Activity- and Ca²⁺-Dependent Modulation of Surface Expression of Brain-Derived Neurotrophic Factor Receptors in Hippocampal Neurons

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is a potent neurotrophic protein that regulates neuronal survival and differentiation (Lewin and Barde 1996). Signal transduction and neurotrophic functions of BDNF are mediated primarily by TrkB, a high affinity receptor tyrosine kinase (Kaplan and Stephens 1994). BDNF is also capable of binding to the low affinity receptor p75 (p75NR) and eliciting apoptotic function in certain populations of neurons (Casaccia-Bonnefil et al. 1996, Casaccia-Bonnefil et al. 1998; Frade et al. 1996). Evidence accumulated in the last few years suggests that BDNF is involved in synapse development and plasticity, in addition to its traditional role in neuronal survival and differentiation (Thoenen 1995; Bonhoeffer 1996; Berninger and Poo 1996; Lu and Chow 1999; McAllister et al. 1999). BDNF has been shown to exert complex modulation of dendritic and axonal growth in the brain, particularly in the visual system (Cohen-Cory and Fraser 1996; Cohen-Cory and Lom 1999; McAllister et al. 1995, McAllister et al. 1996, McAllister et al. 1997). BDNF is also involved in activity-dependent synaptic competition and formation of ocular dominance columns in the visual cortex (Maffei et al. 1992; Gu et al. 1994; Cabelli et al. 1995; Riddle et al. 1995; Galuske et al. 1996; Huang et al. 1999). BDNF is capable of rapidly regulating synaptic transmission at the neuromuscular junction and central nervous system (CNS) synapses (Lohof et al. 1993; Knipper et al. 1994; Lessmann et al. 1994; Levine et al. 1995; Takei et al. 1997). In the hippocampus, BDNF promotes tetanus-induced long-term potentiation (LTP; Korte et al. 1995, Korte et al. 1996; Figurov et al. 1996; Patterson et al. 1996; Kang et al. 1997). Moreover, BDNF selectively enhances high frequency but not low frequency synaptic transmission (Tanaka et al. 1997; Frerking et al. 1998; Gottschalk et al. 1998). Recent experiments using BDNF knockout mice demonstrate that BDNF enhances high frequency synaptic transmission by facilitating synaptic vesicle docking in the hippocampus, possibly by increasing the levels of the vesicle protein synaptobrevin in the presynaptic terminals of CA1 synapses (Pozzo-Miller et al. 1999).

Based on the above discoveries, BDNF has been proposed to participate in several forms of activity-dependent plasticity in the CNS (Thoenen 1995; Bonhoeffer 1996; Lu and Chow 1999). A critical element of such proposition is that BDNF acts preferentially on active neurons. Indeed, blockade of neuronal activity and synaptic transmission prevents the increase of dendritic arborization induced by BDNF (McAllister et al. 1996). BDNF cannot enhance the survival of retinal ganglion neurons unless they are depolarized by high K⁺ or glutamate agonists, or their intracellular cAMP is increased (Meyer-Franke et al. 1995). Presynaptic depolarization greatly facilitates the BDNF modulation of synaptic transmission at the neuromuscular junction (Boulanger and Poo 1999). In the hippocampus, the effect of BDNF on CA1 synapses is observed only when presynaptic neurons are stimulated at high frequency (Gottschalk et al. 1998). These results support the notion that certain levels of neuronal activity are required for neuronal responsiveness to BDNF.

As a diffusible molecule, how does BDNF distinguish active and inactive neurons or synapses, and restrict its action preferentially on active neurons/synapses? One possible mechanism is that cellular responsiveness of neurons to BDNF is enhanced by neuronal activity. Thus, whether or how well a neuron can respond to BDNF may depend on its activity levels. It is unclear, however, how activity-dependent regulation of BDNF responsiveness is achieved. Neuronal activity could increase the number of BDNF receptors on the cell surface, facilitate the internalization of BDNF–receptor complex, or facilitate the signaling mechanisms for BDNF. Depolarization or cAMP elevation has been shown to increase the levels of the BDNF receptor TrkB on the cell surface of retinal ganglion cells and spinal neurons (Meyer-Franke et al. 1998). Here we investigate whether physiologically relevant stimuli such as electric stimulation can modulate the BDNF receptors on the cell surface of neurons in the hippocampus, where activity-dependent plasticity is most commonly observed. Using three independent approaches (biotinylation, receptor binding, and immunocytochemistry), we show that high frequency tetanic stimulation, but not low frequency stimulation or simple depolarization, can rapidly enhance the insertion of TrkB into the cell surface. We also demonstrate that the activity-dependent modulation requires Ca²⁺ influx through NMDA and Ca²⁺ channels, and involves Ca²⁺/calmodulin-dependent kinase II (CaMKII). Not only may these findings provide insights into the mechanistic link between activity-dependent and neurotrophic modulation of CNS neurons and synapses, but they may also have general implications in the cell biology of growth factor signaling.

