ADDITIVE EFFECT OF GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR AND NEUROTROPHIN-4/5 ON RAT FETAL NIGRAL EXPLANT CULTURES M. MEYER, a;c E. R. MATARREDONA, b R. W. SEILER, a J. ZIMMER c and H. R. WIDMER a * a Department of Neurosurgery, University of Bern, Inselspital, Freiburgstrasse, CH-3010 Bern, Switzerland b Department of Physiology, University of Ca¤diz, Ca¤diz, Spain c Department of Anatomy and Neurobiology, Institute of Medical Biology, University of Southern Denmark, Odense University, Odense, Denmark AbstractöTransplantation of embryonic dopaminergic neurons is an experimental therapy for Parkinson’s disease, but limited tissue availability and suboptimal survival of grafted dopaminergic neurons impede more widespread clinical application. Glial cell line-derived neurotrophic factor (GDNF) and neurotrophin-4/5 (NT-4/5) exert neurotrophic e¡ects on dopaminergic neurons via di¡erent receptor systems. In this study, we investigated possible additive or synergistic e¡ects of combined GDNF and NT-4/5 treatment on rat embryonic (embryonic day 14) nigral explant cultures grown for 8 days. Contrary to cultures treated with GDNF alone, cultures exposed to NT-4/5 and GDNF+NT-4/5 were signi¢cantly larger than controls (1.6- and 2.0-fold, respectively) and contained signi¢cantly more protein (1.6-fold). Treatment with GDNF, NT-4/5 and GDNF+NT-4/5 signi¢cantly increased dopamine levels in the culture medium by 1.5-, 2.5- and 4.7- fold, respectively, compared to control levels, and the numbers of surviving tyrosine hydroxylase-immunoreactive neurons increased by 1.7-, 2.1-, and 3.4-fold, respectively. Tyrosine hydroxylase enzyme activity was moderately increased in all treatment groups compared to controls. Counts of nigral neurons containing the calcium-binding protein, calbindin- D28k, revealed a marked increase in these cells by combined GDNF and NT-4/5 treatment. Western blots for neuron- speci¢c enolase suggested an enhanced neuronal content in cultures after combination treatment, whereas the expression of glial markers was una¡ected. The release of lactate dehydrogenase into the culture medium was signi¢cantly reduced for GDNF+NT-4/5-treated cultures only. These results indicate that combined treatment with GDNF and NT4/5 may be bene¢cial for embryonic nigral donor tissue either prior to, or in conjunction with, intrastriatal transplantation in Parkinson’s disease. ß 2001 IBRO. Pub- lished by Elsevier Science Ltd. All rights reserved. Key words: tissue culture, dopamine, Parkinson’s disease, calbindin. Pretreatment of fresh or cultured nigral donor tissue with neurotrophic factors or co-grafting of genetically engi- neered cell lines as well as intracerebral infusions of neu- rotrophic factors may improve survival of grafted dopaminergic neurons (Haque et al., 1996; Espejo et al., 2000; Mendez et al., 2000). Glial cell line-derived neurotrophic factor (GDNF) belongs to a distant branch of the transforming growth factor-L superfamily (Lin et al., 1993, 1994), which also includes neurturin (Kotzbauer et al., 1996), persephin (Milbrandt et al., 1998) and the recently described artemin/enovin/neublas- tin (Baloh et al., 1998; Masure et al., 1999; Rosenblad et al., 2000). The mechanism of the GDNF action is not completely understood, but recent work demonstrates that the phys- iological response of GDNF requires glycosyl-phospha- tidylinositol (GPI)-linked proteins designated GFR-K1 and GFR-K2 which are expressed on GDNF responsive cells and bind GDNF with high and moderate a/nity, respectively (Jing et al., 1997; Glazner et al., 1998; Horger et al., 1998). GDNF has been found to form a complex with GFR-K1 or GFR-K2 and the receptor ty- rosine kinase Ret to induce receptor tyrosine autophos- phorylation and activation (Jing et al., 1996; Treanor et al., 1996; Trupp et al., 1996). In cultures of fetal midbrain, GDNF stimulates sur- vival and di¡erentiation of dopaminergic neurons and increases high-a/nity dopamine uptake (Lin et al., 273 *Corresponding author. Tel.: +41-31-6322770; fax: +41-31- 3822414. E-mail address : hanswi@insel.ch (H. R. Widmer). Abbreviations : BDNF, brain-derived neurotrophic factor ; CB, cal- bindin; DAB, 3,3P-diaminobenzidine ; E, embryonic day ; FFRT, free-£oating roller-tube ; GDNF, glial cell line-derived neurotro- phic factor ; GFAP, glial ¢brillary acidic protein ; HBSS, Hanks’ balanced salt solution ; HEPES, N-(2-hydroxy ethyl) piperazine- NP-(2-ethane sulfanic acid) ; HPLC, high-performance liquid chromatography ; IR, immunoreactive ; LDH, lactate dehydroge- nase ; NADH, nicotinamide adenine dinucleotide ; NSE, neuron- speci¢c enolase; NT-4/5, neurotrophin-4/5; PBS, phosphate-bu¡- ered saline ; PD, Parkinson’s disease ; PVDF, polyvinylidene di£uoride; SDS, sodium dodecyl sulfate; TH, tyrosine hydroxy- lase. NSC 5259 26-11-01 www.neuroscience-ibro.com Neuroscience Vol. 108, No. 2, pp. 273^284, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain PII:S0306-4522(01)00418-3 0306-4522 / 01 $20.00+0.00