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The Journal of Neuroscience, March 10, 2004, 24(10):2380-2393; doi:10.1523/JNEUROSCI.4112-03.2004
Previous Article | Next Article
Development/Plasticity/Repair
Postsynaptic TrkB-Mediated Signaling Modulates Excitatory and Inhibitory Neurotransmitter Receptor Clustering at Hippocampal Synapses
Sarina B. Elmariah,1 Mark A. Crumling,2 Thomas D. Parsons,2 andRita J. Balice-Gordon1 1Department of Neuroscience, University of Pennsylvania School of Medicine, and 2Department of Clinical Science, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania 19104
Abstract
Top
Abstract
Introduction
Tyrosine receptor kinase B (TrkB)-mediated signaling modulates synaptic structure and strength in hippocampal and other neurons, but the underlying mechanisms are poorly understood. Full-length and truncated TrkB are diffusely distributed throughout the dendrites and soma of rat hippocampal neurons grown in vitro. Manipulation of TrkB-mediated signaling resulted in dramatic changes in the number and synaptic localization of postsynaptic NMDA receptor (NMDAR) and GABAA receptor (GABAAR) clusters. BDNF treatment resulted in an increase in the number of NMDAR and GABAAR clusters and increased the proportion of clusters apposed to presynaptic terminals. Downregulation of TrkB signaling resulted in a decrease in receptor cluster number and synaptic localization. Examination of the time course of the effects of BDNF on receptor clusters showed that the increase in GABAAR clusters preceded the increase in NMDAR clusters by at least 12 hr. Moreover, the TrkB-mediated effects on NMDAR clusters were dependent on GABAAR activation. Although TTX, APV, and CNQX treatment had no effect, blockade of GABAARs with bicuculline abolished the BDNF-mediated increase in NMDAR cluster number and synaptic localization. In contrast, application of exogenous GABA prevented the decrease in NMDAR clusters induced by BDNF scavenging. Together, these results suggest that TrkB-mediated signaling modulates the clustering of postsynaptic GABAARs and that receptor activity is required for a subsequent upregulation of NMDAR clusters. Therefore, TrkB-mediated effects on postsynaptic neurotransmitter clusters may be part of a mechanism that balances inhibitory and excitatory synaptic transmission in developing neural circuits.
Key words: neurotrophin; TrkB; NMDA receptor; GABAA receptor; activity; synapse formation
Introduction
Neurotrophins and the family of tyrosine kinase receptors (Trk) [Nerve Growth Factor (NGF)/TrkA, Brain derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4)/TrkB, NT-3/TrkC] play many roles during nervous system development, regulating neuronal survival, morphology, and synaptic connectivity (for review, see Schuman, 1999; Cohen-Cory, 2002
Several lines of evidence from in vivo and in vitro experiments suggest that neurotrophins can modulate neuronal excitation and inhibition by acting presynaptically as well as postsynaptically. Acute BDNF-induced enhancement of glutamatergic synaptic transmission among hippocampal neurons has been shown to be, in part, attributable to an increase in presynaptic glutamate release (Carmignoto and Vicini, 1992
Here, we asked whether TrkB-mediated signaling modulates postsynaptic neurotransmitter receptor clusters at CNS synapses, using hippocampal synapses in vitro as a model system. We focused on times in vitro when both excitatory and inhibitory synapses are being formed. Our results suggest that TrkB-mediated signaling increases the number and synaptic localization of NMDAR as well as GABAAR clusters, and that the TrkB-mediated effects on NMDARs require GABAAR activity. Thus, TrkB-mediated signaling modulates postsynaptic neurotransmitter receptor clustering as part of a mechanism that balances inhibitory and excitatory synaptic transmission in developing neural circuits.
Materials and Methods
Cell culture. Primary cell cultures were prepared as described previously (Goslin et al., 1988Growth factor and pharmacological treatments. Neurons were treated with 0-50 ng/ml BDNF (Upstate Biotechnology, Lake Placid, NY) or 0-2 µg/ml TrkB-IgG (gift from Regeneron Pharmaceuticals, Tarrytown, NY) at 1-3 d in vitro (DIV) and 8-11 DIV. The time course and dose dependence of the BDNF-mediated effects on NMDAR clusters were evaluated at each age. No changes in the pattern of NMDAR immunoreactivity were observed after treatment at 1-3 DIV (Dalva et al., 2000
Dendrite number and length were measured using interactive software (MetaMorph; Universal Imaging, Downingtown, PA) and were compared between manipulated and control cultures. Treatment with BDNF at 8 DIV for 48 hr produced no change in pyramidal neuron dendritic number or length [number, 8.2 ± 0.2 (40), 8.6 ± 0.3 (42); length, 37.2 ± 1.4, 36 ± 1.8 µm in BDNF-treated and untreated controls, respectively; not significantly different; Student's t test]. Similarly, treatment with TrkB-IgG produced no change in dendritic number or length in pyramidal neurons [number, 9 ± 0.4 (35); length, 36 ± 1.7 µm; not significantly different from untreated controls; Student's t test]. These data are consistent with previous reports in hippocampal neurons maintained in vitro for 7-11 d or longer (Vicario-Abejon et al., 1998
To examine effects of NT-3 or NGF, cultures were treated with different neurotrophin concentrations (0.5-50 ng/ml) and variable treatment durations (1 min to 48 hr) at 1-3 or 8-11 DIV. No effects of NT-3 or NGF on NMDAR or GABAAR cluster formation or maintenance were observed (see Fig. 2B-E; Table 1).
