Antibodies to BDNF and pro-BDNF reveal similar staining patterns
Hippocampal sections prepared from 8-wk-old animals were incubated either with the monoclonal BDNF antibody Mab#9 (anti-BDNF; ) or with polyclonal antibodies recognizing the BDNF prodomain (anti–pro-BDNF; ). Tissues from age-matched mice engineered to delete BDNF from neurons (cbdnf ko
; Rauskolb et al., 2010
) were used as a negative CON (). In addition, sections from a knockin mouse line expressing Bdnf-Myc
(; Matsumoto et al., 2008
) were incubated with Myc antibodies (anti-Myc), with wild-type (WT) tissue used as a CON (). These three unrelated antibodies yielded strikingly similar staining patterns (). In particular, BDNF-, pro-BDNF–, and Myc-immunoreactivity (IR) were each most prominently distributed in the cell bodies and axon terminals of the mossy fiber projection pathway, whereas the layers comprising the CA1 area were only weakly stained, especially in the septal hippocampus.
Figure 1. α-BDNF, α-Myc, and α–pro-BDNF antibodies all generate similar staining patterns. (A) A schematic representation of the BDNF precursor pro-BDNF and the two cleavage products pro-peptide and BDNF. (B) Low-power view of a (more ...)
BDNF-IR is detected in subsets of neurons
Low-power examination of BDNF-IR in the dentate gyrus (DG) revealed a subset of immunopositive granule cells in the supra- and infrapyramidal blades (). BDNF-IR varied in intensity among the labeled cells, with staining concentrated in the somal apex (, inset). Anti–pro-BDNF staining was confined to exactly the same subset of granule cells containing BDNF-IR (), with somal pro-BDNF–IR also concentrated at the apex (, inset). In addition, the hilar region, which contains mossy fiber collateral axons of the granule cells, was intensely stained (). In the CA3 region, a subset of pyramidal neurons also showed both BDNF-IR () and pro-BDNF–IR (). High-resolution examination of sections labeled with anti-Myc and anti–pro-BDNF, along with antibodies against the Golgi matrix protein GM130, revealed Myc-IR throughout the soma and the initial dendritic segments (), whereas pro-BDNF–IR showed a similar, albeit more punctate, distribution (). Comparison with GM130-IR showed pro-BDNF–positive puncta closely associated with the Golgi apparatus (). In line with this, anti–pro-BDNF immunogold labeling localized the protein to the Golgi complex of CA3 somata (). In CA1, BDNF and pro-BDNF costaining was also detected in a small number of pyramidal neurons in temporal hippocampus sections (unpublished data).
Figure 2. Detection of BDNF-IR and pro-BDNF–IR in subsets of principal neurons. (A–C) Low-power confocal stack of the DG. The same subset of granule cells (arrows) coexpresses BDNF-IR (A) and pro-BDNF–IR (B). Boxed areas are enlarged in (more ...)
BDNF-IR and pro-BDNF–IR are both detected in presynaptic terminals
The granule cells give rise to mossy fiber axons, whose targets include the complex spines on proximal dendrites of CA3 neurons. Mossy fibers project through and terminate in stratum lucidum (SL) and are characterized by prominent specialized endings known as mossy fiber boutons (MFBs). Accordingly, strong BDNF-IR and pro-BDNF–IR were observed within SL ( and ). Using high-resolution confocal microscopy, both BDNF-IR and pro-BDNF–IR were found to be colocalized to the same subset of MFBs ().
Figure 3. Presynaptic localization of BDNF-IR. (A–C) High-resolution optical slice of SL showing Myc-IR (A) and pro-BDNF–IR (B) in the same subset of MFBs (arrows). (C) Note the significant colocalization of red and green puncta in the overlay. (more ...)
Additional markers were then applied to identify the type of vesicles containing BDNF-IR and to compare the distribution of BDNF with other peptides known to be anterogradely transported by granule cells. As expected, BDNF-IR did not colocalize with the synaptic vesicle markers synaptophysin (SYP; ) or VGLUT-1 (). We then tested possible colocalization with Met-enkephalin (Met-enk), an opioid peptide also derived from a larger precursor protein and stored in dense core vesicles (DCVs; Cheng et al., 1995
). In a small proportion of granule cells and their axons, Met-enk–IR was detected throughout the soma and initial dendritic segment (). Although Met-enk–positive granule cells invariably coexpressed BDNF-IR (), the immunoreactive signals of these two precursor-derived molecules remained separate, suggesting that they do not reside together in the same secretory vesicles (). Similar conclusions were reached with cholecystokinin (CCK), a neuropeptide transported along the mossy fiber projection pathway of the ventral mouse hippocampus (Gall et al., 1986
). Double labeling with anti-CCK and -BDNF revealed complete segregation of the two peptides within MFBs ().
