Brain injury does not alter the intrinsic differentiation potential of adult neuroblasts

Abstract

Neuroblasts produced by the neural stem cells of the adult subventricular zone (SVZ) migrate into damaged brain areas after stroke or other brain injuries, and previous data have suggested that they generate regionally appropriate new neurons. To classify the types of neurons produced subsequent to ischemic injury, we combined BrdU or virus labeling with multiple neuronal markers to characterize new cells at different times after the induction of stroke. We show that SVZ neuroblasts give rise almost exclusively to calretinin-expressing cells in the damaged striatum, resulting in the accumulation of these cells during long term recovery after stroke. The vast majority of SVZ neuroblasts as well as newly born young and mature neurons in the damaged striatum constitutively express the transcription factor Sp8, but do not express transcription factors characteristic of medium-sized spiny neurons, the primary striatal projection neurons lost after stroke. Our results suggest that adult neuroblasts do not alter their intrinsic differentiation potential after brain injury.

Figure 1.

Dcx⁺ immature neurons are abundant in the damaged striatum after stroke. A, B, Photomicrographs of Dcx (A) and Dcx/NeuN double (B) immunostaining in the damaged striatum 2 weeks after stroke. C–E, Higher magnification of the boxed areas in B showing Dcx⁺/NeuN⁺ cells. F, G, BrdU was injected intraperitoneally twice daily during days 3–7 after stroke and BrdU/Dcx double immunostaining was performed at 2 weeks recovery from stroke showing BrdU⁺/Dcx⁺ cells in the contralateral (F) and ipsilateral (G) striatum. Scale bars: (in A) A, B, 100 μm; (in C) C–G, 10 μm.

Figure 2.

New CR⁺ cells are generated in the damaged striatum after stroke. A–H, BrdU was injected intraperitoneally twice daily on days 3–7 of recovery from stroke and BrdU was combined with immunostaining for neuronal markers at 6 weeks after stroke. BrdU⁺/NeuN⁺ (A) and BrdU⁺/CR⁺ (H), but not BrdU⁺/DARPP-32 (B), BrdU⁺/CB⁺ (C), BrdU⁺/PV⁺ (D), BrdU⁺/SOM⁺ (E), BrdU⁺/ChAT⁺ (F), or BrdU⁺/TH⁺ (G) cells are found in the damaged striatum. I–N, BrdU⁺/NeuN⁺/CR⁺ cells (arrows) in the damaged striatum 6 weeks after stroke. Note that NeuN protein is lightly expressed in one newborn CR⁺ cell (J). O, P, BrdU was injected intraperitoneally twice daily during days 1–14 after stroke and BrdU was combined with immunostaining for neuronal markers at 6 weeks after stroke. BrdU⁺/NeuN⁺ cells (O, arrows) and BrdU⁺/CR⁺ cells (P, arrows) are more abundant in the damaged striatum. Scale bar: (in P) A–P, 10 μm.

Figure 3.

Retrovirus-labeled SVZ-derived cells in the damaged striatum 6 weeks after stroke express CR. A, B, To label dividing cells, replication-incompetent retroviruses encoding the marker gene alkaline phosphatase (AP) were bilaterally injected into the SVZ 1 d before inducing stroke. AP histochemistry staining in the contralateral (A) and ipsilateral (B) SVZ and AP/Dcx double immunostaining (C, D) were performed 2 d after injection. Note that most AP-labeled cells are located within the SVZ of the contralateral hemisphere (A), whereas some AP-labeled cells have migrated out of the SVZ and into the damaged striatum of the ipsilateral hemisphere (B, D) 2 d after injection. Myelin is weakly stained with the BCIP/NBT in the contralateral (intact) striatum. E–J, AP⁺/CR⁺ cells in the damaged striatum 6 weeks after stroke. Note that two AP⁺/CR⁺ cells (J, arrows) are morphologically alike, and may have been generated from a single progenitor cell. Scale bars: (in B) A, B, 100 μm; (in J) C–J, 10 μm.

Figure 4.

