KCl (3h K nife) and an almost complete inhibition at 20 mM KCl (3h KK nife). C, TTX strongly inhibits the MDDL increase for TrkB mRNA in 10 mM KCl (3h K TTX). The glutamate receptor antagonist kinurenic acid reduced the 10 mM KCl depolarization effects (3h K kinu) more effectively than at 20 mM KCl (3h KK kinu). D, In contrast, nifedipine almost completely abolishes the effects induced by 20 mM KCl (3h KK nife) and only partially inhibits those induced by 10 mM KCl depolarization (3h K nife). Error bars represent SE.
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Hippocampal neurons express glutamate receptors of the NMDA and AMPA types. To ascertain the contribution of glutamatergic synaptic transmission to the KCl-induced increase in dendritic localization of BDNF and TrkB mRNA, kinurenic acid (1 mM), which blocks both NMDA and AMPA glutamate receptors, was added to cell cultures. The addition of kinurenic acid to either the 10 or 20 mM KCl medium partially counteracts the increase in MDDL for BDNF mRNA: the resulting MDDL increase is 1.32-fold (57% inhibition) for 10 mM KCl and 1.26-fold (55% inhibition) for 20 mM KCl (Fig. 7A, Table 1). In contrast, for TrkB mRNA, the inhibitory effect of kinurenic acid is more effective at 10 mM KCl, resulting in an MDDL increase of only 1.23-fold (80% inhibition), whereas at 20 mM KCl it has an effect similar to that observed for BDNF mRNA (1.48-fold increase; 53% inhibition) (Fig. 7C, Table 1). Thus, glutamatergic synaptic transmission is involved in the KCl-induced increase in MDDL at the two tested concentrations in a similar way for the BDNF mRNA, and the TrkB mRNA is more relevant at 10 than at 20 mM KCl.
Glutamate receptors of the NMDA type are known to be important for the Ca2+ influx in the neurons during synaptic activity. Another important Ca2+ entry route is represented by the voltage-sensitive Ca2+ channels, in particular those of the L-type. To test the contribution of these channels to the regulation of mRNA targeting, nifedipine (1 µM), a specific blocker of the L-type voltage-dependent Ca2+ channel, was used. When nifedipine is added to the 10 mM KCl medium, the MDDL increase is 1.38-fold for BDNF mRNA (50% inhibition) (Fig. 7B, Table 1) and 1.67-fold (58% inhibition) (Fig. 7D, Table 1) for TrkB mRNA. In contrast, the MDDL increase observed in 20 mM KCl for both mRNAs is almost completely abolished by nifedipine (96% and 83% inhibition for BDNF and TrkB, respectively) (Fig. 7B,D, Table 1). Taken together, these data suggest that the MDDL increase of BDNF and TrkB mRNAs observed in 20 mM KCl is almost totally dependent on Ca2+ entry through the L-type voltage-sensitive Ca2+ channels, whereas in 10 mM KCl the MDDL increase appears to depend only partially on the activation of these channels. Dendritic TrkB and BDNF immunoreactivity is rapidly increased by electrical activity
The finding that the dendritic targeting of TrkB and BDNF mRNA is increased in hippocampal cultured neurons after KCl stimulation leads naturally to the question of whether the dendritic content of the corresponding proteins also would be subjected to modulation by electrical activity. To answer this question, the distribution of the BDNF and TrkB proteins in hippocampal cells was studied by immunofluorescence and confocal microscopy analysis with anti-BDNF and anti-TrkB antibodies, in control cultures and in cultures treated for 10 min with 10 mM KCl. For these experiments we used antibodies that were shown in previous studies to detect BDNF (Goodman et al., 1996
Fig. 8. A short incubation in high potassium increases BDNF and TrkB protein levels in the dendritic compartment. Immunohistochemistry on cultured hippocampal neurons. Each picture represents the integration in a single projection of a series of five optical sections obtained with a confocal microscope, as described in Materials and Methods. A, Anti-BDNF immunostaining of control cultures. B, Anti-BDNF immunostaining after 10 mM KCl depolarization for 10 min. C, Anti-BDNF staining after a preincubation with nocodazole for 6 hr followed by 10 min of 10 mM KCl depolarization in the continuous presence of nocodazole. D, Staining with anti-TrkB antibody in control conditions. E, Staining with anti-TrkB antibodies after 10 min depolarization in 10 mM KCl. F, Anti-TrkB staining after a preincubation with nocodazole for 6 hr followed by 10 min of 10 mM KCl depolarization in the continuous presence of nocodazole. Scale bar (shown in F): 20 µm for A-F.