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Figure 2. BDNF increases the number, size, and synaptic localization of NMDAR clusters in hippocampal neurons. Hippocampal neurons at 8 DIV were treated with 0.5-50 ng/ml BDNF for 48 hr and were immunostained at 10 DIV with an antibody against NR1 and SP. A, Treatment with 50 ng/ml BDNF (right) leads to an increase in the number of NMDAR clusters compared with untreated control neurons (left). Scale bar, 10 µm. Areas within white boxes are shown below at higher magnification. Scale bar, 2 µm. B, Quantification of dose-dependent BDNF-mediated increase in NMDAR clusters per 20 µm dendrite segment. Asterisk indicates significant increase compared with no treatment (Table 1). C, Quantification of dose-dependent BDNF-mediated increase in NMDAR cluster size. Cumulative histograms of NMDAR cluster size for each treatment are shown. Asterisk indicates significant increase compared with no treatment (Table 1). D, Treatment with 50 ng/ml BDNF (middle left) leads to an increase in the synaptic localization of NMDAR clusters compared with untreated control neurons (left). No changes in NMDAR number or synaptic localization were observed after treatment with NT-3 (middle, right) or NGF (right). Top, NMDAR clusters visualized with antibody against NR1. Middle, Presynaptic terminals visualized with an antibody against SP. Bottom, Overlay of NMDAR (red) and SP (green). Scale bar, 2 µm. E, Quantification of BDNF-mediated increase in synaptic localization of NMDAR clusters. Asterisk indicates significant increase compared with no treatment (Table 1). F, AMPARs are distributed diffusely in the dendrites of pyramidal neurons; few AMPAR clusters are present (arrowhead), and none are synaptically localized. Treatment with 50 ng/ml BDNF (right) does not alter AMPAR cluster number or synaptic localization compared with no treatment (left). Top, AMPAR clusters were visualized with an antibody against GluR1. Middle, Presynaptic terminals with an antibody against SP. Bottom, Overlay of GluR1 (red) and SP (green). Scale bar, 2 µm. G, Quantification of the number of AMPAR clusters in BDNF-treated and control neurons. No significant differences were observed between BDNF-treated and control neurons. H, Quantification of synaptic localization of AMPAR clusters. No significant differences were observed between BDNF-treated and control neurons.
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Table 1. TrkB-mediated modulation of NMDAR cluster number, size, and synaptic localization
For activity manipulation experiments, dilutions of APV (Sigma, St. Louis, MO), bicuculline methiodide (Sigma), CNQX (Sigma), GABA (Sigma), or TTX (Calbiochem, La Jolla, CA) were made fresh on the day of the experiment and added during delivery of neurotrophins or IgG proteins. Dose-response curves were performed for the following pharmacological agents: TTX (0-2.0 µM), bicuculline (0-50 µM), and GABA (0-10 mM). For experiments requiring selective blockade of excitatory transmission, APV (50 µM) and CNQX (10 µM) were used (cf. Rao and Craig, 1997Delivery of neurotrophin and Trk constructs to hippocampal neurons. Recombinant, replication-defective adenoviruses, encoding FL or truncated (t) Trks (TrkB.FL, TrkC.FL, t-TrkA, TrkB.t1, t-TrkC) and enhanced green fluorescent protein (GFP; Clontech, Palo Alto, CA), under control of the cytomegalovirus (CMV) promoter and an internal ribosomal entry site sequence (Gonzalez et al., 1999
Immunostaining and confocal microscopy. In experiments in which anti-NMDAR, PSD-95 (postsynaptic density-95), and synaptophysin (SP) immunostaining were performed, hippocampal neurons were fixed and permeabilized with methanol at -20°C for 10 min. For anti-GABAAR-
2/3 and GAD immunostaining, neurons were fixed in 4% paraformaldehyde and 4% sucrose at room temperature for 15 min. For all other experiments, neurons were fixed in 4% paraformaldehyde for 4 min, rinsed in Optimem (Invitrogen) with 0.2% Triton X-100, and blocked in Optimem containing 2% BSA. To examine surface TrkB expression, primary antibody diluted in Optimem with 2% BSA was applied to neurons before fixation. Cells were subsequently fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and incubated with an anti-synaptophysin antibody. Double and triple labeling were performed with combinations of primary antibodies: anti-NMDAR1 (1:500, rabbit polyclonal; Sigma),
Western analysis. After neurotrophin manipulation, total cell lysates were harvested into Laemmli buffer from low-density hippocampal cultures at 10 DIV. After SDS-PAGE, samples were transferred to nitrocellulose membranes and probed for antibodies to NR1 (1:2000, rabbit polyclonal; Chemicon) or, as a loading control, Kv1.2 (1:5000, monoclonal; Upstate Biotechnology). AP-conjugated goat anti-rabbit or anti-mouse antisera (1:5000; Applied Biosystems, Foster City, CA) were used, and signals were visualized using chemiluminescence (WesternStar detection system; Applied Biosystems). Films were digitally scanned, and the signal was quantified using MetaMorph (Universal Imaging) software.