Lentivirus-labeled SVZ-derived cells in the damaged striatum 2 weeks poststroke express Dcx. A, Self-inactivating lentiviruses expressing GFP were injected into the SVZ 1 d before inducing stroke and GFP/Dcx double immunostaining was performed 2 weeks after stroke showing that most GFP-labeled cells in the damaged striatum express Dcx. B–G, Higher magnification of GFP⁺/Dcx⁺ cells in A (arrows). Scale bars: A, 100 μm; (in B) B–G, 10 μm.

Figure 5.

Lentivirus-labeled SVZ-derived cells in the damaged striatum 6 weeks after stroke express CR. A, Self-inactivating lentiviruses expressing GFP were injected into the SVZ 1 d before inducing stroke and GFP/CR double immunostaining was performed 6 weeks after stroke showing that most GFP-labeled cells in the damaged striatum express CR. B–G, Higher magnification of GFP⁺/CR⁺ cells in A (arrows). Note that one GFP⁺ cell in E lightly expresses CR protein. H–J, Photomicrographs of A GFP⁺/GFAP⁺ cell (arrow in J) in the SVZ 6 weeks after stroke. K–O, Photomicrographs of GFP⁺/Dcx⁺ cells in the SVZ (arrow in M) and RMS (arrow in O) 6 weeks after stroke. Scale bars: A, 100 μm; (in B) B–O, 10 μm.

Figure 6.

Newly born young neurons in the damaged striatum express CR. A, Dcx/CR double immunostaining in the damaged striatum 2 weeks after stroke. B–E, Higher magnification of the boxed areas in A showing Dcx⁺/CR⁺ cells in the corpus callosum (B) and damaged striatum (C–E). F, G, Photomicrographs of CR (F) and Dcx/CR double (G) immunostaining in the RMS and OB of the normal adult rat brain (10 weeks old). H–K, Higher magnification of the boxed areas in G showing Dcx⁺/CR⁺ cells in the RMS and its surroundings (H–J) and the OB (K, arrows). Scale bars: (in A) A, F, G, 100 μm; (in B) B–E, H–K, 10 μm.

Figure 7.

CR⁺ cells are more abundant in the ipsilateral striatum 6 months after stroke, compared with the contralateral striatum. A, B, Photomicrographs of CR immunostaining in the contralateral (A) and ipsilateral (B) striatum 6 months after stroke. C, Higher magnification of the boxed area in (A). D, E, Higher magnification of the boxed areas in B showing morphology of CR⁺ cells in the damaged striatum. Scale bars: (in B) A, B, 200 μm; (in E) C–E, 50 μm.

Figure 8.

Newly born neurons in the damaged striatum express Sp8. A–D, Dcx⁺ cells in the adult rat SVZ express Sp8. E, F, Dcx⁺ cells in the normal (E) and damaged (F) striatum express Sp8. G, H, CR⁺/Sp8⁺ cells are small interneuron-like cells in the normal (G) and damaged (H) striatum. Note that a CR⁺ medium-sized aspiny neuron does not express Sp8 (arrow in G). I–L, Photomicrographs of a BrdU⁺/CR⁺/Sp8⁺ cell in the damaged striatum 6 weeks after stroke. Scale bar: (in L) A–L, 10 μm.

Figure 9.

The majority of Dcx⁺ cells in the adult OB express Sp8. A–L, Photomicrographs of Dcx⁺/Sp8⁺ cells in the glomerular layer and external plexiform layer (A–D), superficial (E–H) and deep (I–L) granular cell layer of the OB. Scale bar: (in L) A–L, 10 μm.

Figure 10.

Model of neurogenesis in the damaged striatum after stroke. Neuroblasts generated by neural progenitor cells (including type B neural stem cells and type C transit-amplifying progenitor cells) in the adult SVZ migrate into the damaged striatum and then differentiate into CR⁺ cells after stroke. The transcription factor Sp8 is constitutively expressed during the entire process from the neuroblast stage through differentiation into mature CR⁺ interneuron-like cells.