Variant brain-derived neurotrophic factor (Val66Met) alters adult olfactory bulb neurogenesis and spontaneous olfactory discrimination

Abstract

Neurogenesis, the division, migration, and differentiation of new neurons, occurs throughout life. Brain derived neurotrophic factor (BDNF) has been identified as a potential signaling molecule regulating neurogenesis in the subventricular zone (SVZ), but its functional consequences in vivo have not been well defined. We report marked and unexpected deficits in survival but not proliferation of newly born cells of adult knock-in mice containing a variant form of BDNF [a valine (Val) to methionine (Met) substitution at position 66 in the prodomain of BDNF (Val66Met)], a genetic mutation shown to lead to a selective impairment in activity-dependent BDNF secretion. Utilizing knock-out mouse lines, we identified BDNF and tyrosine receptor kinase B (TrkB) as the critical molecules for the observed impairments in neurogenesis, with p75 knock-out mice showing no effect on cell proliferation or survival. We then localized the activated form of TrkB to a discrete population of cells, type A migrating neuroblasts, and demonstrate a decrease in TrkB phosphorylation in the SVZ of Val66Met mutant mice. With these findings, we identify TrkB signaling, potentially through activity dependent release of BDNF, as a critical step in the survival of migrating neuroblasts. Utilizing a behavioral task shown to be sensitive to disruptions in olfactory bulb neurogenesis, we identified specific impairments in spontaneous olfactory discrimination, but not general olfactory sensitivity or habituation to olfactory stimuli in BDNF mutant mice. Through these observations, we have identified novel links between genetic variant BDNF and adult neurogenesis in vivo, which may contribute to significant impairments in olfactory function.

Figure 1.

Loss of BDNF signaling via TrkB impacts the survival but not proliferation of neuroblasts. a, b, Quantification of the density of BrdU-labeled cells in the SVZ (a) counted 2 h after BrdU administration (BDNF^+/+, n = 4; BNDF^+/−, n = 4) and the granule cell layer of the OB (gcOB) (b) counted 28 d after BrdU administration (BDNF^+/+, n = 4; BDNF^+/−, n = 4). c, Quantification of the percentage of overlap between BrdU-positive and NeuN-positive immunofluorescent double labeling in the gcOB from mice 28 d after injection (n = 820 cells from 8 mice). Data are presented as the percentage of double-labeled cells for BDNF^+/+ and BDNF^+/− mice. d, e, Quantification of BrdU cell density in the (d) SVZ of wild-type (n = 8), p75 knock-out (p75^−/−, n = 4), and TrkB heterozygous (TrkB^+/−, n = 4) mice 2 h after BrdU administration and (e) BrdU cell density in the gcOB 28 d after BrdU injection (WT, n = 4; p75^−/−, n = 4; TrkB^+/−, n = 4). f, g, Quantification of the density of BrdU-positive cells in BDNF Val66Met knock-in mice (f) in the SVZ 2 h after BrdU administration (BDNF^+/+, n = 4 and BDNF^Met/Met, n = 4) or (g) or in the gcOB 28 d after BrdU administration (BDNF^+/+, n = 6 and BDNF^Met/Met, n = 6). Asterisks indicate statistically significant difference: *p < 0.05; **p < 0.01; ***p < 0.001. Error bars depict the SEM.

Figure 2.

BDNF distribution and regional survival of newly born cells in the OB in BDNF and receptor mutant mice. a–c, Quantification of the density of surviving BrdU-positive cells 28 d after BrdU injection plotted by location within the olfactory bulb (caudal, central, rostral) of (a) BDNF^+/+ and BDNF^+/− mice, (b) WT, TrkB^+/−, and p75^−/− mice, and (c) BDNF^+/+, BDNF^+/M, and BDNF^Met/Met mice. d, Histogram depicting BDNF protein levels as assayed by ELISA for each of three regions of the OB (caudal, central, and rostral) as well as for the hippocampus. Statistical comparison for hippocampus relative to the olfactory bulb was with average BDNF levels across the three regions of the OB. Asterisks indicate statistically significant difference, *p < 0.05.

Figure 3.

Specificity of phosphorylated TrkB receptor antibodies. Affinity purification of polyclonal antibodies raised against a short peptide in the C-terminal region of rat TrkB containing tyrosine 816, which is phosphorylated in response to BDNF, was performed as described in Materials and Methods. a, To assess specificity of purified antibodies, lysates (40 μg each well) obtained from 293 cells (left) stably expressing TrkB receptors and cultured hippocampal neurons (right) treated with 50 ng/ml BDNF for 10 min were probed with affinity-purified serum as well as purified sera that was preincubated with a phosphorylated TrkB peptide competitor. b, Confocal images of phospho-TrkB receptor antibody-labeled cells within the SVZ, RMS, and selOB counterstained with TO-PRO3 (blue). c–f, Specificity of pTrkB receptor immunofluorescent labeling of cells in the RMS was determined with pTrkB alone (c) or in presence of phosphorylated (pTrkB) peptide (d), or nonphosphorylated (TrkB) peptide (e), and with IgG control alone (f). g, h, Specificity of pTrkB receptor immunofluorescent labeling of cells in the RMS was further confirmed by the comparison of labeling in the RMS of TrkB^+/+ (g) and TrkB^+/− mice (h). Scale bars, 100 μm.