Quantification and statistical analysis. For each condition, a minimum of 6-10 neurons was examined on each of three coverslips in three to six independent experiments. Neurons used for analysis were randomly selected. Pyramidal neurons were distinguished from interneurons by pyramidal morphology and lack of anti-GAD immunoreactivity. In all experiments, the number, size, and synaptic localization of NMDARs, GABAARs, or other postsynaptic components were determined from confocal images using interactive software (MetaMorph, Universal Imaging). Confocal images of neurons were thresholded, and the number and area of individual clusters were determined. Values are presented as mean ± SEM (number of cells).
Values for cluster number were compared using the Kruskal-Wallis nonparametric ANOVA test followed by Dunn's pairwise multiple comparison test. Cluster size values were plotted as cumulative histograms for each treatment, and distributions were compared using the Kolmolgorov-Smirnov two-sample test. To quantify synaptic localization, NMDAR or PSD-95 clusters with >30-40% pixel overlap with synaptophysin clusters were considered synaptic. The percentage of synaptic localization in manipulated and control cultures was compared using Student's t test.
Electrophysiology. Patch-clamp recordings were performed at room temperature (20-25°C) on pyramidal neurons at 8-11 DIV and 3-4 weeks in vitro. Gigaseal recordings were performed using borosilicate patch pipettes with resistances of 2-4 M
. Cellular responses were amplified (Axopatch 200B, Axon Instruments, Foster City, CA; or EPC10, Heka Elektronik, Lambrecht/Pfalz, Germany), low-pass filtered at 2-2.9 kHz, and sampled at 10 kHz. Coverslips were placed in a flow-through chamber driven by a peristaltic pump that allowed perfusion and washout of drugs. The bath solution contained the following (in mM): 145 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 8 glucose, pH 7.4 (300-310 mOsm). To record IPSCs in the whole-cell configuration, the bath solution contained APV (50 µM) and CNQX (10 µM) to block NMDA and AMPA receptor currents, respectively, and the internal pipette solution contained the following (in mM): 152 KCl, 1 CaCl2, 4 Mg-ATP, 1 EGTA, 10 HEPES, and 30 glucose, pH 7.4 (290-305 mOsm). To record EPSCs, the bath solution contained bicuculline (50 µM) to block GABAAR currents and gluconate substituted for Cl- in the internal pipette solution (in mM): 130 potassium gluconate and 10 KCl, pH 7.4 (290-305 mOsm). Membrane potential was held at -70 mV.
To detect spontaneous action potential activity with minimal cellular perturbation, action currents were recorded in the cell-attached configuration. The patch pipette contained bath solution (without drugs) and was voltage clamped at 0 mV after achieving a gigaseal. In the resulting records, current flowing into the patch pipette is plotted as a negative deflection (see Fig. 7A).
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Figure 7. Low levels of spontaneous activity are present during the first week in vitro. Patch-clamp recordings were performed on hippocampal neurons at 8-11 DIV to examine spontaneous and evoked activity. A, Representative traces from a neuron in cell-attached voltage-clamp mode. Spontaneous action currents were observed at a low frequency. Calibration: 20 pA, 1 sec. B, Depolarizing current steps (20 pA) elicited action potentials (top) that were abolished by the Na+ channel blocker TTX (1 µM; middle) and subsequently recovered after washout (bottom). Calibration: 40 mV, 100 msec. C, Representative recordings from three neurons in whole-cell voltage-clamp mode. Spontaneous IPSCs were observed at a low frequency in some neurons (top, middle) and were not observed in the majority of neurons (bottom). Calibration: 40 pA, 1 sec. D, Focal application of 5 µM GABA elicited GABAA receptor-mediated currents (top) that were blocked by 50 µM bicuculline (middle) and recovered after washout (bottom). Calibration: 200 pA, 2 sec.
To evaluate whether functional GABAA and NMDA receptors were expressed at 8-11 DIV and to validate the effects of pharmacological agents used in the morphological studies, neurotransmitters were focally applied to neuron somata in the presence and absence of receptor antagonists. Application of GABA (5-100 µM) evoked currents that were reversibly blocked by 50 µM bicuculline (see Fig. 7D). In bath solution containing 10 µM CNQX, application of glutamate (5-100 µM) evoked NMDAR-mediated currents that were attenuated by 50 µM APV (data not shown) (cf. Brunig et al., 2001To determine whether GABA was excitatory or inhibitory, perforated patch recordings with gramicidin D were performed to determine the GABAA reversal potential at 4 and 8-11 DIV. Patch pipettes were tip-filled with internal solution without gramicidin and then backfilled with internal solution containing gramicidin (20 µg/ml). The internal pipette solution contained the following (in mM): 154 KCl, 9 NaCl, 1 MgCl2, 0.2 EGTA, and 10 HEPES, pH 7.4 (290-305 mOsm). The bath contained 50 µM APV, 10 µM CNQX, and 25 µM saclofen, a GABAB receptor antagonist. To minimize voltage errors induced by high series resistance, recordings were performed in current-clamp configuration, and only those recordings with series resistances <100 M
Results
TrkB localization in hippocampal neurons in vitro
To study the effects of neurotrophin signaling on neurotransmitter receptor cluster formation and maintenance, we first characterized the distribution of NMDARs and GABAARs in low-density embryonic rat hippocampal neuronal cultures maintained in defined medium for 3-4 weeks. Immunostaining showed that NMDAR and GABAAR clusters gradually increase in number and become localized to postsynaptic densities and synaptic sites during the first 3 weeks in vitro, consistent with previous reports (Rao and Craig, 199710-15% of NMDAR clusters and 35-40% of GABAAR clusters were apposed to presynaptic terminals immunostained with antibodies against SP or SV2 (Fig. 1A). By the end of the second week in vitro, approximately one-third of NMDAR clusters and approximately two-thirds of GABAAR clusters were synaptically localized. Thus, to determine whether TrkB signaling plays a role in the clustering or maintenance of neurotransmitter receptors, we examined cultures during the highly dynamic period of synaptogenesis, which occurs between the first and second weeks in vitro.
