The glial sling is a migratory population of developing neurons

Abstract

For two decades the glial sling has been hypothesized to act as a guidance substratum for developing callosal axons. However, neither the cellular nature of the sling nor its guidance properties have ever been clearly identified. Although originally thought to be glioblasts, we show here that the subventricular zone cells forming the sling are in fact neurons. Sling cells label with a number of neuronal markers and display electrophysiological properties characteristic of neurons and not glia. Furthermore, sling cells are continuously generated until early postnatal stages and do not appear to undergo widespread cell death. These data indicate that the sling may be a source of, or migratory pathway for, developing neurons in the rostral forebrain, suggesting additional functions for the sling independent of callosal axon guidance.

Fig. 1

Neuronal markers label the subcallosal sling. (A–I) Coronal sections from E17 C57Bl/6J mouse brains stained with either Cresyl Fast Violet (A) or antibodies directed against the molecules indicated. In C–F NeuN labeling is red (nuclear labeling) and GFAP and TUJ1 are green. Scale bar in F: 400 μm (A,B); 120 μm (C); 200 μm(D,E); 35 μm (F). Scale bar in I: 100 μm.

Fig. 2

Patch-clamp recordings of sling neurons demonstrate that these cells fire action potentials. Current-clamp recordings in response to intracellular injection of depolarizing current pulses in (A,B) identified sling cells and (C) wedge cells. (A) Responses to 55 pA (black) and 75 pA (red), 100-msecond long current pulses of a sling cell at E18. The Na⁺-dependent spike (solid arrow) is suppressed after 8 minutes of TTX application, and the Ca²⁺-dependent spike (dotted arrow) is abolished in the absence of Ca²⁺ (0 Ca²⁺). At P2 (B) sling cells evoke TTX-sensitive Na⁺ spikes. Inset depicts spontaneous action potentials recorded, at rest, from the same cell; all spontaneous spikes were reversibly abolished by TTX. By contrast, identified glial wedge cells (C) respond to depolarizing currents with nearly linear increases in voltage responses, and evoke no regenerative spikes. (D) Phase-interference contrast micrograph showing the sling lying directly underneath the corpus callosum (CC). A patch pipette (P) is in place to record from an identified sling cell. Sling cells were identified morphologically directly below the corpus callosum as they form a stream of tightly connected cells, with their leading processes pointing toward the midline (on the left). Only one focal plane is visible in this image, therefore surrounding sling cells are not in focus. Scale bar: 20 μm.

Fig. 3

Cellular morphology of sling and glial wedge cells filled with biocytin during current-clamp recordings. In cellular recordings of the type shown in Fig. 2, the recording pipette was used to fill cells with biocytin. (A,B) E17 filled cells (red) within the sling (A) and glial wedge (B, arrow). Slices were doubled labeled with GFAP to identify the glial wedge (green labeling in B). (C,D) Cells at P0 within the sling showed a more mature neuronal morphology with multiple dendrites (red). The cell in D is shown double labeled with NeuN (arrow indicates a yellow nucleus). Scale bar: 20 μm.

Fig. 4

Birth dating of the sling. Coronal sections of brains from animals injected with BrdU on either E14 (A) E15 (B) or E16 (C) and sacrificed at E17. Animals were also injected at E17 (D) or E18 (E) and sacrificed at P0 or injected at P2 and sacrificed at P3 (F). All sections were processed for BrdU immunohistochemistry using a fluorescent (Cy3) secondary antibody. Arrows in B and F show the position of the sling. Scale bar: 250 μm in all panels.

Fig. 5

Migration of the sling cells from the SVZ to the cortical midline. Individual litters were injected with BrdU at either E16 (A–F) or P3 (G–J) and then pups or whole litters were sacrificed at the times shown. Brains were sectioned coronally and processed for BrdU immunohistochemistry (red labeled cells in all panels) and counterstained with Sytox green (green cells in all panels except F). In F BrdU-labeled sections were double-labeled with PCNA which demonstrated the presence of proliferating cells within the sling (yellow cell in F). By using BrdU labeling sling cells could be seen migrating successively toward the midline (arrows in A–E and H and I). Scale bars: 200 μm in C for A–F and in J for G–J.

Fig. 6

Postnatal development of the sling. Coronal sections of P0 (A,B), P5 (C,D) or P10 (E,F) mouse brain were processed for NeuN immunohistochemistry (A,C,E) or for Cresyl Violet staining (B,D,F). The sling is present at P0 (arrows in A and B) and P5 (arrows in C,D), but absent by P10. Scale bar: 250 μm for all panels.

Fig. 7

TUNEL labeling of the sling. Coronal cryostat sections at 10 μm were labeled using TUNEL and an FITC secondary antibody. TUNEL-positive cells were present within the sling at E17 and E18 (arrows in A and B respectively) but no TUNEL-positive cells were present at P0 (C) or P3 (D), despite the continuing generation of the sling cells at these ages (see Fig. 3). Even at P5 (E) and P10 (F) when the sling structure disappears there is no TUNEL labeling. Scale bar: 200 μm in all panels.