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A quantification of these results was performed with confocal microscopy by measuring the immunofluorescence signal within the dendrites. For these measurements only the principal dendrites of the cells (i.e., >100 µm in length) were chosen. The immunofluorescence signal was integrated over five different confocal sections taken across the entire width of the dendrites. The integrated images of the dendrites were longitudinally subdivided into two regions, which were contoured manually: a proximal region (from the base of the dendrite up to 30 µm from the cell soma) and a distal region (from 30 µm up to 90 µm from the cell soma). In control conditions, the majority of dendrites >100 µm in length contained BDNF and TrkB mRNAs in both the proximal and distal regions described above (Fig. 4C,E). The fluorescence density (see Materials and Methods) over the proximal and distal regions was quantified under different experimental conditions. The 10 mM KCl depolarization induces in the proximal region a 1.30-fold increase of the BDNF fluorescence density and a stronger increase of 1.78-fold in the distal region (Fig. 9A). Also for TrkB, after 10 min in 10 mM KCl an increase in fluorescence density in the distal dendrites is found (1.75-fold), whereas no significant variation is found in the proximal region (Fig. 9C). These experiments demonstrate that electrical activity affects BDNF and TrkB protein levels by rapidly increasing their amount in the dendrites. 
Fig. 9. Quantitative analysis of BDNF and TrkB protein levels in proximal and distal regions of the dendrites. Immunofluorescence for BDNF or TrkB was acquired by five confocal sections and integrated in a single projection, in control conditions and after 10 min depolarization with 10 mM KCl, in the presence or absence of cycloheximide (A, C) and of nocodazole (B, D). Fluorescent density of BDNF (A, B) and TrkB (C, D) was determined in proximal and distal regions of the projections of labeled dendrites as described in Materials and Methods. Bars represent the mean fold increase of the fluorescence density of 45 dendrites, with respect to the controls (=1.0). Error bars represent SE. A, Incubation of cells in 10 mM KCl for 10 min leads to a strong increase of BDNF fluorescence density in both proximal and distal regions (10
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The activity-dependent increase in dendritic BDNF and TrkB immunoreactivity requires protein synthesis
To determine whether new protein synthesis is involved in the KCl-induced increase of BDNF and TrkB dendritic immunostaining, cultures were preincubated for 30 min with the protein synthesis inhibitor cycloheximide, before the depolarizing stimulus. Cultures were then incubated for 10 min either in 10 mM KCl or in control medium, in the continuous presence of cycloheximide. In unstimulated cultures, cycloheximide does not change the basal levels of fluorescence density of BDNF or TrkB, with respect to those of control conditions; the only exception is a slight decrease of TrkB in the proximal dendrites (Fig. 9A,C). This shows that under basal conditions the half-life of the BDNF and TrkB proteins, in all compartments for BDNF and at least in the distal dendrites for TrkB, is >40 min. In KCl-stimulated cultures, cycloheximide completely prevents the increase in BDNF and TrkB immunoreactivity in the distal dendritic compartment (Fig. 9A,C). For BDNF, the cycloheximide treatment also fully inhibits the increase found in the proximal region. Taken together, these results demonstrate that the electrical activity-dependent increase in BDNF and TrkB immunostaining observed in distal and proximal dendrites requires new protein synthesis.The activity-dependent increase in dendritic BDNF and TrkB immunoreactivity occurs also under dendritic transport blockade
The kinetics of fast protein transport in dendrites (Kiss et al., 1977; Feig and Lipton, 1993
To ascertain whether the immunofluorescence increase observed in the distal dendrites would reflect an accumulation caused by transport of newly synthesized BDNF and TrkB protein from the perikaryon, cells were depolarized in the presence of nocodazole (1 µg/ml), which inhibits the protein transport in dendrites, by affecting the amount of polymerized tubulin (Dotti and Banker, 1991
DISCUSSION
Neurotrophins are localized to the dendritic processes of neurons, from which they can be secreted in an activity-dependent way (Blöchl and Thoenen, 1996
