Abstract
Organotypic cultures from the ventral mesencephalon (VM) are widely used to model Parkinson's disease (PD). In this method, neurotoxic compounds have traditionally been applied to the media to induce a uniform dopaminergic (DAergic) cell death in the tissue slices, regardless of the variation existing among slices. This study demonstrates a refinement of the toxic induction technique. We show that unilateral application of 6-hydroxydopamine (6-OHDA) at the tissue surface by means of a microelectrode causes a precisely localized cell death that closely resembles an in vivo stereotactic model. This technique introduces an internal control that accounts for variation between slices and enables a precise quantification of the cell loss due to the toxin in use. We characterized organotypic VM cultures in terms of effects of 6-OHDA toxicity and number of DAergic neurons as judged by immunofluorescence and Western blots. Our findings illustrate that this new application technique greatly improves the representativeness of organotypic cultures as a model for PD.We characterized organotypic VM cultures in terms of effects of 6-OHDA toxicity and number of DAergic neurons as judged by immunofluorescence and Western blots. Our findings illustrate that this new application technique greatly improves the representativeness of organotypic cultures as a model for PD.
Highlights
Parkinson’s disease (PD) is a progressive neurodegenerative disease characterized by a selective loss of dopaminergic (DAergic) cells in the substantia nigra pars compacta (SNpc) in the midbrain and a subsequent deficiency of striatal dopamine (DA)
By means of a unilateral toxic application technique in which the toxin is applied at the tissue surface at the main DAergic cell groups in one hemisphere of the ventral mesencephalon (VM), namely the A8 DAergic cell group in the retrorubral field (RRF), A9 in the SNpc, and A10 in the ventral tegmental area (VTA), we demonstrate a method for precise quantification of the DAergic cell number in the two hemispheres of the same slice
In order to demonstrate how natural variation among tissue slices might bias the effect of toxic compounds, we identified the irregular distribution of DAergic cells along the rostrocaudal axis
Summary
Parkinson’s disease (PD) is a progressive neurodegenerative disease characterized by a selective loss of dopaminergic (DAergic) cells in the substantia nigra pars compacta (SNpc) in the midbrain and a subsequent deficiency of striatal dopamine (DA). DA-selective neurotoxic compounds, such as 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), and rotenone, have been widely used to model PD, as they induce rapid DAergic cell death and subsequent motor deficits in both rodents and primates[1,2,3,4]. Stahl et al.: Modeling Parkinson's Disease In Vitro. In vivo, such neurotoxins are stereotactically injected into the SN or striatum in one hemisphere to induce unilateral DAergic cell death[5,6,7,8]. These models convey high representativeness, as they maintain the whole biological system intact along with all systemic parameters. In vivo models are time consuming and implicate suffering of the animals involved
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