Localization of brain-derived neurotrophic factor to distinct terminals of mossy fiber axons implies regulation of both excitation and feedforward inhibition of CA3 pyramidal cells

Abstract

Hippocampal dentate granule cells directly excite and indirectly inhibit CA3 pyramidal cells via distinct presynaptic terminal specializations of their mossy fiber axons. This mossy fiber pathway contains the highest concentration of brain-derived neurotrophic factor (BDNF) in the CNS, yet whether BDNF is positioned to regulate the excitatory and/or inhibitory pathways is unknown. To localize BDNF, confocal microscopy of green fluorescent protein transgenic mice was combined with BDNF immunohistochemistry. Approximately half of presynaptic granule cell-CA3 pyramidal cell contacts were found to contain BDNF. Moreover, enhanced neuronal activity virtually doubled the percentage of BDNF-immunoreactive terminals contacting CA3 pyramidal cells. To our surprise, BDNF was also found in mossy fiber terminals contacting inhibitory neurons. These studies demonstrate that mossy fiber BDNF is poised to regulate both direct excitatory and indirect feedforward inhibitory inputs to CA3 pyramdal cells and reveal that seizure activity increases the pool of BDNF-expressing granule cell presynaptic terminals contacting CA3 pyramidal cells.

Figure 3.

BDNF-immunoreactive giant mossy fiber boutons and mossy fiber bouton filipodia. Top, GFP in granule cell giant mossy fiber boutons (arrows) and mossy fiber bouton filipodia (arrowheads). Middle, BDNF-immunoreactive clusters that colocalize with giant boutons are denoted with arrows. Arrowheads denote the regions of the filipodia that are immunoreactive for BDNF (middle panel). Bottom, Merged GFP and BDNF images with double-labeled regions showing up as blue. Images are maximum projections (showing the regions of highest intensity) of several confocal scans through the pictured MFB but excluding the regions above and below the bouton. Scale bar, 3 μm.

Figure 2.

Composite confocal image showing a dentate granule cell giant mossy fiber bouton (white arrow), a dentate granule cell MFB filipodia (green arrowhead), dentate granule cell en passant terminals (yellow arrowheads, EPT), a CA3b pyramidal cell apical dendrite with thorny excrescences (blue arrows, Th. Exc.), and one of the individual spine heads that makes up an excrescence (blue arrows, Th. Exc. Spine head). Scale bar, 3 μm.

Figure 1.

Confocal reconstruction showing GFP immunoreactivity in the hippocampus of the Thy1 GFP-expressing mouse. In these sections, numerous dentate granule cells are labeled. Their axons can be seen projecting into the mossy fiber pathway (hilus, stratum lucidum over CA3). A single labeled CA3 pyramidal cell is also pictured. Scale bar, 250 μm.

Figure 4.

A, BDNF-immunoreactive mossy fiber en passant terminals. Top, GFP-expressing giant mossy fiber bouton (arrow) and en passant terminals (arrowheads) in the stratum lucidum. Circled regions denote BDNF-immunoreactive particles within axons. Middle, BDNF immunoreactivity in the same fields as shown in the top panels. The mossy fiber bouton (arrow) and the top en passant terminal (arrowhead) are BDNF immunopositive. The bottom en passant terminal is negative. Bottom, Merged GFP and BDNF images with double-labeled regions showing up as blue. Images are maximum projections (showing the regions of highest intensity) of several confocal scans through the pictured en passant terminals but excluding the regions above and below the terminal. B, BDNF-immunoreactive particles are found in dentate granule cell mossy fiber axons. Circled regions denote BDNF-immunoreactive particles within axons. The GFP image (top) is a maximum projection of the axon (showing the regions of highest intensity), and the BDNF image (middle) is a projection of three focal planes showing the BDNF particles within the axon. Scale bars: A, 3 μm; B, 1 μm.

Figure 5.

Confocal scan of a single focal plane reveals BDNF immunoreactivity surrounding CA3 pyramidal cell thorny excrescences. Left column, Thorny excrescence enveloping a BDNF-immunoreactive cluster. Right column, Thorny excrescence spine head surrounded by but not containing BDNF immunoreactivity (left of asterisk). GFP-immunoreactive thorny excrescences are shown in the top row; BDNF immunoreactivity in the same fields is shown in the middle row; and merged images are shown in the bottom row. Scale bar, 2 μm.

Figure 6.

Colocalization within scans from single focal planes reveals BDNF-immunoreactive CA3 pyramidal cell thorny excrescences. A, Maximum projection displaying the region of highest intensity from six focal planes taken at 0.4 μm increments of the thorny excrescence shown in B. B, Three scans of the same thorny excrescence taken at three different focal planes, each plane being separated by 0.4 μm. The scans show BDNF immunoreactivity colocalzing with the thorny excrescence spine heads. GFP immunoreactivity is shown in the top row; BDNF immunoreactivity in the same fields is shown in the middle row; and merged images are shown in the bottom row. Moving left to right, note how the GFP and BDNF signals from the spine denoted by the arrowhead disappear simultaneously in the right image. (In the right image, the GFP and BDNF signals from the spine become faint and “fuzzy” because they are no longer in the focal plane). Similarly, a second spine (arrow), which is just out of focus in the left image, appears simultaneously with BDNF immunoreactivity in the middle and right images. d, Dendrite. Scale bar, 2 μm.

Figure 7.

BDNF immunoreactivity is localized to astrocytes in the mossy fiber pathway. Top row, GFP-expressing astrocytes located at the CA3b stratum lucidum. Middle row, BDNF-immunoreactivity in the same fields as shown in the top row. Bottom row, Merged GFP and BDNF images with double-labeled regions showing up as blue. A, Confocal scan from a single focal plane showing BDNF immunoreactivity in the cell body of an astrocyte. B, Stratum lucidum astrocyte containing numerous BDNF-immunoreactive particles in its processes. C, Magnified view of the boxed region shown in B. Images are a maximum projection of 20 confocal scans of focal planes separated by 0.2 μm increments. The maximum projection shows the regions of highest intensity (the maximum) from the 20 scans. SR, Stratum radiatum; SL, stratum lucidum. Scale bars: A, 5 μm; B, 10 μm; C, 2 μm.

Figure 8.

Confocal scans from single focal planes showing astrocytic processes enveloping BDNF-immunoreactive clusters. Top row, GFP-expressing astrocytic processes. Middle row, BDNF immunoreactivity in the same fields as shown in the top row. Bottom row, Merged GFP and BDNF images. Note the close apposition but negligible colocalization (shown as blue) between the processes of the astrocytes and the BDNF-immunoreactive clusters. Scale bar, 3 μm.

Figure 9.

Left graph, The percentage of BDNF-immunoreactive giant MFBs was significantly increased 48 hr after 3 hr of pilocarpine-induced status epilepticus (SE) relative to saline-treated controls (C). The numbers of BDNF-immunoreactive mossy fiber bouton filipodia and mossy fiber en passant terminals (EPT) were not significantly increased. ^**p < 0.01, rank-sum test. Right graph: The percentage of CA3 pyramidal cell thorny excrescence spine heads with BDNF immunoreactivity adjacent to the spine head was significantly increased after status epilepticus relative to control animals. ^*p < 0.05, rank-sum test.