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Figure 1. Localization of neurotransmitter receptor clusters and TrkB in hippocampal neurons in vitro. Hippocampal neurons at 10 DIV were immunostained and analyzed with confocal microscopy. A, Immunostaining with an antibody against NR1 (red) shows that the majority of NMDAR clusters in the dendrites of pyramidal neurons are colocalized with PSD-95 (green), a molecule that is localized to postsynaptic specializations (left), whereas few NMDAR clusters are localized to synapses, as visualized with an antibody against SP (green), a molecule localized to synaptic vesicles (middle). Immunostaining for the GABAAR subunit
During this time period in vitro, immunostaining using an antibody against all TrkB isoforms showed that the receptor is expressed in all hippocampal neurons, present both diffusely and in clusters, and differentially distributed among dendrites and axons. Double labeling using antibodies against TrkB and the dendritic microtubule associated protein MAP2 showed that TrkB is distributed diffusely in somata and dendrites, with some TrkB clustered in the dendritic and somatic membrane (Fig. 1B). TrkB was also localized to the proximal portions of some axons, visualized using an antibody against tau, but was largely absent from growth cones and distal axon compartments (Fig. 1B). A similar localization was observed using antibodies specific to full-length TrkB containing the intracellular tyrosine kinase domain (Fig. 1B). In these cultures, similar distributions were observed in hippocampal neurons with pyramidal morphology as well as in GAD+ interneurons. As reported previously (Muragaki et al., 1995TrkB signaling modulates the number and synaptic localization of NMDAR clusters
We first examined the effects of TrkB-mediated signaling on NMDAR clusters in cultures treated at 8-10 DIV with BDNF for 48 hr. BDNF treatment increased the number, size, and synaptic localization of NMDAR clusters in a dose-dependent manner (Fig. 2). A comparison of the number of NMDAR clusters in randomly selected pyramidal neurons from control and BDNF-treated cultures showed that NMDAR cluster density increased nearly twofold, from a mean of 5.0 ± 0.4 clusters per 20 µm dendritic segment in untreated neurons to 9.4 ± 0.7 clusters per segment in neurons treated with 50 ng/ml BDNF (Fig. 2B, Table 1). After 48 hr of BDNF treatment, the size of NMDAR clusters was also increasedIn addition to effects on cluster number and size, BDNF treatment also affected the localization of NMDAR clusters with other synaptic components. After BDNF treatment, a significantly higher proportion of NMDAR clusters was apposed to SP-labeled presynaptic terminals (Fig. 2D,E; Table 1) and thus synaptically localized. Surprisingly, no change in the number of SP-stained terminals was observed after BDNF treatment [5.6 ± 0.3 per 20 µm dendritic segment (86 neurons); 4.9 ± 0.7 in untreated neurons (88); not significantly different; Student's t test]. This finding indicates that the increase in synaptic NMDAR clusters was not simply attributable to an increase in presynaptic boutons and thus synapses. Rather, this observation suggests that BDNF induces a dynamic change in the localization of NMDAR clusters, increasing the number of NMDARs apposed to presynaptic terminals. In contrast, the number of postsynaptic densities, labeled using an antibody against PSD-95, increased
Immunostaining at 8-10 DIV for AMPA receptor subunits GluR1 and GluR2 showed that AMPARs were diffusely distributed in dendrites, with few AMPAR clusters formed at this stage of development (Fig. 2F). In contrast to the robust upregulation of NMDAR clusters, no changes were observed in the number or synaptic localization of AMPAR clusters after treatment with BDNF (Fig. 2F-H), suggesting that BDNF specifically modulates NMDAR clusters at excitatory synapses.
