Glial cell line-derived neurotrophic factor is essential for postnatal survival of midbrain dopamine neurons

Abstract

Glial cell line-derived neurotrophic factor (GDNF) is one of the most potent trophic factors that have been identified for midbrain dopamine (DA) neurons. Null mutations for trophic factor genes have been used frequently for studies of the role of these important proteins in brain development. One problem with these studies has been that often only prenatal development can be studied because many of the knockout strains, such as those with GDNF null mutations, will die shortly after birth. In this study, we looked at the continued fate of specific neuronal phenotypes from trophic factor knockout mice beyond the time that these animals die. By transplanting fetal neural tissues from GDNF -/-, GDNF +/-, and wild-type (WT) mice into the brain of adult wild-type mice, we demonstrate that the continued postnatal development of ventral midbrain dopamine neurons is severely disturbed as a result of the GDNF null mutation. Ventral midbrain grafts from -/- fetuses have markedly reduced DA neuron numbers and fiber outgrowth. Moreover, DA neurons in such transplants can be "rescued" by immersion in GDNF before grafting. These findings suggest that postnatal survival and/or phenotypic expression of ventral mesencephalic DA neurons is dependent on GDNF. In addition, we present here a strategy for studies of maturation and even aging of tissues from trophic factor and other knockout animals that do not survive past birth.

Fig. 1.

A, B, Tyrosine hydroxylase (TH) immunohistochemistry on sections from mouse midbrain; C, D, TH staining from the striatum of adult mice. The average TH staining pattern in mice that have been lesioned with MPTP (A, C) compared with the staining pattern seen in an intact adult wild-type mouse (B, D) is demonstrated in this figure. Note the sparse distribution of TH-positive neurons in the MPTP-treated substantia nigra (A) and striatum (C), compared with the nontreated control (B, D). E andF represent two transplants from a GDNF −/− donor (E) and a wild-type donor (F) at 8 weeks after grafting. These cresyl violet-stained sections demonstrate the most common placement of the transplants as well as the relative size of the two graft types (transplants from wild-type donors were on average twice as large as transplants from GDNF −/− fetuses). Arrows, Graft/host border; Str, striatum; cc, corpus callosum; tp, transplant; lv, lateral ventricle. Scale bar (shown in F):A, B, 50 μm;C–F, 100 μm.

Fig. 2.

TH immunocytochemistry of grafts from WT (A, B), GDNF +/− (C, D), and GDNF −/− (E, F) donors. D andF are larger magnifications of the grafts shown inC and E, respectively, whereasB represents a high magnification of a wild-type graft other than the one seen in A. Arrowsdelineate grafts, and the magnified areas are demarcated with corners in C and E. Note the much greater TH-immunoreactive cell numbers and fiber outgrowth in WT compared with −/− grafts, which were virtually devoid of TH-positive neurons and neurites and, additionally, contained a large number of macrophages. The +/− grafts had an intermediate number of TH-positive cells and fibers. Scale bar (shown in F): A, 70 μm; C, E, 100 μm;B, D, F, 30 μm.

Fig. 3.

Bar graph depicting both the number of TH-positive cells per section (left) and the staining intensity in a 1 mm halo surrounding the graft/host border (right). The staining intensity (right graph) is expressed as a mean value subtracted from background on a standardized gray scale ranging from 0 (white) to 256 (black). Theleft graph depicts the mean number of neurons per section from transplants in the different groups. The group legends are depicted underneath the graph. As can be seen from this bar graph, transplants from GDNF −/− fetuses contained significantly fewer TH-positive neurons than the other groups and also had a marked decrease in the TH staining intensity surrounding the graft/host border (right). Only four transplants of −/− tissue contained any TH-positive neurons. However, GDNF −/− transplants treated with GDNF in the preincubation buffer (striped bars) did not differ from WT controls, either in number of cells per section or in staining density. Thus, GDNF treatment appeared to normalize these two parameters of dopamine cell survival in the −/− grafts. Heterozygous grafts (Hetero) contained fewer mean cells per section than the WT controls, but the innervation density was similar. Statistical results were obtained with ANOVA and Scheffé's post hoc analysis. **p < 0.01; ***p < 0.001.

Fig. 4.

Effects of 6 nm GDNF pretreatment on WT grafts shown by TH immunocytochemistry.Arrows delineate transplants. A,B, WT grafts after GDNF pretreatment. C,D, WT grafts without GDNF pretreatment. The areas shown in higher magnification (B, D) areoutlined with corner markers in A andC. Note the increased TH cell survival and fiber outgrowth after GDNF exposure, even in wild-type grafts. Scale bar (shown in D): A, C, 100 μm; B, D, 40 μm.

Fig. 5.

Effects of 6 nm GDNF pretreatment on −/− grafts shown by TH immunocytochemistry.A, Low-power photomicrograph showing −/− transplants without GDNF immersion (left graft) and with GDNF immersion (right graft) within the same brain.B, C, GDNF −/− grafts with GDNF pretreatment. D, E, GDNF −/− grafts without GDNF pretreatment. The areas shown at higher magnification inC and E are outlined with corner markers in B and D, respectively. Note markedly increased numbers of TH-positive cells as well as fiber outgrowth after GDNF pretreatment. The −/− transplant pretreated with vehicle was virtually devoid of TH-positive cells and fiber outgrowth. The sparse plexus of TH-immunoreactive neurites in the striatum of the host inD and E most likely originates from spared innervation from the host nigra, because MPTP produces only a partial dopamine denervation in mice. The GDNF −/− transplant shown in D and E contained numerous larger cells reminiscent of macrophages (D, bottom arrow). Arrows delineate transplants. Scale bar in (shown in E): A, 200 μm;B, 100 μm; D, 65 μm;C, E, 40 μm.