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.2012 Apr 18;32(16):5398-413.
doi: 10.1523/JNEUROSCI.4515-11.2012.

Distinct subsets of Syt-IV/BDNF vesicles are sorted to axons versus dendrites and recruited to synapses by activity

Affiliations

Distinct subsets of Syt-IV/BDNF vesicles are sorted to axons versus dendrites and recruited to synapses by activity

Camin Dean et al. J Neurosci..

Abstract

BDNF plays a critical role in the regulation of synaptic strength and is essential for long-term potentiation, a phenomenon that underlies learning and memory. However, whether BDNF acts in a diffuse manner or is targeted to specific neuronal subcompartments or synaptic sites to affect circuit function remains unknown. Here, using photoactivation of BDNF or syt-IV (a regulator of exocytosis present on BDNF-containing vesicles) in transfected rat hippocampal neurons, we discovered that distinct subsets of BDNF vesicles are targeted to axons versus dendrites and are not shared between these compartments. Moreover, syt-IV- and BDNF-harboring vesicles are recruited to both presynaptic and postsynaptic sites in response to increased neuronal activity. Finally, using syt-IV knockout mouse neurons, we found that syt-IV is necessary for both presynaptic and postsynaptic scaling of synaptic strength in response to changes in network activity. These findings demonstrate that BDNF-containing vesicles can be targeted to specific sites in neurons and suggest that syt-IV-regulated BDNF secretion is subject to spatial control to regulate synaptic function in a site-specific manner.