To determine whether the effect of BDNF on NMDAR cluster number, size, and synaptic localization are specific to TrkB signaling, the effects of TrkC or TrkA activation were examined in parallel experiments. TrkC, which binds the neurotrophin NT-3 with high affinity, is expressed in hippocampal neurons in vitro and in vivo, but little TrkA is expressed (Martinez et al., 1998
To determine whether TrkB-mediated modulation of NMDAR clusters persists in older cultures after synapses have been formed, cultures were treated at 15-17 and 22-24 DIV with BDNF for 48 hr. BDNF treatment at both 2 and 3 weeks in vitro induced an increase in NMDAR cluster number [10.6 ± 0.5 per 20 µm dendritic segment (63); 5.9 ± 0.7 in untreated neurons (50); p < 0.001; Student's t test] and synaptic localization [71.8 ± 2.3 per 20 µm dendritic segment (63); 32.5 ± 5.0 in untreated neurons (50); p < 0.001; Student's t test] similar to that observed at 1 week in vitro. Moreover, no changes were observed in NMDAR clusters after treatment with NT-3 in cultures examined after 2-3 weeks in vitro [6.4 ± 0.7 clusters per 20 µm dendritic segment; 25.0 ± 4.3% synaptic localization (37)]. Together, these results demonstrate that TrkB signaling modulates NMDAR cluster number and synaptic localization during at least the first 3 weeks in vitro. In subsequent experiments, we focused on TrkB-mediated signaling during synaptogenesis between 8 and 10 DIV when the effects of BDNF on neurotransmitter cluster number and synaptic localization first become apparent.
Postsynaptic TrkB signaling is necessary for NMDAR cluster maintenance
To determine whether TrkB signaling is necessary for the maintenance of NMDAR clusters at hippocampal synapses, levels of endogenous BDNF were reduced by adding a TrkB-IgG fusion protein to the culture medium. This protein binds and sequesters neurotrophin, preventing its binding to and activation of surface TrkB receptors (cf. Binder et al., 1999
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Figure 3. Scavenging endogenous BDNF decreases the number, size, and synaptic localization of NMDAR clusters in hippocampal neurons. Hippocampal neurons at 8 DIV were treated with 0.5-2.0 µg/ml of TrkB-IgG or control IgG for 48 hr and were analyzed after immunostaining at 10 DIV with an antibody against NR1 and SP. A, Treatment with 2.0 µg/ml TrkB-IgG (top) leads to a decrease in the number of NMDAR clusters compared with neurons treated with IgG (bottom). Scale bar, 10 µm. Areas within white boxes are shown below at higher magnification. Scale bar, 2 µm. B, Quantification of TrkB-IgG dose-dependent decrease in NMDAR clusters per 20 µm dendrite segment. Asterisk indicates significant decrease compared with no treatment (Table 1). C, Quantification of TrkB-IgG dose-dependent decrease in NMDAR cluster size. Cumulative histograms of NMDAR cluster size for each treatment are shown. Asterisk indicates significant decrease compared with no treatment (Table 1). D, Treatment with 2 µg/ml TrkB-IgG (right) leads to a decrease in the synaptic localization of intact NMDAR clusters compared with untreated control neurons (left). Top, NMDAR clusters visualized with antibody against NR1. Middle, Presynaptic terminals visualized with an antibody against SP. Bottom, Overlay of NMDAR (red) and SP (green). Scale bar, 2 µm. E, Quantification of TrkB-IgG-induced decrease in the synaptic localization of NMDAR clusters compared with untreated control neurons. Asterisk indicates significant decrease compared with no treatment (Table 1).
In addition, NMDAR cluster localization with other synaptic components was altered by scavenging BDNF. Although the number of SP-stained presynaptic terminals observed remained unchanged after TrkB-IgG treatment compared with untreated neurons [4.9 ± 0.3 (88), 4.9 ± 0.7 (88), respectively; not significantly different; Student's t test], a smaller proportion of NMDAR clusters was apposed to presynaptic terminals (Fig. 3D,E; Table 1). No change was observed in the number of PSD-95 clusters per dendritic segment after TrkB-IgG treatment compared with untreated neurons [6.2 ± 0.4 (88), 6.4 ± 0.4 (88), respectively; not significantly different; Student's t test]. However, of the few NMDAR clusters present after scavenging, >95% remained associated with PSD-95. In addition, no changes were observed in the number or synaptic localization of AMPAR clusters per dendritic segment after TrkB-IgG treatment compared with untreated neurons [1.9 ± 0.5 (76), 2.2 ± 0.4 (91), respectively; not significantly different; Student's t test]. These results suggest that in the absence of BDNF, NMDARs are selectively dispersed from clusters and removed from postsynaptic sites.At neuromuscular synapses, postsynaptic TrkB signaling is required to maintain AChR clusters in muscle fiber membranes (Gonzalez et al., 1999
Overexpression of truncated TrkB in hippocampal neurons at 7-10 DIV resulted in a significant, approximately eightfold decrease in the number of NMDAR clusters (Fig. 4A,B; Table 1). NMDAR cluster size was also decreased approximately twofold after overexpression of truncated TrkB (Fig. 4C, Table 1). In addition, few if any of the remaining NMDAR clusters were synaptically localized in neurons overexpressing truncated TrkB (Fig. 4D,E; Table 1), and no change was observed in the number of SP-stained presynaptic boutons in these neurons (Fig. 4D) [5.2 ± 0.3 per 20 µm dendritic segment (36); 5.0 ± 0.5 in uninfected neurons (40); not significantly different; Student's t test]. No changes were observed in NMDAR cluster number or synaptic localization in neighboring uninfected neurons from the same coverslip (Fig. 4A-C). Infection with control adenoviruses encoding LacZ and GFP or truncated TrkA and GFP did not alter the number or size of NMDAR clusters (Fig. 4A-C; Table 1). Moreover, in neurons overexpressing truncated TrkB, decreases in NMDAR cluster number were partially reversed, and decreases in size were completely reversed, with the addition of exogenous BDNF (Fig. 4B,C; Table 1). That the addition of exogenous BDNF could partially rescue the effects on NMDAR clusters resulting from overexpression of truncated TrkB suggests that dominant-negative downregulation of Trk signaling occurs via scavenging of ligand in addition to heterodimerization with endogenous full-length TrkB. Together, these results show that postsynaptic TrkB signaling is necessary for the maintenance of NMDAR clusters at hippocampal synapses.
