The chemokine stromal cell-derived factor-1 regulates GABAergic inputs to neural progenitors in the postnatal dentate gyrus

Abstract

Stromal cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) are important regulators of the development of the dentate gyrus (DG). Both SDF-1 and CXCR4 are also highly expressed in the adult DG. We observed that CXCR4 receptors were expressed by dividing neural progenitor cells located in the subgranular zone (SGZ) as well as their derivatives including doublecortin-expressing neuroblasts and immature granule cells. SDF-1 was located in DG neurons and in endothelial cells associated with DG blood vessels. SDF-1-expressing neurons included parvalbumin-containing GABAergic interneurons known as basket cells. Using transgenic mice expressing an SDF-1-mRFP1 (monomeric red fluorescence protein 1) fusion protein we observed that SDF-1 was localized in synaptic vesicles in the terminals of basket cells together with GABA-containing vesicles. These terminals were often observed to be in close proximity to dividing nestin-expressing neural progenitors in the SGZ. Electrophysiological recordings from slices of the DG demonstrated that neural progenitors received both tonic and phasic GABAergic inputs and that SDF-1 enhanced GABAergic transmission, probably by a postsynaptic mechanism. We also demonstrated that, like GABA, SDF-1 was tonically released in the DG and that GABAergic transmission was partially dependent on coreleased SDF-1. These data demonstrate that SDF-1 plays a novel role as a neurotransmitter in the DG and regulates the strength of GABAergic inputs to the pool of dividing neural progenitors. Hence, SDF-1/CXCR4 signaling is likely to be an important regulator of adult neurogenesis in the DG.

Figure 1.

Expression of SDF-1 and CXCR4 in the adult mouse dentate gyrus. a–d, CXCR4, SDF-1, nestin, and DCX are all expressed in the dentate gyrus of EGFP BAC reporter mice (4 weeks). In c, the arrowhead illustrates an example of a nestin-EGFP-expressing type 1 cell. Arrow illustrates an example of a nestin-EGFP-expressing type 2 cell. e, f, RT-PCR analysis of CXC chemokine receptor expression by FACS derived cells isolated from the DG of nestin-EGFP (e) and DCX-EGFP (f) transgenic BAC reporter mice. Note the robust CXCR4 expression in both populations of cells. g, Colocalization of BrdU labeling (red, immunohistochemistry) with CXCR4-EGFP-expressing cells (arrowheads) in the DG of CXCR4-EGFP BAC reporter mice (4 weeks). Scale bars: a–d, g, 50 μm; c, insets I, II, 20 μm.

Figure 2.

SDF-1 is expressed in synaptic vesicles in the adult mouse dentate gyrus. a, SDF-1-mRFP1 expression in numerous vesicles like structures throughout the dentate gyrus from an SDF-1-mRFP1 BAC transgenic mouse (4 weeks). The arrows show a blood vessel. The arrowheads illustrate SDF-1-mRFP1-containing vesicles within the cell body of a basket cell. The asterisks illustrate unlabeled cell bodies surrounded by SDF-1-mRFP1-containing synaptic vesicles. GrDG, Granule cell layer of DG; PoDG, polymorphic layer of the DG. a′, The same section stained with parvalbumin (pv). a″, Merged image. b, Confocal microscopy reveals expression of SDF-1-mRFP1 in association with blood vessels (tomato lectin, green) in the DG. c, Confocal microscopy reveals that SDF-1-mRFP1 is localized to vesicles within a pv-expressing neuron (basket cell) in the DG. d, e, SDF-1-mRFP1-labeled nerve terminals (red puncta) are localized in close proximity to dividing cells in the DG labeled by BrdU incorporation (d) and Ki67 staining (e) (both green). Scale bars: a–a″, 20 μm; b, c, 32 μm; d, e, 10 μm.

Figure 3.