Cultures of hippocampal neurons were prepared according to the published procedure (Feng et al. 1999) with minor modifications. In brief, hippocampus was dissected from embryonic day 18 rats, dissociated in Ca²⁺- and Mg²⁺-free HBSS containing 0.125% tyrosine for 15 min, triturated in DMEM/10% FBS, and plated at 2 × 10⁵ cells per well in 12-well plates. Cells were grown at 37°C, 5% CO₂ and 95% humidity, first in 10% FBS/DMEM, and 1 d later switched to serum-free medium Neurobasal plus B27 (Life Technologies). Cultures were grown in serum-free medium for 11–14 d before being used for experiments, and the medium was changed every 3 d. Fresh medium was applied 24 h before each experiment. These cultures yielded virtually pure neurons (data not shown). Drugs were applied immediately before electric stimulation. In some cases, serum-free medium was replaced with Ca²⁺-free medium (Ca²⁺-free DMEM; Life Technologies) for 30 min before electric stimulation.

Hippocampal neurons were stimulated using a method similar to one that has been described previously (Bito et al. 1996; Deisseroth et al. 1996; Fields et al. 1997). Field electric stimulation was applied across a 12-well dish through a homemade lid, which contained platinum wires contacting the medium in each well. Each stimulation pulse (1 msec, 2–8 V) was sufficient to elicit action potentials in these cultured neurons (see Fig. 1 A). The entire electric stimulation was performed in a 37°C, 5% CO₂ incubator. The following stimulation paradigms were used. (i) TBS: each episode consisted of four bursts, each with five biphasic pulses at 100 Hz (10-msec interval), separated by an interburst interval of 200 msec. One episode was given every 5 s throughout the whole incubation period. (ii) Tetanic stimulation: 1 s, 100 Hz, given every 10 min for for 30 min. (iii) Low frequency stimulation: 0.16 Hz during the entire incubation period. (iv) Long-term depression (LTD)-inducing stimulation: 4 min, 5 Hz. Whole-cell recording was performed under the current-clamped or voltage clamped conditions as previously described (Kim et al. 1994). Data were collected by an Axopatch 200B amplifier, filtered at 5 kHz, digitized at 10 kHz, and analyzed by P-clamp software (Axon Instruments).

Surface TrkB receptors was measured by biotinylation followed by Western blot using either a TrkB antibody or a p75NR antibody, as described elsewhere (Meyer-Franke et al. 1998). In brief, various blockers were added to the hippocampal cultures, and electric stimulation was applied immediately in a 37°C incubator. At the end of electric stimulation (60 min), ice-cold PBS, pH 7.4, with Ca²⁺ and Mg²⁺, pH 7.4; Life Technologies) was added to the cultures to prevent receptor internalization. After three washes with ice-cold PBS, cells were incubated in Sulfo-NHS-LC-biotin (0.25 mg/ml in cold PBS; Pierce) for 30 min. The surface biotinylation was stopped by removing the above solution and incubating the cells in 10 mM ice-cold glycine in PBS for 20 min. Cells were then washed three times with cold PBS and lysed by RIPA buffer, which contains 20 mM Hepes, pH 7.4, 100 mM NaCl, 1 mM EGTA, 1 mM Na₃VO₄, 50 mM NaF, 1% NP-40, 1% deoxycholate, 0.1% SDS, 1 mM [4-(2-aminoethyl)-benzenesulfluoride hydrochloride], 10 μg/ml leupeptin, and 1 μg/ml aprotinin. Biotinylated proteins (160 μg) were precipitated with 100 μl of ImmunoPure Immobilized Streptavidin (Pierce). Western blots were performed by separating the biotinylated protein precipitates on SDS-PAGE gel and transferring the proteins to Immobilon P membrane. The membranes were probed with a monoclonal anti-TrkB antibody (1:250; Transduction Laboratories), or an anti-p75NR antibody (1:250; Upstate Biotechnology), followed by peroxidase-conjugated goat anti–rabbit IgG (1:10,000; Vector Laboratories). Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech). The ECL signal intensities were quantified by NIH Image program. To measure the total amount of TrkB, cultured hippocampal neurons were simply harvested by RIPA buffer and processed for Western blot. Quantitation for each experimental condition was based on three to six independent experiments (samples), each was repeated at least two to three times. The results were pooled and averaged, and presented as mean ± SE.