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Figure 4. Dominant-negative disruption of TrkB signaling decreases the number, size, and synaptic localization of NMDAR clusters. Hippocampal neurons at 7 DIV were infected with recombinant adenoviruses encoding TrkB.t1ha, t-TrkAha, LacZ, and GFP. At 10 DIV, neurons were immunostained with antibodies against NR1 (top), or the epitope tag ha or
TrkB-mediated signaling does not affect expression of NMDARs
Despite the loss of NMDAR clusters, NMDAR immunoreactivity was not abolished after either BDNF scavenging with TrkB-IgG or overexpression of truncated TrkB (Figs. 3, 4). A consistent, qualitative increase in diffuse membrane staining was observed throughout the somata and dendritic processes of a majority of neurons. To determine whether these qualitative changes in the pattern of NMDAR immunoreactivity reflected changes in overall NMDAR expression or changes in the membrane distribution of receptors, cultures were treated with BDNF, TrkB-IgG, or control medium for 48 hr. After treatment, total cell homogenates were collected, and Western blot analysis was performed. No significant changes were observed in NMDAR expression compared with Kv1.2 expression as an internal standard after treatment with BDNF or TrkB-IgG (Fig. 5). These results suggest that BDNF may promote the assembly of NMDARs into clusters. Conversely, in the absence of BDNF, NMDAR clusters may be disassembled or are removed from the cell surface, but expression levels are not appreciably altered. Because the number of PSD-95 clusters does not change after BDNF scavenging, these results suggest that scavenging BDNF disperses NMDARs from clusters or prevents the accumulation of NMDARs at postsynaptic sites but does not affect the integrity of the postsynaptic density. Together, these data suggest that TrkB signaling is necessary for the maintenance of NMDAR clusters and promotes the assembly of existing NMDARs into clusters.
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Figure 5. TrkB signaling modulates the clustering of existing NMDARs in hippocampal neurons. Hippocampal neurons at 8 DIV were incubated with 50 ng/ml BDNF, 2 µg/ml TrkB-IgG, or control medium for 48 hr. Total cell lysates were harvested at 10 DIV. Top, Western blot analysis of total cell homogenate performed using an antibody against NR1. Bottom band is nonspecific and demonstrates loading of each lane. Bottom, Quantification of relative band intensity demonstrates no significant differences across treatments.
TrkB signaling modulates the number and synaptic localization of postsynaptic GABAAR clusters
In the cultures used here, both glutamatergic pyramidal neurons and GABAergic interneurons are present (cf. Rao and Craig, 1997BDNF treatment resulted in an approximately twofold increase in the number of GABAAR clusters compared with untreated control neurons (Fig. 6A,B; Table 2) but had no effect on cluster size (Table 2). BDNF treatment also significantly increased the synaptic localization of GABAAR clusters by approximately twofold (Fig. 6A,C; Table 2). This effect was specific to BDNF, because no change in GABAAR cluster number or size was observed with NT-3 or NGF (data not shown). In contrast, reduction of endogenous BDNF by treatment with TrkB-IgG for 48 hr reduced GABAAR cluster number by
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Figure 6. TrkB-mediated modulation of postsynaptic GABAAR cluster number and synaptic localization precedes modulation of NMDAR clusters. Hippocampal neurons at 8 DIV were treated with 50 ng/ml BDNF for 24-48 hr and were analyzed after immunostaining at 9 or 10 DIV. A, Time course of the effects of BDNF on GABAAR clusters. Treatment with BDNF for 36 hr (middle right) and 48 hr (right) increases the number of GABAAR clusters compared with untreated neurons (left). Top, GABAAR clusters visualized with antibody against GABAA
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Table 2. TrkB modulation of GABAR cluster number, size, and synaptic localization
TrkB-mediated modulation of postsynaptic GABAAR clusters temporally precedes modulation of NMDAR clusters
The data presented above extend previous reports of the effects of BDNF on channel conductance (Rutherford et al., 1997Cultures were treated with BDNF for 24, 36, and 48 hr, and GABAAR and NMDAR cluster number and synaptic localization were analyzed by immunostaining and confocal microscopy. Twenty-four hours of BDNF treatment resulted in a
