Glial Cell Line-Derived Neurotrophic Factor Stimulates the Morphological Differentiation of Cultured Ventral Mesencephalic Calbindin- and Calretinin-Expressing Neurons Hans R. Widmer,* Benoı ˆt Schaller,* Morten Meyer,† and Rolf W. Seiler* *Department of Neurosurgery, University of Bern, CH-3010 Bern, Switzerland; and †Department of Anatomy and Neurobiology, SDU-Odense University, Odense, Denmark Received October 18, 1999; accepted January 15, 2000 Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for mesencephalic dopa- minergic neurons. Subpopulations of these neurons express the calcium-binding proteins calbindin (CB) and calretinin (CR). Understanding the specific effects of GDNF on these neurons is important for the devel- opment of an optimal cell replacement therapy for Parkinson’s disease. To investigate the effects of GDNF on the morphological complexity of mesence- phalic tyrosine hydroxylase (TH)-immunoreactive (-ir), CB-ir, and CR-ir neurons, dissociated cultures of embryonic (E14) rat ventral mesencephalon were pre- pared. Chronic administration of GDNF (10 ng/ml) for 7 days promoted the survival of TH-ir and CB-ir neu- rons but did not alter the density of CR-ir neurons. Total fiber length/neuron and number of branching points/neuron of CB-ir and CR-ir cells were signifi- cantly increased after GDNF treatment (2 for CB-ir cells and 1.4 and 1.7, respectively, for CR-ir cells), which resulted in a significantly larger size of neurite field/neuron (2.9 and 1.5 for CB-ir and CR-ir neu- rons, respectively). The number of primary neurites/ neuron of CB-ir neurons was found to be 1.5 larger, while no difference could be detected for CR-ir cells. Assessment of the effects of GDNF on TH-ir neurons unveiled a similar outcome with an increased total fiber length/neuron (1.5), an increased number of pri- mary neurites/neuron (1.6), and a twofold larger size of neurite field/neuron. In conclusion, our findings rec- ognize GDNF as a neurotrophic factor that stimulates the morphological differentiation of ventral mesence- phalic CB-ir and CR-ir neurons. © 2000 Academic Press Key Words: GDNF; ventral mesencephalon; tyrosine hydroxylase; cell morphology; cell culture; rat; Parkin- son’s disease. INTRODUCTION Intracerebral grafting is an experimental therapy for patients with severe Parkinson’s disease. However, ethical concerns related to the use of fetal human tis- sue as well as a moderate tissue availability combined with a suboptimal survival of grafted dopaminergic neurons limit so far a more widespread application (11, 12, 25, 51, 69). Pretreatment of nigral donor tissue with neurotro- phic factors (5, 26, 83), coimplantation of cells geneti- cally engineered to release specific neurotrophic factors (61), and intracerebral infusions (27, 59, 64) may im- prove the survival and the functional potential of grafted dopaminergic neurons. Glial cell line-derived neurotrophic factor (GDNF) is a member of the trans- forming growth factor- superfamily (42) which also includes neurturin (39), persephin (48), and artemin/ neublastin (8, 60). GDNF is expressed throughout the rat CNS with relatively high levels in the developing striatum (16, 67), low levels in the adult striatum (66), and low levels in the ventral mesencephalon (16, 62). The precise mechanism of the GDNF action is not known, but the physiological response of the growth factor requires the presence of glycosylphosphatidyl- inositol (GPI)-linked proteins (designated GFR-) which are expressed on GDNF-responsive cells and which bind GDNF with high affinity (35). GDNF– GFR- forms a complex with the membrane-associated receptor Ret— considered as the signaling compo- nent—thereby inducing receptor tyrosine autophos- phorylation and activation (13, 34, 70, 71). It was re- cently suggested that calcium might be required for the complex formation between Ret and GDNF (50) and that Ret and the GFR-’s may be colocalized in the same tissues or expressed separately in projecting and target tissues, indicating the existence of at least two distinct modes of interaction (82). Moreover, the com- plementary and overlapping expression of ligand, Ret, and GFR- points to the existence of multiple mecha- nisms to imply trophic action for different neuronal populations (72). It is hence of particular interest that a recent report described Ret-dependent and -indepen- dent mechanisms of GDNF-induced signaling in neu- Experimental Neurology 164, 71– 81 (2000) doi:10.1006/exnr.2000.7418, available online at http://www.idealibrary.com on 71 0014-4886/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.