Binding assays were performed in hippocampal cultures in a 37°C, 5% CO₂ incubator in quadruplicates. In brief, cells were washed three times with warm DMEM, and then incubated in binding buffer (DMEM plus 0.5 mg/ml protamine sulfate and 10 mM Hepes, pH 7.4) containing I¹²⁵-BDNF (2,200 Ci/mmol, 5 × 10⁻¹¹ M; NEN Life Science Products) with or without excess cold BDNF (5 × 10⁻⁸ M; provided by Regeneron Pharmaceuticals, Inc.) for 30 min. During the entire period of incubation, the hippocampal neurons were electrically stimulated in the incubator in the presence or absence of various blockers. After incubation, the 12-well dishes were placed on ice to prevent receptor internalization. Nonspecifically bound BDNF was removed by washing three times with 1 ml of ice-cold PBS. The I¹²⁵-BDNF bound to cell surface was obtained by a 10-min acid wash on ice (0.2 M acetic acid, pH 2.2, 0.5 M NaCl, 0.5 ml), and the counts were used as the measure for BDNF surface binding. An LKB γ counter was used to count the radioactivity. Raw data (quadruplicates) from a specific experimental condition were normalized to the mean in control condition. The results in several experiments were pooled and averaged, and presented as mean ± SE.

To visualize surface TrkB, cultured hippocampal neurons were fixed with 2% paraformaldehyde, 120 mM sucrose in PBS at room temperature for 3 min. After paraformaldehyde was quenched with 0.1 M glycine in PBS, the nonspecific binding was blocked with 50% goat serum, 1% BSA, and 100 mM lysine in PBS for 40 min. The cells were then incubated with a chicken antibody against extracellular domain of TrkB (a gift from Dr. Louis Reichardt, University of California, San Francisco, CA) in blocking solution overnight at 4°C, or in room temperature for 40 min. The secondary antibody was Cy3-conjugated goat anti–chicken Y antibody (1:100; Jackson ImmunoResearch Laboratories). After several washes, cells were mounted with mounting medium Vectashield (Vector Laboratories). To visualize both surface and intracellular TrkB, the cells were fixed with 4% paraformaldehyde, 120 mM sucrose in PBS for 20 min at room temperature, followed by quenching with 0.1 M glycine in PBS. The cells were permeabilized and nonspecific binding was blocked with 10% goat serum, 0.4% Triton X-100 in PBS for 40 min at room temperature. The cells were stained with rabbit anti-TrkB (1:50; Chemicon) overnight at 4°C. After several washes the cells were incubated with Cy3-conjugated anti–rabbit antibody (1:200; Jackson ImmunoResearch Laboratories) in 5% goat serum in PBS for 1 h at room temperature. The cells were washed three times and then mounted with Vectashield. Fluorescence images were acquired by a MicroMax 1300 cool CCD camera mounted on a Nikon Eclipse E800 microscope, and assigned to a pseudo color (green or red). In some cases, immunostained cells were examined using a confocal microscope (MRC1024; Bio-Rad). The images were processed by IPLab software. Each experimental condition was repeated at least three times.

The autocamtide-2–related inhibitory peptide (AIP) was as a specific inhibitor for CaMKII (Ishida et al. 1995). To facilitate the translocation of AIP across the cell membrane, we fused AIP to a 16-residue antennapedia homeopeptide (antp-AIP; Prochiantz 1996; Passafaro et al. 1999). A control peptide was made using antp and scrambled AIP sequences (antp-AIPscm). This scrambled sequence was analyzed by Program Blastp, and no significant similarity was found in the current database. The sequences of the peptides are listed below:

[H]-RQIKIWFQNRRMKWKKALRRRAVEDAL—Antp-AIP

[H]-RQIKIWFQNRRMKWKKRLAAADLVERR—Antp-AIPscm

The peptides were synthesized and HPLC-purified by Princeton Biomolecules. To allow sufficient penetration of the peptides into the cytoplasm of hippocampal neurons, the cells were pretreated with Antp-AIP or Antp-AIPscm (20 μM) 3 h before the biotinylation or binding assay was performed.