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Figures

Figure 1.
Figure 1.
Spatial accuracy of photoactivation and identification of axons versus dendrites.A, A slide streaked with photoactivatable fluorescein was photoactivated with 40% laser power 405 nm light in rectangular ROI 1, followed by bleaching with 10% 488 nm laser power in ROI 2, and 40% 488 nm laser power in ROI 3. Scale bar is 5 μm.B,C, Verification of axons versus dendrites in a low-magnification image of a GFP-transfected neuron where the axon can be traced back to the cell body and identified in higher-magnification imaging experiments (B) and by retrospective immunostaining with the dendritic marker MAP-2 (C). Scale bar is 10 μm.D, Time lapse images of trafficking GFP-tagged BDNF vesicles imaged by confocal (left panels) and epifluorescence (right panels). Scale bar is 3 μm in left panels and 5 μm in right panels.
Figure 2.
Figure 2.
Syt-IV- and BDNF-harboring vesicles are not shared between axons and dendrites.A–C, Images of a hippocampal neuron cotransfected with sytIV-PAGFP and mCherry before and after photoactivation of the cell body (A), axon (B), or dendrites (C) in the indicated regions.D–E, Neuron cotransfected with BDNF-PAGFP/mCherry before and after photoactivation of the cell body (D), axon (E), or dendrites (F).G, Quantitation of fluorescence increase in the cell body, dendrites, and axons of sytIV-PAGFP-transfected neurons following photoactivation in the indicated regions. For axon photoactivation experiments, fluorescence intensity was calculated in a region within (in) the photoactivated area, in an area outside (out) just distal to the photoactivated area, in the cell body, and in proximal dendrites. For dendrites, fluorescence intensity was calculated in a region within (in) the photoactivated area, in a different dendrite (where the vesicles would have to travel from the photoactivated dendrite, into the cell body, and then into another dendrite) designated “dendrite (out),” in the cell body, and in the proximal region of the axon.H, Quantitation of fluorescence in the cell body, dendrites, and axons of BDNF-PAGFP-transfected neurons following photoactivation.I, Time course of fluorescence increase in cell bodies following photoactivation of axons or dendrites (n = 12 time lapse experiments for each condition; 3 cultures/transfections; error bars indicate SEM). Scale bars are 10 μm in all panels.
Figure 3.
Figure 3.
BDNF-PAGFP transfer to neighboring cells. BDNF-PAGFP is secreted and taken up by neighboring neurons; photoactivation of the cell body of a hippocampal neuron cotransfected with mCherry and BDNF-PAGFP is shown. Note that photoactivated BDNF-PAGFP is taken up by the soma of a neighboring cell (arrows). Scale bar is 10 μm.
Figure 4.
Figure 4.
BDNF-PAGFP trafficked from the cell body accumulates at synaptic sites following increased neuronal activity.A, Time course of fluorescence decrease in cell bodies (indicating transport of BDNF-PAGFP or syt-IV-PAGFP out of the cell body and into neuronal processes) immediately following photoactivation of the cell body in the presence or absence of 200 μm glycine. Tau values of single exponential fits of the data for the indicated condition are: syt-IV-PAGFP = 7.3 ± 0.7, syt-IV-PAGFP + glycine = 8.0 ± 0.9, BDNF-PAGFP = 8.2 ± 1.0, and BDNF-PAGFP + glycine = 8.1 ± 1.0.B, Representative images of a hippocampal neuron cotransfected with BDNF-PAGFP and mCherry and treated with 200 μm glycine for 15 min before and throughout and imaging before and after photoactivation of the cell body by illumination of the indicated region.C, Retrospective immunostain of the same neuron with an anti-synaptophysin antibody (using a Cy5 secondary antibody, pseudocolored red) to mark synaptic sites. BDNF-PAGFP accumulates at synaptic sites (arrowheads).D, Time course of accumulation of BDNF-PAGFP at synapses (arrows) along the indicated neuronal process following photoactivation of the cell body. Line scans of intensity of synaptophysin (red line) and BDNF-PAGFP (green lines) along the neuronal process are shown. Dashed line indicates the border of the photoactivated region. Scale bar is 10 μm.
Figure 5.
Figure 5.
