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First published online February 23, 2005
doi: 10.1242/10.1242/jcs.01694

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The chemokine SDF-1 differentially regulates axonal elongation and branching in hippocampal neurons

Fabien Pujol, Patrick Kitabgi and Hélène Boudin^*,

INSERM E0350, Hospital St Antoine, 184 rue du Fg St Antoine, 75571 Paris CEDEX 12, France

View larger version (36K): [in a new window]   Fig. 1. Western blot analysis of CXCR4 in cultured hippocampal neurons. Cultured neurons and rat brain membrane homogenates were immunoblotted with anti-CXCR4 antibody. The antibody revealed a 45 kDa band in both cultured neurons and rat brain samples, corresponding to the expected molecular mass for CXCR4. Molecular weight markers are indicated on the right.

View larger version (81K): [in a new window]   Fig. 2. CXCR4 is distributed in a non-uniform manner both in the somatodendritic and axonal domains, but is preferentially associated with axons. Hippocampal neurons at day 10 in culture were fixed and immunostained for CXCR4 (A1,B1) and either the dendritic marker MAP2 (A2) or the synaptic marker SV2 (B2). CXCR4 immunoreactivity was detected within neuronal cell bodies and a subset of nerve cell processes identified as MAP2-positive and -negative processes, indicating a somatodendritic and axonal localization of CXCR4 (A1-A3). The extent of axonal CXCR4 immunostaining was however more prominent than the dendritic labelling. CXCR4 immunoreactivity was not uniformly distributed but was often observed in large clusters along the cell body and processes (arrows in insets corresponding to higher magnification of boxed regions in A3). Double labelling with SV2 (B3) shows that these CXCR4 clusters did not overlap with the synaptic marker, indicating that CXCR4 clusters were localized at nonsynaptic sites. Bar, 20 µm.

View larger version (66K): [in a new window]   Fig. 3. The somatodendritic and axonal CXCR4 receptors are functionally coupled to intracellular Ca2+ mobilization. (A) Phase contrast and pseudocolour images of a field containing dendrites and axons. Neurons were loaded with Fura-2 AM and exposed to 20 nM SDF-1. Pseudocolour images were taken 5 seconds before SDF-1 stimulation (–5 sec), 7 seconds and 40 seconds after SDF-1 stimulation. Most processes displayed SDF-1-induced [Ca2+]i increase. The colour calibration bar shows pseudocolour mapping of the ratio of fluorescence emission at 340 and 380 nm. (B) Higher magnification of the boxed regions shown in A show a dendrite and an axon based on morphological features analysed by phase-contrast microscopy. (C) Traces of variations of [Ca2+]i recorded in the boxed regions depicted in A. A rise in [Ca2+]i levels was observed with a similar time course for dendrites and axons, 3 seconds after SDF-1 exposure as symbolized by asterisks, and recovered to near basal levels within 1 minute. Bar, 10 µm.

View larger version (93K): [in a new window]   Fig. 4. Developmental distribution of CXCR4 in hippocampal neurons from day 2 to day 7. Hippocampal cultures were fixed at day 2 (A1,A2), day 4 (B1-B4) and day 7 (C1,C2) and immunostained for endogenous CXCR4 at all stages and for MAP2 at day 4 (B3,B4) or were transfected at the time of plating with CXCR4-GFP (D1,D2) or the parent vector GFP (E1,E2). At day 2 and day 4, the tips of the axon and the other neurites visualized in the phase-contrast images (A1,B1) were frequently enriched in CXCR4 immunoreactivity as seen in the paired immunolabelled images (A2,B2). This CXCR4 accumulation at the leading edge of processes was observed in the absence (A1-A2, arrows) and in the presence of contacts with other neurons (B1-B4, arrows). At day 4, the tips of two processes originating from the same cell body were highly immunopositive for CXCR4 at the site of contact with neighbouring neurons (B1-B4, arrows). At day 7 (C1,C2), CXCR4 immunoreactivity was no longer enriched at growing tips, but was rather detected along a subset of processes. At day 2, in neurons transfected with CXCR4-GFP cDNA the fluorescence was preferentially concentrated at the tips of processes (D1,D2), whereas in neurons transfected with the parent GFP vector the fluorescence was diffusely distributed throughout the cell (E1,E2). Bar, 20 µm.

View larger version (58K): [in a new window]   Fig. 5. SDF-1 regulates axonal patterning without influencing the other neurites. Hippocampal neurons were treated at day 1 for 16 hours with (B) or without (A) 50 nM SDF-1 and were fixed at day 2 for analysis by phase-contrast microscopy. Quantification of the length of the axon and the other neurites in the presence and absence of the glial feeder layer (C), the growth cone area (D), the soma area (E), the number of processes emerging from the cell body (F), the number of primary branch points per 100 µm of axon and other neurites (G) and the number of growth cones per process (H) was carried out. The analysis was performed on control cells, cells treated with 50 nM SDF-1, cells treated with 50 nM SDF-1 in the presence of 1 µM bicyclam, a CXCR4 antagonist, and cells treated with bicyclam alone. Data are presented as the mean±s.e.m. of two to three independent experiments. **P<0.01, ***P<0.001 (Student's t-test) compared to corresponding measurements in control cells. Bar, 12 µm.

View larger version (117K): [in a new window]   Fig. 6. SDF-1 secreted from transfected COS cells acts as an extracellular signal regulating axonal patterning of CXCR4-GFP-expressing neurons. (A,B) COS cells transfected with CXCR4-GFP were incubated with or without 100 nM SDF-1 for 2 hours. In unstimulated cells, CXCR4-GFP was diffusely distributed throughout the cells (A) whereas SDF-1 stimulation induced the formation of CXCR4-GFP-containing intracellular clusters (B). (C) COS cells were separately transfected with either CXCR4-GFP (green) or SDF-1 (red, identified with an anti-SDF-1 antibody), and were subsequently co-cultured. CXCR4-GFP distribution exhibits a typical internalization pattern only if an SDF-1-expressing cell was located in the proximity of the former. By contrast, in cells distant from any SDF-1-transfected cells, CXCR4-GFP was distributed diffusely over the cell. (D,E) Hippocampal neurons were transfected at the time of plating with CXCR4-GFP. COS cells previously transfected with SDF-1 were added to the neuron culture at day 1 and the co-culture was fixed at day 2. CXCR4-GFP-expressing neurons in contact with SDF-1-expressing COS cells showed a reduced axonal length (E1,E2) compared to neurons distant from any SDF-1-transfected cells (D1,D2). Bar, 20 µm.

View larger version (40K): [in a new window]   Fig. 7. Quantitative analysis of the effect of SDF-1-expressing COS cells on the morphology of CXCR4-GFP-transfected hippocampal neurons. Hippocampal neurons transfected with CXCR4-GFP were co-cultured for 16-20 hours with SDF-1-transfected COS cells. For morphometric analysis, CXCR4-GFP-expressing neurons were classified into the control group or the SDF-1-stimulated group depending on their distance from a SDF-1-expressing COS cell. Quantification for the number of processes emerging from the cell body (A), the length of the axon and the other neurites (B), the number of branch points per 100 µm of axon and other neurites (C), the number of growth cones per process (D) were performed for the two groups. Data are presented as the mean±s.e.m. of two independent experiments. *P<0.05, **P<0.01 (Student's t-test) compared to corresponding measurements in control cells.

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