Representative images from 6-OHDA-lesioned rats treated with TGFα for 2 weeks. Under low magnification, two distinct nestin+ (green) waves of cells were visible (a). Whereas one wave appears to arise from the ipsilateral (IL) SVZ and migrates through the IL striatum toward the TGFα infusion site, the other wave appears to arise from the contralateral (CL) SVZ and migrates through the septum toward the TGFα infusion site. Within the lateral ventricle SVZ of the TGFα-treated rats, ubiquitous hypercellular nodules were present that contained both GFAP+(red) and nestin+/GFAP+colocalized (yellow) cells (b). Under higher magnification, the striatal wave of cells is composed of nestin+cells (c,d). The nestin+cells exhibit a high nuclear-to-cytoplasmicratio and possess numerous fine processes that extend considerable distances(d).*,Hypercellularnodule; CC, corpus callosum; LV, lateral ventricle; S, septum; Str, striatum. Scale bars: b,50 μm; c,20μ m; d,10 μm.

Representative images of SVZ in 6-OHDA-lesioned rats treated with TGFα or vehicle for 2 weeks. On examination of the bilateral SVZ at low power (a-d), more nestin+ (green) cells are present in the lesioned TGFα-treated SVZ (a,b) than in the lesioned PBS-administered SVZ (c,d). Within the expanded SVZ of TGFα-treated rats (e), β-catenin accumulated compared with PBS-administered SVZ (f). *, Lateral ventricle; CC, corpus callosum; S, septum; Str, striatum. Scale bars: a-d, 200 μm; e,f, 50 μm.

Intrastriatal TGFα administration increases nestin+ SVZ cells. High-magnification images of thelesioned TGFα-treated SVZ(a) and lesioned PBS-administered SVZ (b) depict a thickened SVZ when TGFα was administered. The thickened SVZ contained numerous nestin+/GFAP+(yellow) cells that exhibited an oval soma with elongated processes (1) accompanied by numerous nestin-/GFAP+ (red) cells (2). The lesioned PBS-administered SVZ is within normal limits of cellularity and contains both GFAP+(red) cells (1) and nestin+ (green) cells (3). This difference in the number of nestin+ cells was quantified as cells per millimeter² after 2 and 4 weeks of infusion (c). There were significantly more nestin+cells in the SVZ of the TGFα-treated rats (white) relative to the SVZ in the vehicle control (black) at both 2 and 4 weeks (t test; p < 0.05). *, Lateral ventricle; IL, ipsilateral. Scale bar, 20 μm.

Representative images of SVZ in 6-OHDA-lesioned rats treated with TGFα or vehicle for 2 and 4 weeks. Vehicle-administered rats exhibited scattered nestin+ (red) cells among numerous GFAP+ (blue) cells in the SVZ with an expected number of PCNA+(green) nuclei homogeneously distributed throughout the lateral wall(a-d). The ipsilateral (IL) and contralateral (CL) SVZ in vehicle-administered rats exhibited similar features at both 2 (a,b) and 4 (c, d) weeks of infusion. After 2 weeks of TGFα infusion, both ipsilateral (e) and contralateral SVZ (f) contained numerous nestin+ (red) cells with PCNA+ (green) nuclei. The hypercellular nodules present in both SVZ were composed entirely of PCNA+ nuclei. After 4 weeks of TGFα infusion, the ipsilateral SVZ (g) contained numerous nestin+ cells that were also present in the adjacent striatum. The ipsilateral hypercellular nodules appeared to become confluent, forming a thickened SVZ (g). The contralateral SVZ (h) was also thickened but lacked any nestin+ cells in the adjacent striatum. The hypercellular nodules in the contralateral SVZ remained prominent (h). *, Lateral ventricle; CC, corpus callosum; Str, striatum. Scale bar, 250 μm.

Representative images of BrdU-labeled cells in 6-OHDA-lesioned rats after 2 weeks infusion of TGFαor vehicle. Intrastriatal infusion of TGFα produced numerous BrdU+ cells in the ipsilateral SVZ (a, b) compared with vehicle-administered rats (c, d). The hypercellular SVZ nodules (e) that extend into the lateral ventricle (*) contain numerous nestin+cells (red), but only a fraction of these cells are also BrdU+(green). The majority of the intensely stained BrdU+nuclei reside at the SVZ-nodule interface. At higher magnification(f),a proportion of the nestin+cells (red) are BrdU+(green).GFAP(blue)andβ-tubulin (red)werealso examined in the SVZ(g). Isolated GFAP+/BrdU+and β-tubulin+/BrdU+cells were present. At higher magnification (h), the isolated β-tubulin+(red)/BrdU+(green) cell was confirmed by three-dimensional confocal imaging. Str, Striatum. Scale bar, 50μm.

Intrastriatal TGFα administration reduces PSANCAM+ SVZ cells. Rats that received an intrastriatal infusion of vehicle demonstrated numerous PSANCAM+cells in the SVZ (a), whereas rats treated with TGFα possessed relatively few PSANCAM+cells in the SVZ (b). This difference was quantified as cells per millimeter after 2 and 4 weeks of infusion (c). There were significantly fewer PSANCAM+cells in the SVZ of the TGFα-treated rats (white) relative to the SVZ in the vehicle control (black) (t test; p<0.05). IL, Ipsilateral. Scale bar, 20μm.

Characterization of the cell migratory waves. In the parenchyma (gray), ipsilateral (IL; dark green) and contralateral (CL; light green) cell, migratory waves radiated away from the respective SVZs (dark and light blue) during intrastriatal infusion of TGFα (orange) (a). After 2 weeks, the cell migratory waves were only present anterior (A) to the infusion site, and cell densities reduced at the anterior tip of the waves (b). The cell migratory waves were predominantly composed of nestin+/PCNA+cells exhibiting a fusiform morphology (c-h). After 4 weeks of TGFα infusion, dopamine-denervated rats showed prominent nestin+cellular waves in the contralateral prelimbic and orbital cortex and olfactory bulb with a hypercellular thickening of the ipsilateral olfactory ventricle lining (i, j). D, Dorsal; V, ventral; P, posterior; AC, anterocaudal. Scale bar, 25 μm.

Lack of new DA neurons. None of the TH (red)-immunopositive neurons in the SNc of any animal in any group had nuclear incorporation of BrdU (green), although small infrequent BrdU+nuclei were found in the region (a). The lack of striatal or nigral DA neurogenesis was reflected by an absence of improvement in amphetamine-induced side bias in either TGFα-or vehicle-administered rats (b). Scale bar, 20 μm.