Syt-IV is recruited to synapses by activity.A–E, Immunostains of syt-IV and VGluT and VGAT (to mark excitatory and inhibitory synapses, respectively) in hippocampal neurons in control conditions (A) and following treatment with 200 μm glycine (gly) for 15 min (15′) (B) or 1 h (C), 50 μm forskolin (fsk) for 4 h (D), or 50 μm MG132 for 4 h (E).F, Quantitation of increase in syt-IV average intensity within thresholded VGluT and VGAT puncta expressed as percent of control. Pearson'sr for syt-IV/VGluT = 0.10 ± 0.03 (control), 0.27 ± 0.02 (15′ gly), 0.47 ± 0.08 (1 h gly), 0.42 ± 0.07 (fsk), and for syt-IV/VGAT = 0.16 ± 0.07 (control), 0.37 ± 0.09 (15′ gly), 0.41 ± 0.08 (1 h gly), 0.09 ± 0.21 (fsk) (n = 10 images; 2 coverslips each from 5 cultures; error bars indicate SEM). Insets: Western blot of syt-IV in control and treated cultures; equal amounts of protein (12 μg) were loaded per lane.G, Quantitation of the fraction of syt-IV-positive synapses. VGluT and VGAT puncta were thresholded and the fraction of synapses with detectable syt-IV signal within VGlut or VGAT-positive puncta was calculated for each condition (n = 10 images; 2 coverslips each from 5 cultures; error bars indicate SEM. Significance was determined by a Student'st test; *p < 0.05, **p < 0.01, and ***p < 0.001. Significance relative to control is shown except for indicated comparisons between VGluT and VGAT). Scale bars are 10 μm in all panels.
Figure 6.
Figure 6.
BDNF is recruited to synapses by activity concomitantly with syt-IV.A, Hippocampal cultures immunostained for syt-IV, BDNF, and synaptophysin to mark synapses following treatment with 200 μm glycine (gly) for1 h; cntrl, control.B, Quantitation of the intensity of syt-IV and BDNF at synapses marked by synaptophysin. Synaptophysin was thresholded to mark all visible puncta, and the average intensity of syt-IV or BDNF fluorescence within these puncta was calculated and expressed as percentage of control. Pearson'sr for syt-IV/synaptophysin = 0.05 ± 0.06 (cntrl), 0.22 ± 0.08 (1 h gly), and Pearson'sr for BDNF/synaptophysin = 0.02 ± 0.06 (cntrl l), 0.21 ± 0.04 (1 h gly) (n = 10 images; 2 coverslips/5 cultures; error bars indicate SEM).C, Quantitation of the percentage of syt-IV and BDNF puncta at synaptic sites in control and glycine-treated cultures (n = 9 images; 3 coverslips/ 3 cultures; error bars indicate SEM).D, Quantitation of syt-IV and BDNF average intensity at synapses marked by synaptophysin in control and glycine, APV, or glycine/APV-treated cultures (n = 9 images; 3 coverslips/3 cultures; error bars indicate SEM).E, Quantitation of syt-IV average intensity at synapses marked by synaptophysin and percent syt-IV-positive synapses in control and 45 mm KCl-treated cultures (n = 9 images; 3 coverslips/3 cultures; error bars indicate SEM).F, Time lapse images of GFP-syt-17 (arrowheads indicate a mobile vesicle transiting along an axonal process).G, Representative images and quantitation of syt-17 average intensity at synapses in control and glycine-treated cultures. Pearson'sr for syt-17/synaptophysin = 0.09 ± 0.03 (cntrl), 0.07 ± 0.03 (1 h gly) (n = 9 images; 3 coverslips/ 3 cultures; error bars indicate SEM). *p < 0.05, **p < 0.01. Scale bars are 10 μm in all panels.
Figure 7.
Figure 7.
Syt-IV-harboring vesicles are distinct from synaptic vesicles at synapses.A, Representative example of a STED image of synaptic vesicles in dissociated hippocampal neurons immunostained for synaptophysin (syp). Left, Confocal image where synapses appear as single fluorescent puncta. Middle, STED image of the same field, where individual synaptic vesicles, clustered at synaptic sites, can be resolved. Right, Enlarged image of the boxed region indicated in the middle panel, showing single vesicles clustered at synaptic sites.B, Left, Confocal image of a hippocampal process labeled with an anti-synaptophysin antibody to mark synaptic sites. Middle, Dual color confocal image of synaptophysin and syt-IV. Note that the synaptophysin and syt-IV signals overlay in this image. Right, STED image of the same field in which anti-synaptophysin- and anti-syt-IV-labeled vesicles are distinct but are both present at synaptic sites.C, Enlarged images of the boxed region indicated in the right panel ofB.D, STED image of synaptic vesicles colabeled with syt-I and synaptophysin, where the signals are largely colocalized.E, STED image of vesicles colabeled with syt-IV and BDNF, where a subset of syt-IV-positive puncta colocalize with BDNF. Scale bars are 300 nm in all panels.
Figure 8.
Figure 8.
