The role of brain-derived neurotrophic factor receptors in the mature hippocampus: modulation of long-term potentiation through a presynaptic mechanism involving TrkB

Abstract

The neurotrophin BDNF has been shown to modulate long-term potentiation (LTP) at Schaffer collateral-CA1 hippocampal synapses. Mutants in the BDNF receptor gene trkB and antibodies to its second receptor p75NTR have been used to determine the receptors and cells involved in this response. Inhibition of p75NTR does not detectably reduce LTP or affect presynaptic function, but analyses of newly generated trkB mutants implicate TrkB. One mutant has reduced expression in a normal pattern of TrkB throughout the brain. The second mutant was created by cre-loxP-mediated removal of TrkB in CA1 pyramidal neurons of this mouse. Neither mutant detectably impacts survival or morphology of hippocampal neurons. TrkB reduction, however, affects presynaptic function and reduces the ability of tetanic stimulation to induce LTP. Postsynaptic glutamate receptors are not affected by TrkB reduction, indicating that BDNF does not modulate plasticity through postsynaptic TrkB. Consistent with this, elimination of TrkB in postsynaptic neurons does not affect LTP. Moreover, normal LTP is generated in the mutant with reduced TrkB by a depolarization-low-frequency stimulation pairing protocol that puts minimal demands on presynaptic terminal function. Thus, BDNF appears to act through TrkB presynaptically, but not postsynaptically, to modulate LTP.

Fig. 1.

Targeting disruption of thetrkB gene. A, Schematic diagrams of thetrkB gene, the targeting construct, and the targetedtrkB locus. The probes used for screening and the expected Southern blot fragments are indicated. The homology arms are represented in thick lines. B,BamHI; Bs, multiple BamHI sites; C, ClaI; H,HindIII; K, KpnI;X, XbaI. B, Southern blot analyses of representative tail DNA sample. DNA was digested withBamHI and blotted with probe A or probe C. Using probe A, 10.5 and 7.5 kb bands are generated by digestion of the wild-typetrkB and the targeted trkB alleles, respectively. Probe C does not detect any band from the wild-type allele but detects a 1.3 kb band from the floxed trkBallele. C, Northern blot analysis of trkBmRNAs. Fifteen micrograms of total brain RNA were loaded onto each lane. +/+, Wild-type; fBZ/fBZ, homozygous for thefBZ allele. Note the presence of a single RNA from the floxed trkB allele. D, Western blot analysis of TrkB protein. Protein extracts were prepared from the brains of wild-type and fBZ/fBZ homozygous mice. Forty micrograms of protein were loaded onto each lane.

Fig. 2.

Pattern of trkB recombination in the brain. X-gal staining of representative hippocampi fromfBZ/+;CaMKcre/+ mice is shown. The ages of the mice are indicated. The section shown in A was counterstained with nuclear fast red. Note that the X-gal staining in the hippocampus is essentially limited to the CA1 region in both P29 and P68 animals.cc, Corpus callosum; DG, dentate gyrus;Ntx, neocortex; SN, substantia nigra;Th, thalamus.

Fig. 3.

Lack of trkB expression in CA1 pyramidal neurons. A–C, Immunofluorescent staining for αCaMKII and β-galactosidase in the CA1 region of a P55trkB CA1-KO (fBZ/fBZ;CaMKcre/+) mouse. Note that all neurons positive for αCaMKII are also positive for β-galactosidase immunoreactivity. D, E, In situ hybridization of trkB mRNAs from 3-month-old fBZ/fBZ (D) andtrkB CA1-KO (E) mice. Thearrow in E indicates some positive cells in the CA1 ventral side. Scale bar: A–C, 20 μm;D, E, 100 μm.

Fig. 4.

Normal hippocampal structure intrkB mutants. Histological stainings were performed on sagittal sections of mouse brains with genotypes as indicated onright. A–C, Nissl-stained hippocampi of adult mice. D–I, The hippocampal CA1 regions of adult mice were stained immunohistochemically for parvalbumin (D–F) and calbindin (G–I). Note that there are no significant differences among three genotypes of animals in the gross anatomical structure of the hippocampus and the number and morphology of interneurons positive for calbindin or parvalbumin. Scale bar:A–C, 200 μm; D–I, 50 μm.

Fig. 5.

Normal dendritic morphologies of CA1 pyramidal neurons in the trkB CA1-KO. Histological stainings were performed on sagittal sections of mouse brains with genotypes as indicated. The hippocampal CA1 regions of adult mice were stained immunocytochemically for MAP2 (A–C) and β-galactosidase (D, E). Note that there are no significant differences in the dendritic structure of CA1 pyramidal neurons revealed by MAP2 and β-galactosidase immunohistochemistry among control mice and trkB mutants. Scale bar:A–C, 20 μm; D, E, 50 μm.