BDNF receptors on the surface of cultured hippocampal neurons were determined by a biotinylation assay. All membrane proteins were biotinylated, followed by precipitation with ImmunoPure Immobilized Streptavidin and Western blot analysis using antibodies against the high affinity TrkB receptors or the p75NRs. Field electric stimulation was applied to the culture dishes in a 37°C incubator to induce neuronal firings (Bito et al. 1996; Deisseroth et al. 1996; Fields et al. 1997). Whole-cell current clamp recording indicated that field stimulation reliably elicited action potentials (Fig. 1 A). The firing patterns of the neurons followed well with either TBS (Fig. 1 A) or 100 Hz tetanus (data not shown). When equal amounts of biotinylated proteins were loaded into the SDS gel, significant differences in the amount of both full-length (145 kD, or p145) and truncated (95 kD, or p95) forms of TrkB receptors on the cell surface were observed between active and inactive hippocampal neurons (Fig. 1B and Fig. C). Neurons stimulated with TBS exhibited significantly more surface TrkB as compared with those in unstimulated cultures (Fig. 1, B1 and C). To control for any nonspecific effects of electric stimulation on surface expression of TrkB, we performed most of our experiments in cultures stimulated with TBS either alone (active) or in the presence of activity blockers (inactive). The stimulation alone group is referred to as control (Ctr). Tetrodotoxin (TTX; 1 μM), which blocks Na⁺ channels and therefore all neuronal action potentials, significantly reduced the amount of surface TrkB (Fig. 1 C). TBS had no effect on surface expression of p75NRs (Fig. 1 D), suggesting that the effect of TBS is specific for TrkB receptors. In these cultures, neurons were well connected, and electric stimulation often elicited excitatory synaptic transmission (data not shown). Inhibition of excitatory transmission, either by the general glutamate receptor antagonist kynurenic acid (kyn; 1 mM) or a combination of the non-NMDA receptor antagonist 6-cyano-7-nitroquinozaline-2,3-dione (CNQX; 100 μM) and the NMDA receptor antagonist MK801 (80 μM), significantly attenuated the TBS-induced increase in surface TrkB (Fig. 1, B1 and C). Blockade of high frequency transmission by kyn had no effect on surface p75NR (Fig. 1 D). Thus, high frequency neuronal activity modulates TrkB, but not p75NR, on the surface of hippocampal neurons, and this effect appears to require action potentials coupled to excitatory synaptic transmission.

The increase of surface TrkB could be due to an increase in TrkB insertion into the cell surface, a decrease in TrkB internalization, or an increase in TrkB synthesis. Several pieces of evidence argue against a general increase in TrkB synthesis. First, there was no difference in the total amount of TrkB between active (TBS) and inactive (TBS plus TTX or Kyn) hippocampal neurons (Fig. 1 B2). Second, inhibition of protein synthesis by anisomycin (10 μg/ml) or cycloheximide (10 μg/ml) did not decrease the levels of surface TrkB in neurons stimulated with TBS (Fig. 1 B3). Thus, the synthesis of TrkB receptors is not enhanced by high frequency neuronal activity. Finally, electric stimulation of hippocampal neurons resulted in an increase, rather than a decrease in TrkB internalization (data not shown). These results, together with the immunocytochemistry experiments (see below), suggest that TBS facilitates the insertion of TrkB onto the surface membrane, rather than its production, in hippocampal neurons.

A previous study showed that depolarization induced by high concentration of K⁺ (50 mM) resulted in a significant increase in the surface TrkB in retinal ganglion cells (Meyer-Franke et al. 1998). In our study, we found that simple depolarization by high K⁺ did not affect the amount of surface TrkB in hippocampal neurons. The levels of surface TrkB were the same in neurons treated with 50 mM of K⁺, 50 mM of Na⁺, or nothing at all (Fig. 1 B4 and data not shown). In retinal ganglion cells, the full-length (p145) but not the truncated (p95) form of TrkB was detected (Meyer-Franke et al. 1998). Both the full-length and truncated TrkB receptors were found on the cell surface of hippocampal neurons and both were increased by TBS (Fig. 1 B1). Moreover, very low levels of p75NR were detected in the hippocampal neurons (data not shown), whereas those in the retinal cells are known to be high (Frade et al. 1996; von Bartheld et al. 1996). Thus, modulation of surface TrkB receptors in hippocampal neurons may be different from that in retinal ganglion neurons.