Syt-IV is recruited to presynaptic and postsynaptic sites.A, Neuroligin (Nlg)-expressing HEK cells immunostained for synaptophysin to mark presynaptic terminals and for GluR2 to mark postsynaptic sites. Nlg induced the formation of presynaptic terminals in contacting axons positive for synaptophysin that do not have corresponding postsynaptic structures and thus show no accumulation of GluR2 at the surface of the Nlg-expressing HEK cell.B, There was no accumulation of syt-IV signal on GFP-transfected HEK cells (top). In contrast, a ring of syt-IV in presynaptic terminals surrounded Nlg-IRES-GFP transfected cells (middle). Treatment with 200 μm glycine for 1 h induced the recruitment of syt-IV to these presynaptic terminals (bottom).C, Neurexin (Nrx)-expressing HEK cells immunostained with synapsin to mark presynaptic terminals and PSD-95 to mark postsynaptic sites. Nrx induced postsynaptic “hemisynapses” containing PSD-95 but lacking presynaptic terminals, marked with synapsin.D, A ring of syt-IV surrounding Nrx-GFP transfected HEK cells at postsynaptic sites was evident (top), and this syt-IV signal increased following induction of activity with glycine (bottom) (n = 20 cells for each condition; 3 cocultures/transfections; error bars indicate SEM. Significance was determined by a Student'st test; *p < 0.05, **p < 0.01, and ***p < 0.001). Scale bars are 10 μm.
Figure 9.
Figure 9.
BDNF is recruited concomitantly with syt-IV to pre-existing presynaptic and postsynaptic sites.A, Images of Nlg-expressing HEK cells co-cultured with hippocampal neurons in control and glycine-treated conditions and immunostained for the presynaptic cytoskeletal active zone marker bassoon.B, Images of Nrx-GFP-expressing HEK cells co-cultured with hippocampal neurons in control and glycine-treated conditions and immunostained for the postsynaptic density marker PSD-95.C, A ring of a BDNF signal is observed in presynaptic terminals surrounding Nlg-GFP-expressing HEK cells (top) and at postsynaptic sites surrounding Nrx-GFP-expressing HEK cells (bottom). This BDNF signal increases significantly in presynaptic terminals (quantified at top right) and increases slightly (but not significantly) at postsynaptic sites (quantified at bottom right) following treatment with 200 μm glycine for 1 h.
Figure 10.
Figure 10.
Syt-IV is necessary for scaling of synaptic strength.A, Western blot of syt-IV in hippocampal neurons treated with forskolin for 4 h or TTX for 48 h compared to control.B–E, mEPSC and mIPSC sample traces (B), amplitude (Amp) (C), and frequency (Freq) (D) from wild-type (WT) and syt-IV knockout (KO) neurons.E, mEPSC and mIPSC amplitude and frequency in BDNF knockout and syt-IV/BDNF double-knockout neurons.F, mEPSC and mIPSC amplitude in syt-IV knockout and wild-type neurons treated with TTX for 48 h compared to control, shown inA (for all mini recordings,n = 7–10 neurons; 3–4 coverslips/3–4 cultures; error bars indicate SEM).G, Normalized FM1-43 destaining traces from presynaptic terminals of control and TTX-treated wild-type and syt-IV knockout neurons. τ (sec) = 17.1 ± 1.8 (WT), 9.6 ± 0.8 (syt-IV KO), 11.9 ± 0.8 (WT + TTX), 8.7 ± 1.0 (syt-IV KO + TTX) (n = 9–15 coverslips/4 cultures, 15 boutons per coverslip for each condition; error bars indicate SEM).H, mIPSC frequency in wild-type and syt-IV knockout neurons in TTX-treated and control conditions (n = 7–10 neurons; 3–4 coverslips/ 3–4 cultures; error bars indicate SEM).
Figure 11.
Figure 11.
Syt-IV knockouts exhibit enhanced epileptiform responses compared to wild type.A, Wild-type hippocampal slice with voltage-sensitive dye optical signals from each of 464 photodiodes overlaid 50 ms after stimulation with 12 μA threshold current to induce an epileptiform response. CA1, CA3, subiculum (SB), and entorhinal cortex (EC) are indicated (left). Color-coded amplitude map of the optical signal detected 50 ms after stimulation and normalized to maximum amplitude is shown on the right.B, Syt-IV knockout hippocampal slice with voltage-sensitive dye optical signals overlaid 50 ms after stimulation with 6 μA threshold current to induce an epileptiform response with regions indicated (right) and color-coded amplitude map of the optical signal detected 50 ms after stimulation, normalized to maximum amplitude (left).C, Time course of a single evoked epileptiform response in a brain slice from wild-type (top) compared to syt IV knockout (bottom) normalized to maximum amplitude for comparison of spatial spread. Border between the hippocampus and entorhinal cortex (white curves), and the location of the stimulating electrode (white arrows) are indicated.
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