After 36 hr of BDNF treatment, however, an approximately twofold increase in the number of GABAAR clusters was observed (Fig. 6A,E). At this time, the synaptic localization of GABAAR clusters was also increased by
In contrast, NMDAR cluster number and synaptic localization were not significantly altered after 24-36 hr of BDNF treatment (Fig. 6D-F) or at earlier time points (see Materials and Methods). The BDNF-mediated increase in NMDAR cluster number and synaptic localization was only observed after 48 hr of treatment (Fig. 6D-F). Together, these results show that TrkB-mediated modulation of GABAAR clusters temporally precedes that of NMDAR clusters by
TrkB-mediated modulation of GABAAR and NMDAR clusters is activity dependent
In mature networks with high levels of spontaneous activity, BDNF and TrkB signaling can reverse the effects of action potential blockade with TTX on synaptic structure and function (Rutherford et al., 1997Cell-attached patch-clamp recordings from pyramidal neurons revealed low levels of spontaneous activity at 8-11 DIV. Some neurons fired spontaneous action potentials at an average frequency of
We next determined whether the effects of TrkB on NMDAR and GABAAR clusters required the low levels of spontaneous action potential activity described above by treating neurons for 48 hr with BDNF and 0.5-2.0 µM TTX. In neurons treated with TTX alone, NMDAR cluster number and size were not significantly different compared with untreated neurons (Fig. 8C, Table 1). However, the synaptic localization of NMDARs was significantly increased in TTX-treated compared with untreated neurons, to a level similar to that observed with BDNF plus TTX or BDNF treatment alone (Fig. 8A,C,D; Table 1). Consistent with previous reports of the effects of action potential blockade in 3-4-week-old hippocampal cultures (Rao and Craig, 1997
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Figure 8. Effects of action potential blockade on TrkB-mediated increase in NMDAR and GABAAR cluster number and synaptic localization. Hippocampal neurons at 8 DIV were treated with 50 ng/ml BDNF and 2 µM TTX for 48 hr and were analyzed after immunostaining at 10 DIV. A, The number (top) and synaptic localization (bottom) of NMDAR clusters is unchanged after BDNF plus TTX treatment (right) compared with neurons treated with BDNF alone (left). NMDARs stained with an antibody against NR1 (red) and terminals with SP (green) are shown. Scale bar, 2 µm. B, The number (top) and synaptic localization (bottom) of GABAAR clusters are decreased after BDNF plus TTX treatment (right) compared with neurons treated with BDNF alone (left). GABAARs stained with an antibody against
To evaluate the role of NMDAR activity, neurons were treated with 50 µm APV with or without BDNF. APV alone induced an increase in NMDAR cluster number and synaptic localization compared with untreated controls (Table 1). Moreover, like TTX, APV did not block the BDNF-mediated increase in NMDAR cluster number and synaptic localization (Table 1). AMPAR blockade with 10 µM CNQX had no effect on NMDAR clusters, nor did it prevent the BDNF-induced increase in NMDAR clusters (Table 1). Consistent with previous reports (Rao and Craig, 1997In contrast, in neurons treated with TTX alone, GABAAR cluster number was reduced compared with that observed in untreated neurons (Fig. 8C; Table 2), and the proportion of synaptically localized clusters was unaffected (Fig. 8D, Table 2). Synaptic GABAAR clusters were significantly reduced in neurons treated with BDNF plus TTX compared with neurons treated with BDNF alone (Fig. 8B,C,D; Table 2). These results indicate that TrkB-mediated effects on GABAAR clusters are blocked when action potential activity is abolished by TTX.
The results presented thus far show that action potential blockade prevents the effects of BDNF on GABAAR clusters but can mimic the effects of BDNF on NMDAR clusters. One hypothesis that is consistent with these data is that reducing neuronal excitability, either by action potential blockade or by increasing inhibitory input via the BDNF-mediated increase in synaptically localized GABAAR clusters, leads to a subsequent and perhaps compensatory increase in NMDAR clusters. Assuming that GABA is inhibitory at this age, this hypothesis raises two predictions. First, blockade of GABAAR activity would prevent the BDNF-mediated increase in NMDAR clusters. Second, activation of GABAARs would maintain NMDAR clusters when BDNF is scavenged. These predictions were tested and are described below.