Application of BDNF significantly reduced the amount of surface TrkB, presumably due to ligand-induced receptor internalization (data not shown). It was difficult to determine by the biotinylation assay whether electric activity could still modulate TrkB in the presence of BDNF, a situation more likely to occur in the physiological conditions in vivo. We thus turned to a more sensitive assay using radiolabeled BDNF (I¹²⁵-BDNF). The neuronal cultures were incubated at 37°C with I¹²⁵-BDNF (5 × 10⁻¹¹ M) with or without cold BDNF (5 × 10⁻⁸ M) for 30 min. Surface BDNF receptors were determined by the amount of I¹²⁵-BDNF that can be washed off from the cell surface by mild acid (0.2 M acetic acid, 0.5 M NaCl, pH 2.2). Surface binding of I¹²⁵-BDNF was reduced by 80–90% when an excess amount of cold BDNF was added to the cultures (data not shown). Thus, the binding was specifically mediated by BDNF receptors. Stimulation of the hippocampal neurons with TBS reliably elicited an increase in surface binding of I¹²⁵-BDNF (Fig. 2). Compared with neurons stimulated with TBS alone, I¹²⁵-BDNF surface binding was significantly reduced in neurons stimulated with TBS plus TTX, which completely blocked action potentials in these neurons (Fig. 2 A). The glutamate receptor antagonists kyn or CNQX/MK801 also blocked the effect of TBS (Fig. 2 A), suggesting that the excitatory synaptic transmission is required for TBS-induced increase in surface TrkB.

As with the biotinylation experiments, the enhancement of BDNF receptor binding was dependent on high frequency tetanic stimulation. TBS reliably elicited an increase in I¹²⁵-BDNF surface binding as compared with nonstimulated controls (Fig. 2 B). Another tetanic stimulation (three times, 100 Hz, 1 s every 10 min), which elicited a train of high frequency action potentials, resulted in an increase in BDNF binding similar to the result from TBS (Fig. 2 B). In contrast, low frequency stimulation, such as the LTD-inducing stimuli (5 Hz, 4 min) or a constant low frequency train (0.16 Hz), had no effect on I¹²⁵-BDNF surface binding (Fig. 2 B). It is worth pointing out that the same number of pulses was delivered during 30 min of stimulation in both the 100 Hz tetanic stimulation and the 0.16 Hz stimulation. Thus, the modulation of surface BDNF receptors appears to depend on the stimulation frequency, rather than the number of pulses. Again, simple depolarization induced by high K⁺ had little effect on surface binding (Fig. 2 B).

An immediate consequence of tetanus-induced neuronal activity is Ca²⁺ influx through voltage-gated Ca²⁺ channels or NMDA receptors. To determine the mechanisms underlying TBS-induced increase in the surface expression of TrkB, we studied effects of a number of manipulations known to interfere with Ca²⁺ influx. Using the biotinylation assay, we found that blockade of Ca²⁺ influx by the NMDA receptor blocker MK801 (80 μM) markedly reduced the amount of both full-length and truncated TrkB receptors on the surface of hippocampal neurons stimulated with TBS (Fig. 3 A). Inhibition of Ca²⁺ influx by the general Ca²⁺ channel blockers Cd²⁺ (0.2 mM) had similar effects (Fig. 3 A). These results were further confirmed by the I¹²⁵-BDNF surface binding assays. The surface binding was reduced when TBS was applied together with the general Ca²⁺ channel blockers Cd²⁺ (Fig. 3 B) or Co²⁺ (3 mM, not shown), or NMDA antagonists MK801 (80 μM; Fig. 3 B) or 2-amino-5-phosphonovalerate (50 μM; data not shown). Moreover, surface BDNF receptors were significantly reduced in neurons stimulated by TBS in Ca²⁺-free medium, as compared with that in regular medium (Fig. 3 B). Thus, the TBS modulation of surface expression of BDNF receptors appears to depend on Ca²⁺ influx through voltage-gated Ca²⁺ channels and/or NMDA receptors.