Presynaptic BDNF labeling was also observed in the CA1 region, whereby in stratum radiatum (SR), thin varicose processes labeled with anti-BDNF () and anti–pro-BDNF (not depicted) were sparsely distributed throughout the neuropil, likely corresponding to presynaptic Schaffer collateral axons originating from BDNF-positive CA3 neurons. Importantly, BDNF-IR did not colocalize with the postsynaptic marker synaptopodin (synpo; ).
BDNF and pro-BDNF antibodies label secretory vesicles in presynaptic terminals
Ultrathin sections of SL were then examined at 2,000-fold magnification after immunogold labeling with BDNF and pro-BDNF antibodies. MFBs were identified based on their typical morphological characteristics, namely a high density of synaptic vesicles, numerous synaptic contacts with CA3 complex spines, nonsynaptic puncta adherentia at dendritic shafts, and a relatively large surface area. A subset of MFB profiles labeled with anti-BDNF (), anti-Myc (not depicted), or anti–pro-BDNF () contained distinct aggregates of gold grains; at 10,000-fold magnification, these gold clusters were found to be associated with large vesicles encompassed by an electron-dense membrane. Although the vesicles were sometimes masked by gold grains, fortuitous grain distribution occasionally revealed an electron-dense core (, insets). Within MFB profiles of cbdnf ko sections, gold grains were never specifically associated with any type of organelle (). To assess the density of immunogold particles in MFB profiles, gold clusters and single gold grains were quantified in anti-BDNF and anti–pro-BDNF–labeled tissues and compared between sections from pooled WT/Bdnf-Myc mice and cbdnf ko mice. Anti-BDNF–labeled MFB profiles showed a mean density of 2.67 ± 0.35 clusters/µm2 in WT/Bdnf-Myc animals compared with 0.38 ± 0.12 clusters/µm2 (P < 0.005) in cbdnf ko profiles (). Moreover, single gold grain densities were significantly reduced in cbdnf ko MFB profiles, with 5.63 ± 0.94 grains/µm2 compared with 21.99 ± 2.57 grains/µm2 in WT/Bdnf-Myc (P < 0.005; ). Similarly, pro-BDNF immunogold labeling showed a mean density of 2.01 ± 0.43 clusters/µm2 and 27.44 ± 8.21 single grains/µm2 in MFB profiles from WT/Bdnf-Myc animals, whereas in comparison, MFB profiles from cbdnf ko animals showed significant reductions in cluster (0.14 ± 0.10/µm2; P < 0.05; ) and single grain (5.82 ± 2.06/µm2; P < 0.05; ) densities, respectively.
Figure 4. Ultrastructural localization of BDNF and pro-BDNF immunogold labeling in MFBs. (A) Electron micrograph of an ultrathin section of WT SL prelabeled with anti-BDNF immunogold. Numerous gold clusters (arrows) are associated with large secretory vesicles (more ...)
Although BDNF immunogold labeling was mostly concentrated in presynaptic terminals, labeled vesicles were also occasionally observed within unmyelinated axon segments in SL (), which in fortuitous sections could be seen to give rise to giant MFBs ().
Figure 5. BDNF and pro-BDNF immunogold labeling in preterminal axons. (A) In sections treated with anti-BDNF antibodies, labeled vesicles (arrows and inset) were observed in unmyelinated mossy fiber axons. (B) An electron micrograph showing a mossy fiber terminating (more ...)
Ultrathin sections of SR (CA1) labeled with anti-BDNF and anti–pro-BDNF immunogold were also examined, and, as expected, large cluster-labeled secretory vesicles were observed within small axon terminals (). Depending on the proximal distal level of SR, these infrequent labeled boutons likely correspond to Schaffer collateral terminals or entorhinal terminals. No such labeled terminals were observed in cbdnf ko sections ().
BDNF-IR and pro–BDNF-IR are not detected in dendrites
Next, we examined the possible localization of BDNF in dendrites by analyzing sections double labeled with anti-Myc and anti–microtubule-associated protein–2 (MAP-2). Both in BDNF-positive granule cells and CA3 neurons, Myc-IR only extended as far as the initial dendritic segments, and there was no evidence of colocalization in SL (). Sections were alternatively labeled with antibodies to Arc/Arg3.1, an immediate early gene product up-regulated in somata and dendrites during elevated synaptic activity (Lyford et al., 1995
). Confocal scanning revealed that the majority of granule cells labeled with BDNF-IR and pro-BDNF–IR also expressed Arc/Arg3.1-IR (). Although Arc/Arg3.1-IR was seen throughout the soma and dendritic arbor, strong coexpression of BDNF-IR and pro-BDNF–IR was confined to the cell soma ().