Fig. 6.

Impairment of LTP in hippocampal CA1 synapses of fBZ/fBZ and trkB CA1-KO mice. All data in this figure and Figure 7 are expressed as mean ± SEM.A, Time courses of synaptic potentiation induced by tetanic stimulation in CA1 synapses of hippocampal slices from different genotypes. Field EPSPs were recorded in the CA1 area, and tetanus (2 × 1 sec, 100 Hz, 20 sec apart) was applied to Schaffer collaterals at time 0. Synaptic efficacy (initial slope of EPSPs) is expressed as the percentage of baseline values recorded during the first 20 min before tetanus. Each data point represents the averaged values of recordings at that particular time point. Wild-type, n = 5 mice; fBZ/fBZ,n = 4 mice; trkB CA1-KO,n = 7 mice. B, Percentage of successful LTP recordings for wild-type, fBZ/fBZ, andtrkB CA1-KO mice. LTP was judged successful if, at 45 min after the tetanus, the slope of the EPSP was >125% of the baseline. Wild-type, n = 35 slices from eight mice;fBZ/fBZ, n = 34 slices from six mice; trkB CA1-KO, n = 50 slices from seven mice. C, Effect of p75NTR antibodies on LTP. Slices from wild-type animals were treated with or without p75NTR antibodies (50 μg/ml in ACSF). The magnitude of LTP was expressed as a percentage of the EPSP slopes before and 60 min after tetanus. The control and anti-p75NTR antibody-treated groups (n= 4 and 5 slices, respectively) are not statistically different (two-tailed t test, p = 0.6).D, Magnitude of LTP in slices from wild-type,fBZ/fBZ, and trkB CA1-KO mice andtrkB CA1-KO slices treated with TrkB-IgG. Synaptic efficacies 45 min after the tetanus from each animal are averaged and expressed as the percentage of baseline values. Wild-type,n = 8 mice; fBZ/fBZ,n = 6 mice; trkB CA1-KO,n = 7 mice; trkB CA1-KO + TrkB-IgG,n = 4 mice. *Significantly different from wild-type group, p < 0.01; ^#Significantly different from fBZ/fBZ and trkB CA1-KO groups, p < 0.05. E, Synaptic responses to HFS at CA1 synapses in fBZ/fBZ andtrkB CA1-KO mice. The slope of the 100th EPSP in the train is presented as the percentage of the first EPSP slope. *Significantly different from wild-type, Student's ttest, p < 0.001. F, Synaptic responses to HFS at CA1 synapses of wild-type hippocampal slices treated with or without anti-p75NTR IgG. The slope of the 100th EPSP in the train is presented as the percentage of the first EPSP slope. There is no difference between the two groups [n = 4 for wild-type and n = 5 for p75 antibody (Ab)-treated groups, two-tailed t test,p = 0.33].

Fig. 7.

Postsynaptic contributions to LTP are normal infBZ/fBZ mice. A, Input–output relations for wild-type and fBZ/fBZ mutant mice. Field EPSPs were recorded from the stratum radiatum of hippocampal slices at a range of stimulus intensities. Fiber volley amplitudes were binned, and corresponding EPSP slopes were averaged between slices. Measurements were obtained in ACSF containing 100 μmd-AP-5. Each point represents the mean ± SEM for each bin. Wild-type, n = 8;fBZ/fBZ, n = 7.B, Magnitude of the ratio of NMDA current to AMPA current in CA1 pyramidal cells from fBZ/fBZ and wild-type mice. Cells were clamped at +30 mV, and afferent fibers were stimulated to evoke dual-component EPSCs; 50 μmd-AP-5 was then added to the perfusion medium, and afferent stimulation was continued at the same intensity. The average NMDA-only EPSC was derived by subtracting the average AMPA-only EPSC from the average dual-component EPSC. Wild type, n= 9; fBZ/fBZ,n = 13.Insets, Representative examples of NMDA-only and AMPA-only EPSCs from mice of each genotype. Calibration: 20 pA, 10 msec. C, Time course of synaptic potentiation induced by a “pairing” protocol. Evoked EPSCs were recorded at 0.1 Hz from CA1 pyramidal cells clamped at −60 mV in whole-cell mode. At time 0, the cell was depolarized to 0 mV while afferent fibers were stimulated 100 times at 1 Hz, after which the cell was repolarized to −60 mV, and low-frequency stimulation was resumed. Wild type, n= 8; fBZ/fBZ, n = 10.