To determine the changes in the distribution of TrkB receptors on the cell surface, we performed immunocytochemistry under nonpermeable (no detergent) conditions. Cultured hippocampal neurons were fixed in 2% paraformaldehyde for 3 min, and TrkB antibodies were incubated with the fixed cells in buffers containing no detergent. Under these conditions, an antibody against intracellular domains of the TrkB did not stain hippocampal neurons, suggesting that antibodies or proteins can not penetrate the cells (Fig. 4A and Fig. B). The same antibody was able to detect a substantial amount of TrkB if the staining was performed in the presence of detergent (Fig. 4 C). Under the nonpermeable (no detergent) conditions, surface TrkB were detected using an antibody against extracellular domains of the TrkB. In cultures stimulated with TBS, many TrkB receptors were found on the surface of the cell body. More importantly, a large number of TrkB receptors were distributed along the neuronal processes (Fig. 4 D). In contrast, TrkB receptors were mainly clustered on the cell body and there were very few receptors on the neuronal processes in cultures stimulated with TBS in the presence of kyn and Cd²⁺ (Fig. 4 E). The reduction in surface TrkB in the cultures treated with kyn and Cd²⁺ was not due to a decrease in the number of neuronal processes, which were obviously observed under phase contrast microscopy (data not shown). To better visualize the surface TrkB, we used confocal microscopy. Thin section (2 μm) confocal images revealed a substantial increase in the amount of surface TrkB receptors in active (TBS alone) neurons as compared with the inactive (TBS plus kyn and Cd²⁺) neurons (Fig. 4F and Fig. G). These experiments were repeated many times and striking differences in surface TrkB were always observed between active and inactive neurons. Unstimulated neurons also exhibited less surface TrkB receptors than stimulated neurons (data not shown).

To further investigate the changes in the TrkB receptors inside the cells, we stained the hippocampal neurons under permeable (with detergent) conditions using the antibody against the intracellular domain of TrkB. Both surface and intracellular TrkB were detected. In the stimulated cultures, it appeared that majority of TrkB receptors were on the cell membrane (arrows) and only small amounts of the receptors were inside the cells (Fig. 5 A). Significantly fewer TrkB receptors were found on the cell surface but a lot more receptors were detected inside cells (arrowheads) in cultures stimulated with TBS in the presence of kyn and Cd²⁺ (Fig. 5 B). Confocal microscopy was again used to better separate surface and cytoplasmic TrkB. Indeed, active neurons exhibited a “ring” pattern of staining in sections across the middle of the cell body region, with a lot of TrkB receptors on the cell surface but very little in the cytoplasm (Fig. 5 C). In contrast, a great deal of cytoplasmic TrkB was observed in inactive neurons in similar sections (Fig. 5 D). These results further support the notion that high frequency neuronal activity facilitates the insertion of TrkB receptor into the cell surface.

Ca²⁺ influx is known to activate CaMKII, which has been implicated in the activity-dependent insertion of AMPA-type glutamate receptors into the postsynaptic membrane during LTP (Hayashi et al. 2000). To determine whether CaMKII is also involved in the insertion of the tyrosine kinase receptor TrkB, we measured the amount of surface TrkB in cultures stimulated with TBS in the presence or absence of CaMKII inhibitors. Biotinylation experiments demonstrated that inhibition of CaMKII by either KN62 or KN93 significantly reduced the amount of TrkB receptors in cultures stimulated with TBS, whereas inactive compound KN92 had no effect (Fig. 6A and Fig. B). KN62 and KN93 also significantly inhibited BDNF surface binding on hippocampal neurons (Fig. 6 C). Since KN62 and KN93 have been shown to inhibit other CaM kinases and may cause some nonspecific effects in certain conditions, we used AIP, a peptide known to selectively inhibit CaMKII (Ishida et al. 1995). The NH₂ terminus of AIP or a control peptide with scrambled sequence (AIPscm) was fused to the antennapedia homeopeptide (antp, 16 residues) derived from antennapedia gene to facilitate the translocation of the peptide across the cell membrane of hippocampal neurons (Prochiantz 1996; Passafaro et al. 1999). To determine whether the peptides can penetrate into the hippocampal neurons, we labeled the peptides with biotin, and treated the cells with the biotinylated peptides for a few hours. Cy3-conjugated streptavidin detected the biotinylated peptides inside the hippocampal neurons 3 h after peptide incubation (data not shown). In cultures stimulated with TBS in the presence of antp-AIP, significantly lower levels of surface TrkB were detected as compared with those in cultures stimulated with TBS alone (Fig. 6A and Fig. B). The control peptide antp-AIPscm had no effect (Fig. 6A and Fig. B). Similar results were obtained using the I¹²⁵-BDNF surface binding assay (Fig. 6 C). These results strongly suggest the involvement of CaMKII in the activity-modulation of surface TrkB.