Figure 6. BDNF-IR does not extend beyond proximal dendrites. (A) Myc-IR in the CA3 region showing immunoreactive neuronal somata (asterisks) in the pyramidal cell layer (PCL) and Myc-positive MFB profiles in SL. (B) Anti–MAP-2 staining showing the distribution (more ...)
In ultrathin sections prelabeled either with anti-BDNF or anti–pro-BDNF immunogold, gold grains were sparsely distributed within dendritic profiles (). When profiles were thoroughly scrutinized for specifically labeled vesicles or endosomes, none was found to be stained above the background levels observed in dendrites from cbdnf ko sections (). Gold grain quantification of anti-BDNF–labeled sections () revealed a mean density of 3.56 ± 0.45 grains/µm2 in WT/Bdnf-Myc mice versus 4.72 ± 0.91 grains/µm2 in cbdnf ko mice (P = 0.81). Similarly, in anti–pro-BDNF–stained sections, mean densities were comparable between WT/Bdnf-Myc mice (4.69 ± 1.09 grains/µm2) and cbdnf ko mice (4.08 ± 2.02 grains/µm2; P = 0.31; ).
Localization of BDNF-IR and pro-BDNF–IR in Bsn mutants
Next, we examined the hippocampus of mice lacking the presynaptic protein Bassoon, as these mutants develop episodic generalized seizures (Altrock et al., 2003
) and have enlarged cortices and hippocampi (Angenstein et al., 2007
). Concurrent with the development of seizures, BDNF protein levels become significantly higher than those measured in adult CON littermates (see ; Heyden et al., 2011
). Whereas Bassoon (Bsn
) mutants showed the typical distribution pattern of BDNF-IR and pro-BDNF–IR, a dramatic increase in staining intensity largely confined to the neuropil was observed (compare CON in with Bsn
in ). In contrast, granule cell bodies from Bsn
mutants did not show increases in anti-BDNF () or anti–pro-BDNF () staining intensities, although a much higher proportion of cells was labeled in comparison with CON tissues. Granule cell dendrites in the molecular layer remained unlabeled in Bsn
mutants, whereas in the CA3 region, the stark increase in BDNF-IR () and pro-BDNF–IR () was confined to SL. Closer examination revealed intense presynaptic labeling in MFB profiles (), which was confirmed by a lack of colocalization with the postsynaptic markers synpo () and MAP-2 (), respectively.
Figure 10. Biochemical detection of BDNF, its pro-peptide, and pro-BDNF. IP of hippocampal lysates (500 µg) was performed either with an anti–pro-BDNF antiserum or with the BDNF antibody followed by WB with the antibodies indicated. (A) Both pro-BDNF (more ...)
Figure 7. Elevated BDNF staining in Bsn mutants. (A–D) Representative images from CON tissues showing anti-BDNF labeling in the DG (A) and CA3 area (C) and anti–pro-BDNF–Mab5H8 labeling in the DG (B) and CA3 (D). GCL, granule cell layer; (more ...)
Enhanced BDNF staining in Bsn
mutants was not only confined to the granule cell–CA3 projection pathway. In the CA1 region, where BDNF-IR can usually only be detected at high magnification (compare with and ), a conspicuous band of punctate BDNF-IR and pro-BDNF–IR was observed at the border of SR and stratum lacunosum–moleculare (SL-M; Fig. S1, A–C
), corresponding to a region known to harbor fibers from the entorhinal cortex (Amaral and Lavanex, 2006
). Importantly, these increased signals colocalized (Fig. S1 D), showing no overlap with either synpo-IR (Fig. S1 E) or glial fibrillary acidic protein (GFAP)–IR (Fig. S1 F), which label dendritic spines and reactive astrocytes, respectively. This suggests that entorhinal neurons also represent a possible presynaptic source of BDNF for CA1 neurons.