SDF-1-mRFP1 is localized to GABAergic terminals in close proximity to neural progenitors in the DG. a–c, SDF-1-mRFP/CXCR4-EGFP bitransgenic mice illustrate the presence of SDF-1-mRFP/VGAT-positive nerve terminal vesicles in close juxtaposition to a CXCR4-EGFP-expressing neural progenitor (arrow). d–f, SDF-1-mRFP/nestin-EGFP bitransgenic mice illustrate the presence of SDF-1-mRFP/VGAT-positive nerve terminals close to a nestin-EGFP-expressing type 2 neural progenitor (arrow). c, f, Serial optical sections of cells indicated by a white arrow are in the right panels. Red arrows, Terminals just labeled for SDF-1; blue arrows, VGAT only; pink arrows, both SDF-1/VGAT. Examples are from 4-week-old mice. Scale bars: (in a, d) a–f, 20 μm; c and f right panels, 8 μm.

Figure 4.

SDF-1-mRFP1 is localized to synaptic vesicles in nerve terminals in close proximity to CXCR4-positive cells in SGZ. a–d, SDF-1-mRFP/CXCR4-EGFP bitransgenic mice illustrate the presence of SDF-1-mRFP1 (red, red arrows) in presynaptic terminals (blue, blue arrows), labeled by synaptotagmin (a), synaptoporin (b), SV2 (c), or bassoon (d), in close juxtaposition to a CXCR4-EGFP-expressing neural progenitors (green). Colocalization of SDF-1-mRFP1 with other markers is indicated by the pink arrows. Colocalization is also illustrated in three dimensions in each panel. Examples are from 4-week-old mice. Scale bars, 4 μm. GrDG, Granule cell layer of DG; PoDG, polymorphic layer of DG.

Figure 5.

SDF-1 enhances GABA mediated activation of neural progenitors in the dentate gyrus. Recordings taken from type 2 nestin-EGFP-expressing cells in acutely isolated slices from mouse DG are shown. a, Bicuculline (Bic; 100 μm) inhibited PSCs and produced an outward current. b–d, SDF-1 (40 nm) produced a long-lasting inward current (b), which was reversed by bicuculline (c, d). e, f, The inward current produced by SDF-1 was also observed in the presence of either TTX (0.5 μm, e) or Cd (10 μm, f). g, SDF-1 was ineffective in cells that were pretreated with bicuculline. h, i, SDF-1 increased both the frequency and amplitude of PSCs recorded from type 2 cells (h) and this was also the case in the presence of TTX (i). In a–d, 30 of 42 cells examined exhibited an outward current in response to bicuculline and 49 of 69 exhibited an inward current in response to SDF-1. In addition, 7 of 12 cells exhibited an inward current in response to SDF-1 in the presence of TTX and 5 of 9 did so in the presence of Cd²⁺. SDF-1 increased the frequency and amplitude of 14 of 20 cells examined, and in TTX, 6 of 12 cells. **p < 0.01; ***p < 0.001. Error bars indicate SEM.

Figure 6.

SDF-1 is tonically released in the adult mouse dentate gyrus. Recordings made from nestin-EGFP-expressing type 2 cells in the DG. a, The CXCR4 antagonist AMD3100 (1 μm) produced an outward current. b–f, Outward currents produced by AMD3100 or bicuculline (b, c, 100 μm) were not additive (d, e, f, n = 44 cells). g–i, SDF-1 increased and AMD3100 reduced the frequency and amplitude of PSCs. These effects of AMD3100 were observed after treatment with SDF-1 (g) or in its absence (h). **p < 0.01; ***p < 0.001. i, SDF-1 was unable to produce an effect in the presence of AMD3100, but did so after washout of the antagonist. Error bars indicate SEM.

Figure 7.

Effects of SDF-1 on GABAergic inputs to DCX-EGFP-expressing cells in the adult mouse dentate gyrus from DCX-EGFP BAC transgenic mice. a–d, SDF-1 (40 nm) increased the amplitude and frequency of PSCs recorded in DCX-EGFP-expressing cells with immature morphology and this was reversed by both bicuculline (a, b) and AMD3100 (c, d). SDF-1 produced this effect in 20 of 56 cells. This was reversed by bicuculline in five of seven cells and by AMD3100 in three of five cells examined. **p < 0.01; ***p < 0.001. e, f, Both bicuculline (3 of 4) and AMD3100 (3 of 5) produced an outward current in DCX-EGFP cells and their effects were not additive. g, h, SDF-1 produced an inward current in DCX-EGFP cells and its effects were reversed by bicuculline (g, 4 of 5) and by AMD3100 (h, 3 of 6). Error bars indicate SEM.