A critical but unresolved question in the neurotrophin research is how a diffusible molecule such as BDNF achieves preferential regulation of active neurons or synapses. In our study, we have investigated whether neuronal responsiveness to BDNF is dependent on, or modified by, neuronal activity. Using three independent approaches, we demonstrate that several forms of tetanic stimulation, but not low frequency stimulation or simple depolarization, promotes the insertion of the BDNF receptor TrkB into the cell surface of hippocampal neurons. We also show that excitatory synaptic transmission, Ca²⁺ influx, and activation of CaMKII are important for the cell membrane insertion of TrkB. Thus, activity-dependent increase in the number of surface TrkB receptors may explain why BDNF acts preferentially on active neurons. This study reveals a novel mechanism by which neurotrophin signaling and function may be regulated, and provides a potential link between activity-dependent and BDNF-induced modulation of neuronal and synaptic function in the hippocampus.

In this study, hippocampal neurons were grown for 11–14 d. Electrophysiological recording indicated that these neurons were well connected synaptically (data not shown). We show that excitatory synaptic activity plays an important role in regulating surface expression of the TrkB receptor tyrosine kinase. Electric stimulation is a more physiological form of stimulation that has been successfully used to study activity-dependent regulation of signal transduction and gene transcription in cultured hippocampal neurons (Bito et al. 1996; Deisseroth et al. 1996; Fields et al. 1997). LTP-inducing tetanic stimuli enhanced surface expression of TrkB, whereas blockade of excitatory synaptic transmission inhibited the tetanus-induced insertion. Immunocytochemical studies demonstrate that the increase in surface TrkB induced by TBS occurred mostly on neuronal processes rather than cell bodies. Remarkably, low frequency stimulation such as those used to induce LTD, or continuous 0.16 Hz (which delivers the same number of pulses as the 100 Hz tetanus), had no effect. These results suggest that temporal pattern of neuronal activity and the kinetics of changes in intracellular Ca²⁺ concentrations, rather than the number of action potentials or the total amount of Ca²⁺ influx, are the critical factors for the activity-dependent insertion of TrkB receptors. Consistent with this idea, simple depolarization by high K⁺ (Fig. 1 and Fig. 2) or veratridine (data not shown) had no effect on the number of surface TrkB. Using freshly dissociated retinal ganglion cells as a model, Barres and colleagues demonstrated that depolarization by high K⁺ or glutamate agonists increases the number of cells expressing surface TrkB (Meyer-Franke et al. 1998). The mechanisms underlying the apparent discrepancy remain unclear. A simple explanation is that different types of neurons may use different mechanisms to regulate surface expression of TrkB. In the retinal ganglion cells, simple depolarization by high K⁺ may increase intracellular cAMP concentrations, which in turn facilitate the incorporation of TrkB into the surface through a number of unknown steps (Meyer-Franke et al. 1998). In the hippocampal neurons, high frequency stimulation may induce Ca²⁺ influx that is qualitatively different from that induced by high K⁺, leading to the activation of CaMKII. Alternatively, freshly dissociated retinal ganglion neurons and synaptically connected hippocampal neurons (grown for 11–14 d) may respond differently to high K⁺. In retinal ganglion cells, high K⁺ may be sufficient to generate the intracellular signals needed to facilitate surface expression of TrkB (Meyer-Franke et al. 1998). In the hippocampal neurons, however, high K⁺ could not produce the specific temporal pattern of neuronal activity and Ca²⁺ influx and subsequent CaMKII activation required for the modulation.

Our results suggest that the modulation of TrkB receptors by tetanic stimulation is mediated, at least in part, by high frequency excitatory synaptic transmission. We have also demonstrated that the activity-dependent modulation of TrkB requires Ca²⁺ influx. Since Ca²⁺-free medium and Cd²⁺ not only prevent Ca²⁺ influx into the postsynaptic neurons through Ca²⁺ channels but also inhibit transmitter release from presynaptic terminals, it is unclear whether blockade of Ca²⁺ influx directly affects the insertion of TrkB receptor into the cell surface, or indirectly by blocking excitatory synaptic transmission. However, blockade of NMDA receptors, which are primarily localized in the postsynaptic cells rather than presynaptic nerve terminals, also attenuates surface expression of TrkB (Fig. 3). Moreover, CaMKII appears to be involved in the activity-dependent insertion of TrkB (Fig. 6). Given that CaMKII has been shown to be important for the insertion of AMPA-type receptors postsynaptically during hippocampal LTP (Hayashi et al. 2000), it is conceivable that similar postsynaptic mechanisms may be used for the insertion of the tyrosine kinase receptor TrkB. It is important to point out, however, that tetanic stimulation does not increase all surface molecules. The surface p75NR is not increased in neurons stimulated with TBS (Fig. 1 D).