Immunogold-labeled sections from both WT and Bsn mutants were then examined at 2,000-fold magnification. In comparison with CON animals (), a higher number of labeled MFB profiles containing more BDNF-positive DCVs was observed in sections from Bsn mutants (), with clusters accumulated at the synaptic membrane. This was confirmed by quantification, with anti-BDNF–labeled MFBs containing a significantly higher density of gold clusters (1.7 ± 0.3 clusters/µm2; ) and grains (14.4 ± 2.5 grains/µm2; ) compared with CONs (0.8 ± 0.01 clusters/µm2 with P < 0.05 and 6.9 ± 0.5 grains/µm2 with P < 0.05, respectively). Similarly, anti–pro-BDNF–labeled profiles from Bsn mutants also displayed a higher density of gold clusters (2.02 ± 0.27 clusters/µm2; ) and grains (17.2 ± 2.8 grains/µm2; ) compared with CON profiles (0.96 ± 0.04 clusters/µm2 with P < 0.005 and 10.1 ± 0.9 grains/µm2 with P < 0.05, respectively); these relatively low increases in mean cluster densities are a result of the larger areas of MFB profiles in Bsn mutants.
Figure 8. Higher density of BDNF-positive DCVs in MFBs of Bsn mutant animals. (A and B) Electron micrographs of MFBs from CON sections prelabeled with BDNF (A) or pro-BDNF (B) immunogold. Gold cluster–labeled secretory vesicles were occasionally observed (more ...)
Gold grain distribution and density in dendrites were also compared between Bsn mutant () and CON tissues (). Dendritic profiles were again scrutinized for evidence of labeled vesicles, but none was found in tissues from either group. Density measurements revealed background values in Bsn mutant mice similar to those observed in CON mice, both in tissues labeled with anti-BDNF (5.4 ± 0.4 grains/µm2 for Bsn mutant vs. 5.7 ± 0.5 grains/µm2 for CON; P = 0.66; , left) and anti–pro-BDNF (5.1 ± 0.1 grains/µm2 for Bsn mutant vs. 6.3 ± 0.3 grains/µm2 for CON; P = 0.12; , right). Increased labeling density was not detected in the extracellular space or in nonneuronal cell types such as astrocytes.
Figure 9. BDNF is not targeted to dendrites in Bsn mutants. (A–E) Comparison of dendrites (den) in SL in CON (A and C) versus Bsn mutant tissues (B and D) revealed a lack of difference in the density of gold grains, both in the case of BDNF immunogold (E, (more ...)
Verification of background immunogold labeling
To determine whether background gold labeling is evenly distributed over different subcellular compartments, we extended the quantitative gold grain analysis to dendritic spine profiles and myelinated axon profiles in SL, as they are devoid of cluster-labeled organelles. Quantification of single gold grains overlying these profiles revealed that the density of background gold labeling depends on the type of subcellular compartment (). The mean values for gold grain densities in spine and myelinated axon profiles did not differ between WT/Myc versus cbdnf ko or WT versus Bsn mutant tissues. Therefore, spines and myelinated axons from WT mice do not exhibit anti-BDNF and anti–pro-BDNF immunogold labeling above the background levels observed in cbdnf ko tissues, nor do they contain specifically labeled organelles.
Comparative gold grain densities in subcellular compartments devoid of cluster-labeled organelles
Biochemical detection and quantification of the BDNF pro-peptide
The identity of the molecules recognized by the BDNF antibodies in the immunochemistry experiments was then determined using hippocampal lysates from WT and mutant mice (cbdnf ko
). Immunoprecipitates were analyzed by Western blotting (WB) and probed with either the BDNF polyclonal antibody N-20 or the monoclonal antibody 5H8, which recognizes an epitope in the prodomain of BDNF (). These experiments revealed the presence not only of pro-BDNF (~32 kD) but also of the much more abundant pro-peptide (~17 kD; ; also see ). Both signals were absent in lysates from cbdnf ko
animals (). Quantification of the corresponding signal intensities revealed that the ratio of pro-peptide versus pro-BDNF is ~10.3 ± 2.0 (n
= 3; ), similar to that of BDNF versus pro-BDNF (11 ± 2.0; n
= 3; ). A similar analysis of hippocampal lysates from Bsn
mutants confirmed an approximate threefold increase each in BDNF, its pro-peptide, and pro-BDNF in mutant tissues (). These results also indicate that the respective ratios of BDNF and the pro-peptide versus pro-BDNF are similar to what is observed in WT animals. Of note is that the consistent detection of BDNF pro-peptide was only possible after glutaraldehyde fixation of the transfer membrane (see Materials and methods). Furthermore, under the lysate and incubation conditions used, no measurable proteolysis of recombinant pro-BDNF (500 pg) added to the lysates at the beginning of the extraction procedure could be detected (Fig. S2
). The recovery of added recombinant pro-BDNF was 102.2 ± 5.8% (n