Characterization of organotypic ventral mesencephalic cultures from embryonic mice and protection against MPP⁺ toxicity by GDNF

Abstract

We characterized organotypic ventral mesencephalic (VM) cultures derived from embryonic day 12 (E12) mice (CBL57/bL6) in terms of number of dopaminergic neurons, cell soma size and dopamine production in relation to time in vitro and tested the effects of 1-methyl-4-phenylpyridinium (MPP(+)) and glial derived neurotrophic factor (GDNF) to validate this novel culture model. Dopamine production and dopaminergic neuron soma size increased dramatically with time in vitro, whereas the number of dopamine neurons declined by approximately 30% between week 1 and week 2, which was further reduced after week 4. GDNF treatment (100 ng/mL) increased dopaminergic neuron soma size (up to 43%) and DOPAC production (approximately three-fold), but not the number of dopamine neurons in control cultures. One-week-old cultures were more vulnerable to MPP(+), than three-week-old cultures. The EC(50) for dopamine depletion after 2 days exposure and 15 days of recovery were 0.6 and 7 microm, respectively. Both pre-treatment and post-treatment with GDNF are important to obtain maximal protection against MPP(+) toxicity. In one-week-old cultures (5 microm MPP(+), 2 days) GDNF provided potent neuroprotection with dopamine contents reaching control levels and number of tyrosine hydroxylase (TH)(+) cells up to 80% of control, but in three-week-old cultures (10 microm MPP(+), 2 days) the protective potential of GDNF was markedly reduced. Long recovery periods after MPP(+) exposure are required to distinguish between reversible or irreversible toxic and/or trophic effects.

Effects of glial cell line-derived neurotrophic factor deletion on ventral mesencephalic organotypic tissue cultures

“…It is well documented though that cell death is an early event, which should be bypassed in both the cultures and grafting experiments at the time of evaluation (Barker et al, 1996;Branton and Clarke, 1999;Clarkson et al, 1995;Mahalik et al, 1994). Thus, GDNF may have a greater effect on neurite formation than on cell viability of TH-positive neurons, a hypothesis also supported by others (Jacobsen et al, 2005).…”

Dopamine release in organotypic cultures of foetal mouse mesencephalon: effects of depolarizing agents, pargyline, nomifensine, tetrodotoxin and calcium

“…Organotypic mesencephalon cultures of embryonic day 12 (E12) mice were prepared as described previously (Jakobsen et al. , 2005).…”

RET Is Dispensable for Maintenance of Midbrain Dopaminergic Neurons in Adult Mice

“…Therefore, delineating molecular mechanisms that regulate survival of the SNC dopaminergic neurons may provide an avenue for better treatment or prevention of PD. To this end, numerous studies in vitro, in animal models that recapitulate PD, and in human PD patients, have shown that glial cell-line derived neurotrophic factor (GDNF) family ligands (GFLs) promote increased survival of SNC dopaminergic neurons, reduce functional deficits caused by dopaminergic toxins, and improve parkinsonian symptoms (Gash et al, 1996;Cheng et al, 1998;Date et al, 1998;Horger et al, 1998;Kordower et al, 2000;Tomac et al, 2000;Sun et al, 2004;Jakobsen et al, 2005). GFL ligands (GDNF and Neurturin) have been, or are in clinical trials in the treatment of PD (Gill et al, 2003;Slevin et al, 2005).…”