One of the remarkable features of the nervous system is that neuronal activity can modulate synaptic efficacy and connectivity in a local and synapse-specific manner (Stent 1973; Goodman and Shatz 1993; Katz and Shatz 1996; Constantine-Paton et al. 1990). Recent studies strongly implicate a role of BDNF in activity-dependent synaptic modulation, such as the formation of ocular dominance columns in the visual cortex and hippocampal LTP (Thoenen 1995; Lu and Chow 1999; McAllister et al. 1999). It is important to understand how diffusible factors such as BDNF achieve local and synapse-specific modulation, and how BDNF strengthens active synapses without affecting their neighbors. One such mechanism would be a localized secretion of BDNF at the site of active synapses. Although there is some evidence for an activity-dependent secretion of BDNF (Wang and Poo 1997; Goodman et al. 1996; Heymach et al. 1996), so far local or synapse-specific secretion of any neurotrophins has not been demonstrated. It is difficult to imagine that locally secreted factors would not spread to their neighboring, less active synapses. Our results demonstrate an alternative and more practical strategy. Active neurons may respond better to BDNF, and this is achieved by an activity-dependent control of the number of TrkB receptors on the cell surface. These results provide a molecular basis for the facilitation of BDNF-induced synaptic potentiation when coupled to presynaptic depolarization at the neuromuscular junction (Boulanger and Poo 1999), and the restricted action of BDNF on highly active synapses in the hippocampus (Gottschalk et al. 1998). In this context, it is important to note that the tetanic stimuli such as TBS or tetanic stimulation were capable of modulating TrkB receptors, whereas low frequency stimulation was not. Since all of our experiments were done using cultured neurons, their relevance to the BDNF modulation of hippocampal synaptic plasticity in vivo has yet to be established. Nevertheless, activity-dependent enhancement of the number of surface TrkB receptor may define an important mechanism by which the specificity of BDNF modulation is achieved.

The results in our study may have general implications in the cell biology of tyrosine kinase receptors. First, we have demonstrated an activity-dependent increase in the number of surface TrkB receptors in the hippocampal neurons. This is due to an increase in the insertion of TrkB receptors into the neuronal cell surface, rather than an increase in TrkB synthesis or decrease of TrkB internalization. The mechanisms underlying membrane insertion of TrkB receptors, and tyrosine kinase receptors in general, are largely unexplored. This study may trigger further interests in investigating the mechanisms for membrane insertion of tyrosine kinase receptors. It will be interesting to examine whether molecules important for vesicle fusion, such as NSF and SNAP, are involved in the delivery of tyrosine kinases onto cell membrane. Second, we show that the membrane insertion of the TrkB receptors is enhanced by Ca²⁺ influx. To our knowledge, this is the first report for Ca²⁺-dependent modulation of the number of surface tyrosine kinase receptors. Thus, our results suggest a novel mechanism for cross-talk between Ca²⁺ and tyrosine kinase signaling pathways. Whether the tyrosine kinase activity of the TrkB receptors can be regulated by intracellular Ca²⁺ is an interesting topic for future study. Finally, CaMKII has recently been implicated in the insertion of AMPA-type glutamate receptors onto the postsynaptic membrane of hippocampal neurons (Hayashi et al. 2000). Our study demonstrates that similar mechanisms are used for the tetanus-induced increase in the tyrosine kinase receptor TrkB on the surface of hippocampal neurons. It remains to be established whether CaMKII also regulates the membrane insertion of other tyrosine kinases in neurons and in other cell types.

We thank Dr. Phillip Nelson for his valuable advice throughout this work, Drs. Doug Fields, Serena Dudek, Lin Mei, and Hao-Chia Chen, as well as the members of the Lu laboratory for critical comments on the manuscript. We also thank Regeneron Pharmaceuticals for providing recombinant BDNF.

This work is supported in part by a grant from Shanghai Science and Technology Commission and National Natural Science Foundation of China.