Figure 4.

Activated TrkB receptors are highly localized to migrating neuroblasts (type A cells). a–g, Confocal optical sections were taken, at 63× magnification, of coronal slices of the mouse RMS immunolabeled for pTrkB receptors (green) and coimmunolabeled for markers of the various cell types composing the RMS. Top, Representative coronal sections of the RMS coimmunolabeled for pTrkB (green) and GFAP (a; red), Nestin (b; red), and doublecortin (c; red). Center panels display representative coronal sections of the RMS coimmunolabeled for pTrkB (green) and PSA-NCAM (d; red), isolectin B-4 (e; red), and NeuN (f; red). Bottom, Representative coronal section of pTrkB (green) coimmunolabeled with p75 (g; red). Scale bars, 50 μm.

Figure 5.

p75 receptor immunoreactivity in the RMS is restricted to a subset of nestin positive type C cells. a–i, Confocal optical sections were taken at 63× magnification of coronal slices of the mouse SVZ (a), RMS (b), and selOB (c), immunolabeled for p75 receptor (green) and counterstained with TO-PRO 3 (blue). Middle, Representative coronal sections of the mouse RMS coimmunolabeled for p75 receptor (green) and GFAP (d; red), Nestin (e; red), and Ki-67 (f; red). Bottom, Representative coronal sections of the mouse RMS coimmunolabeled for p75 receptor (green) and doublecortin- (g; red), isolectin B-4- (h; red), or NeuN-positive (i; red) cells adjacent to the RMS. Scale bars, 50 μm.

Figure 6.

BDNF and pTrkB immunoreactivity in neurogenic regions. Optical sections were taken at 40× magnification of coronal slices of the mouse SVZ (a), RMS (b), and selOB (c), immunolabeled for BDNF (red) and counterstained with DAPI (4,6,diamidino-2-phenylindole; blue). Scale bars, 50 μm. Middle, Representative coronal sections of the mouse RMS taken at 20× magnification and coimmunolabeled for pTrkB receptor (d), BDNF (e), and merged (f). Included is an inset from confocal imaging of this same section showing BDNF to be highly localized surrounding pTrkB positive cells. Scale bars, 100 μm. g, Bottom, Representative immunoblots from intraperitoneal Westerns blotted for pTrkB and subsequently for total TrkB proteins in SVZ tissue, as well as quantification of percentage difference in the percentage activated TrkB corrected for total TrkB levels. Quantification is presented as percentage difference from wild-type density, with error bars depicting SEM. Asterisk indicates statistically significant difference (Student's t test, *p < 0.05).

Figure 7.

BDNF haploinsufficient, TrkB haploinsufficient, and BDNF knock-in lines of mice do not spontaneously discriminate between odorants. Olfactory behavioral habituation and cross-habituation data in BDNF knock-out, TrkB knock-out, and BDNF knock-in lines of mice (spontaneous discrimination). a, Habituation profiles for BDNF^+/+ (n = 6) and BDNF^+/− (n = 6) mice (for odor sets, see supplemental Table 1, available at www.jneurosci.org as supplemental material), showing a significant reduction in investigation time across four repeated presentations of a habituation odorant (Hab). Asterisks indicate a highly significant difference (p < 0.001) in investigation time between trials Hab1 and Hab4. b, Investigation times of test odorants increased for BDNF^+/+ (wild-type), but not BDNF^+/− mice, as a function of odor similarity (S1, very similar; S2, moderately similar; D, dissimilar). Asterisks indicate significance values, *p < 0.05 between the test odor and Hab4. c, Habituation profiles for TrkB^+/− (n = 8) mice compared with wild-type littermate controls (n = 8). Wild-type mice habituated normal, but TrkB^+/− mice failed to habituation to repeated odorant presentations. Asterisks are as in a, but indicating p < 0.01. d, Investigation times of test odorants increased for wild-type but not TrkB^+/− mice as a function of odor similarity. TrkB^+/− mice failed to spontaneously discriminate any of the test odorants from the habituation odorant. Asterisks indicate a highly significant difference (p < 0.01) in investigation time between Hab4 and the test odor. e, Habituation profiles for mice carrying either one (BDNF^+/Met, n = 7) or two copies (BDNF^Met/Met, n = 7) of the BDNF Met allele are similar to those of wild-type littermates (BDNF^+/+, n = 7). Asterisks are as in a. f, Investigation times of test odorants increased for BDNF^+/+ (wild-type) as a function of odor similarity, but BDNF^Met/Met and BDNF^+/Met mice failed to spontaneously discriminate any of the test odorants from the habituation odorant. Asterisks indicate significant differences (**p < 0.01; ***p < 0.001) in investigation times between Hab4 and the test odor. Error bars depict SEM.