Turnover of dopaminergic neurons in the substantia nigra. The total number of TH+ substantia nigra pars compacta neurons (bilaterally, identified with immunoperoxidase and cresyl violet counterstain) did not change with increasing age (mean ± SEM) (a), although the same region contained apoptotic TUNEL+ condensed nuclei (green) in TH+ (red) neurons dispersed among TH+ neuropil and larger, nonapoptotic TH+ perikarya (b). Confocal three-dimensional image of a newborn (BrdUrd+, green nucleus) TH+ (red) neuron in the substantia nigra of a 16-week-old mouse after 2 weeks of BrdUrd through drinking water followed by 4 weeks without BrdUrd (c). Two cross sections analyzing the depth of the image (z axis) in c along the y and x axis are shown in the schematic drawing (Lower Right). TH+ (red), BrdUrd+ (green) cells express the neuronal markers (violet-blue) Hu (d) and NeuN (e). In f, a BrdUrd+ (green) cell is shown adjacent to a TH+ (red) cell, which could be mistaken for a newborn neuron if not studied in three dimensions. Newborn [³H]thymidine labeled cresyl violet stained neurons in the medial substantia nigra (g) of a 16-week-old mouse, 6 weeks after a 3-day i.p. infusion of [³H]thymidine. (h) EM image of the same neurons as in g with an enlargement showing a synapse in i, with the position indicated by an arrow in h. The synaptic cleft is shown between two arrowheads (i). Semithin section of the midbrain aqueduct (the lumen indicated by asterisks) after photoconversion of DiI, 1 day after intraventricular injection (j). The box in j delineates the area shown in an EM micrograph obtained from the adjacent ultrathin section (k). Dense staining is visible in the lumen (j) and lysosomes (k, indicated by arrows) in ependymal cells (ep). se, a subependymal cell nucleus; arrowheads, ependymal membranes. Note the absence of labeling of membranes and in cytoplasm of subependymal cells (k). Neurospheres from the midbrain aqueduct and third ventricular recess after in vivo labeling of the ependymal layer with DiI (l) or rhodamine-conjugated latex beads (n) contained label suggesting ependymal origin. Such neurospheres were passaged and induced to differentiate into neurons (Tuj-1+, red) and astrocytes (GFAP+, violet-blue) (m). [Scale bars = 10 μm (b, e, f, and h), 5 μm (c and d), 0.1 μm (i), 10 μm (j), 1 μm (k).]

Newborn substantia nigra neurons project to the striatum. (a) Schematic drawing showing the right intraventricular injection of DiI (red) to label cells lining the ventricular system, and left intrastriatal fluorogold injection (blue) to retrogradely trace nigrostriatal projections. (b–d) Six weeks after DiI injection, a few nigral ependyma-derived cells (red) are retrogradely labeled with fluorogold (white) injected 2 days before analysis. Two DiI-labeled cells are indicated by arrows in b, and the inferior one also contains fluorogold. DiI-labeled neurons are morphologically indistinguishable from surrounding neurons (b–d). Colocalization of BrdUrd and fluorogold in the confocal microscope is shown in d. Colocalization was further analyzed three-dimensionally by confocal imaging (e) of a newborn (BrdUrd+, green nucleus) nigral neuron retrogradely labeled with fluorogold (white), injected 3 days earlier in the striatal nerve terminal field. The 10-week-old mouse received BrdUrd through drinking water for 2 weeks, and the animal was killed 2 months later. (f) Schematic drawing showing injection of eGFP PRV (green) and CTB (yellow) into the right somatosensory cortex. Both the virus and CTB were axonally transported, but the latter was not transferred over synaptic connections. Some nigral ipsilateral neurons contained both CTB and eGFP PRV, whereas contralaterally only eGFP PRV was found. (g) Three-dimensional and single channel registrations of confocal images of a transsynaptically labeled newborn neuron, TH+ (violet-blue), BrdUrd+ (red), and eGFP PRV+ (green) in an adult mouse that received BrdUrd in drinking water for 3 weeks, and was allowed to survive 1 month before eGFP PRV was administered. [Scale bars = 20 μm (b–d) and 5 μm (e and g).]

Newborn substantia nigra neurons derive from cells lining the ventricular system. Six hours after an injection of DiI into the lateral ventricle, the label was restricted to the ependymal layer of the midbrain aqueduct (a). Four days later, animals receiving BrdUrd through drinking water, displayed DiI+ cells (red, b and d) with BrdUrd+ nuclei (green in d). BrdUrd incorporation (red) after six i.p. injections of the nucleotide analogue 12 h before analysis was found along the surface of the aqueduct (c). Cell nuclei are visualized with 4′,6-diamidino-2-phenylindole (violet-blue). Seven days after DiI (e), a DiI-labeled cell (red) was observed to colabel with the immature neuronal marker CRMP-4 (green). A TH+ cell (red) showing the immature cell marker nestin (green) in the cytoplasm is shown in f.In g (see Movie 1, which is published as supporting information on the PNAS web site, www.pnas.org), aTH+ (green), DiI+ cell (red) in the substantia nigra expressing the neuronal marker NeuN (violet-blue) is shown. TH+ (green) neurons containing punctate DiI (red) are shown in the substantia nigra pars compacta 14 days (h) and 55 days (i) after a DiI injection into the contralateral ventricle. Arrows point at cells shown at higher magnification. Note that DiI is also present in punctate clusters outside neurons, putatively labeling precursor cells or glia in the substantia nigra. A TH+ (green) neuron containing rhodamine-conjugated latex beads (red) is shown 6 weeks after an i.c.v. bead injection in the contralateral ventricle (j). In k, the total number of DiI+/TH+ neurons in the adult mouse substantia nigra pars compacta are shown at different time intervals after DiI injection [each black dot corresponds to one animal, n = 29, r² (correlation coefficient) = 0.87, bilateral results are based on cell counts in SNpc contralateral to injection]. The number of DiI+/TH+ neurons was reduced in animals receiving BrdUrd in the drinking water for the first 42 days after DiI (open circles, n = 7). Animals that were infused with the anti-mitotic agent Ara-Cyt (n = 4, indicated by red diamond), completely lacked DiI-labeled TH+ neurons. Images in d–j were collected in the confocal laser scanning microscope. V, ventrally; L, laterally. [Scale bars = 100 μm(a and b), 20 μm (c), 5 μm (d and e), 10 μm (f), 50 μm (h–j).]

MPTP increases neurogenesis in the substantia nigra. MPTP-induced effects on neuronal BrdUrd incorporation in substantia nigra (a, chronic continuous i.c.v. infusion; b, repeated systemic injections for 2 days after which mice were analyzed immediately or 3 weeks later) and hippocampus (c, chronic continuous i.c.v. infusion) and on the total number of nigral DiI+/TH+ neurons (d) are shown as bar diagrams (mean ± SEM). For details on treatment, see Materials and Methods. n = 3–5 in each group; asterisk indicates P < 0.05, Student's unpaired t test with Bonferroni′s procedure to balance for increased type I error after multiple comparisons in the same animal.