TrkB-mediated modulation of NMDAR clusters requires GABAAR activity
As a first step to evaluate these predictions, perforated-patch recordings were used to determine whether GABA is excitatory or inhibitory at 8-11 DIV (Rivera et al., 1999
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Figure 9. TrkB-mediated modulation of NMDAR clusters requires activity of GABAA receptors. A, Perforated-patch recordings from pyramidal neurons were performed to determine the GABA reversal potential at 4 DIV (left) and 8-11 DIV (middle). The GABA reversal potential decreased from -45.0 ± 2.5 mV at 4 DIV to -69.4 ± 3.8 mV by the end of the first week in vitro (right). B, Hippocampal neurons at 8 DIV were treated with 50 ng/ml BDNF and 0-50 µM bicuculline, a GABAA receptor antagonist, for 48 hr and were analyzed after immunostaining at 10 DIV with an antibody against NR1 and SP. Treatment with BDNF plus 10 µM bicuculline led to a decrease in NMDAR cluster number compared with neurons treated with BDNF alone. Scale bar, 10 µm. Areas within white boxes are shown below at higher magnification. Scale bar, 2 µm. C, Quantification of BDNF plus bicuculline effects on NMDAR clusters per 20 µm dendrite segment. Asterisk indicates significant decrease compared with BDNF only (Table 1). D, Quantification of BDNF plus bicuculline decrease in the synaptic localization of NMDAR clusters compared with neurons treated with BDNF or bicuculline alone. Asterisk indicates significant increase compared with no treatment (Table 1). Synaptic localization after treatment with BDNF plus bicuculline and bicuculline alone was significantly decreased compared with BDNF alone and was not significantly different from no treatment (Table 1). E, Hippocampal neurons at 8 DIV were treated with 2 µg/ml TrkB-IgG and 0-10 mM GABA for 48 hr and were analyzed after immunostaining at 10 DIV with an antibody against NR1 and SP. Treatment with TrkB-IgG plus 100 µM GABA led to an increase in NMDAR cluster number compared with neurons treated with TrkB-IgG alone. Scale bar, 10 µm. Areas within white boxes are shown below at higher magnification. Scale bar, 2 µm. F, Quantification of TrkB-IgG plus GABA effects on NMDAR clusters per 20 µm dendrite segment. Asterisk indicates significant increase compared with TrkB-IgG alone (Table 1). G, Quantification of TrkB-IgG plus GABA induced increase in the synaptic localization of NMDAR clusters compared with neurons treated with TrkB-IgG alone or untreated neurons. Asterisk indicates significant decrease compared with no treatment (Table 1).
To determine whether GABAAR blockade prevents the BDNF-mediated increase in NMDAR clusters, hippocampal neurons were treated for 48 hr with BDNF while inhibitory synaptic transmission was blocked with 0.5-50 µM bicuculline (Fig. 9B-D) (cf. Turrigiano et al., 1998To determine whether activation of GABAARs maintains NMDAR clusters when BDNF is scavenged, hippocampal neurons were treated for 48 hr with 2 µg/ml TrkB-IgG and 100 µM to10 mM of GABA. Treatment with exogenous GABA prevented the decrease in NMDAR clusters observed after treatment with TrkB-IgG alone. The number of NMDAR clusters was significantly greater in neurons treated with TrkB-IgG plus 10 mM GABA than in neurons treated with TrkB-IgG alone (Fig. 9E,F; Table 1). Moreover, treatment with TrkB-IgG plus GABA also prevented the decrease in cluster size and synaptic localization observed with TrkB-IgG treatment alone (Fig. 9G, Table 1). No significant changes in NMDAR cluster number, size, and synaptic localization were observed after treatment with GABA alone (Fig. 9F,G; Table 1). These results show that GABAAR activation is sufficient to maintain NMDAR cluster number and synaptic localization in the absence of BDNF and TrkB-mediated signaling.
Discussion
We show that TrkB signaling modulates the number and synaptic localization of neurotransmitter receptor clusters and other postsynaptic proteins during the formation and maturation of hippocampal synapses in vitro. Activation of TrkB signaling by BDNF led to an increase in the number and synaptic localization of NMDAR and GABAAR clusters. Conversely, downregulation of TrkB signaling by scavenging of endogenous BDNF using TrkB-IgG or by overexpression of a dominant-negative truncated TrkB showed that postsynaptic TrkB signaling is necessary for maintaining NMDAR and GABAAR clusters at synapses. Moreover, BDNF effects on GABAAR clusters temporally preceded those on NMDAR clusters by 12 hr. The relationship between activity and BDNF and TrkB signaling on GABAAR and NMDAR clusters was examined by blocking action potential activity with TTX or glutamate receptors with APV or CNQX. Activity blockade had no effect on the BDNF-mediated increase in NMDAR clusters. However, activity blockade alone increased synaptically localized NMDAR clusters, mimicking the effects of BDNF. Although spontaneous activity was observed to be infrequent at 8-11 DIV, activity plays an important role in the localization of NMDAR clusters to synapses.These data suggest that BDNF increases GABAAR clusters localized to synapses and that reduced neuronal excitability leads to a compensatory increase in NMDAR clusters. Consistent with this hypothesis, blocking GABAAR activity prevented the BDNF-mediated increase in NMDAR clusters. Conversely, activation of GABAARs led to the maintenance of NMDAR clusters when BDNF was scavenged. Together, these results show that postsynaptic TrkB signaling modulates GABAAR clusters at inhibitory synapses, and that GABAAR activity is required for the formation and maintenance of NMDAR clusters at glutamatergic synapses. Thus, TrkB-mediated signaling may be part of a homeostatic mechanism that balances excitatory and inhibitory synaptic activity in developing neural circuits.
Postsynaptic localization of full-length TrkB
Neurotrophins and Trks are widely expressed in the developing and adult CNS and have been shown to modulate process growth, synaptic transmission, and plasticity (for review, see Schuman, 1999Postsynaptic TrkB signaling modulates NMDAR and GABAAR clusters at hippocampal synapses
Postsynaptic target-derived neurotrophin binding to presynaptic Trk receptors had been widely considered to be the dominant mechanism for neurotrophin signaling (DiStefano et al., 1992