BDNF and TrkB mRNAs are widely expressed in the rat brain. Notably, the majority of the previous studies did not specifically examine the issue of the subcellular localization of these two mRNAs, because they were focused on their overall expression pattern in the brain, showing pictures at magnification too low to detect the dendrites or lacking a clear identification of the dendritic processes. Only one study reported a dendritic localization for TrkB mRNA (Ugolini et al., 1995
Theoretically, the KCl treatment might induce a general intracellular reorganization, attributable to cytotoxic effects. Previous studies on BDNF expression in cultured hippocampal neurons (Zafra et al., 1990
Electrical activity increases the extent of dendritic BDNF and TrkB mRNA localization, by and large, in a similar way. At both KCl concentrations tested, the increase of the intracellular concentration of Ca2+ emerges as one of the triggering events of the cascade that modulates localization of both mRNAs. However, the regulation of TrkB mRNA localization differs from that of BDNF in some respects. First, for BDNF mRNA the effect of 10 mM KCl is stronger than that of 20 mM KCl, whereas for TrkB the two KCl concentrations have a comparable effect. Second, the increase in dendritic targeting is more rapid for TrkB than for BDNF mRNA. Third, the glutamatergic synaptic transmission seems to be more important in regulating the dendritic localization of TrkB than of BDNF mRNA. The increased localization of BDNF and TrkB mRNAs induced by depolarization is a slow process. It is not clear at present whether the limiting time step is the transport process itself [the rate of mRNA transport in the dendrites is on average 11 µm/hr, (Davis et al., 1990
The modulation of BDNF and TrkB protein levels was studied by using shorter KCl stimulation protocols (10 min). A consistent picture emerges from these experiments: (1) the levels of BDNF and TrkB protein in the distal dendritic regions increase as a result of KCl-induced depolarization; (2) the basal levels of BDNF and TrkB protein in control conditions are not affected by a 40 min cycloheximide incubation; and (3) the KCl-induced increase is totally inhibited by cycloheximide and thus protein synthesis dependent. The increase in distal BDNF and TrkB proteins may be caused, therefore, by an increased translation of a preexisting pool of mRNAs and/or to a reduced degradation of these proteins. The cycloheximide dependence of the observed increase argues in favor of the first possibility, i.e., a translational control. During the 10 min depolarization, the transport of BDNF or TrkB proteins from the cell body may contribute to the observed phenomenon, because the most rapid dendritic transport of newly formed proteins in rat hippocampal cells is 50 µm/min (Kiss et al., 1977). However, after incubation of hippocampal cells for 6 hr with nocodazole that fully inhibits, in a reversible manner, the dendritic transport in hippocampal neurons (Dotti and Banker, 1991
Of note is the fact that in the culture system studied, the large majority of the neurons express both BDNF and TrkB. In accordance with previous studies in vivo (Kokaia et al., 1993
In conclusion, our data suggest that the dendritic transport and/or half-life of the BDNF and TrkB mRNAs is selectively enhanced by the electrical activity and that the mRNA localization can contribute to the local synthesis of these two proteins in response to enhanced synaptic activity.
FOOTNOTES
Received July 23, 1997; revised Sept. 29, 1997; accepted Oct. 3, 1997.
This work was supported by a research grant from the Human Frontier Science Project Organization (HFSPO) (RG93-93) to A.C. E.T. was supported by a postdoctoral fellowship from the International Centre for Genetic Engineering and Biotechnology (ICGEB) of Trieste. We thank Professor E. Cherubini and Dr. L. Domenici (SISSA, Trieste) for helpful suggestions and discussions; Professor E. Ferrero and Dr. P. Giulianini (University of Trieste) for help with the imaging system; and Drs. S. Hunt and K. Smith (Medical Research Council Laboratory of Molecular Biology, Cambridge, UK) for advice on confocal analysis. We thank Dr. L. Fasulo for precious advice and I. Masi for excellent technical help with cell cultures. We are greatly indebted to Dr. P. Andjus (SISSA, Trieste) for the electrophysiological recordings and to those cited in the text for providing plasmids.
Correspondence should be addressed to Dr. A. Cattaneo, International School for Advanced Studies (SISSA), Neuroscience Program, Via Beirut 2/4-34014 Trieste, Italy.
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