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

Abstract

Organotypic mesencephalic cultures provide an attractive in vitro alternative to study development of the nigrostriatal system and pathophysiological mechanisms related to Parkinson's disease. However, dopamine (DA) release mechanisms have been poorly characterized in such cultures. We report here endogenous DA release (assessed by high-performance liquid chromatography) in organotypic cultures of foetal mouse (E12) midbrain following single or multiple challenges (1-h incubations) with high K(+) or veratridine in the presence or absence of pargyline, nomifensine, calcium and/or tetrodotoxin (TTX). Basal (i.e. spontaneous) DA release was only detected in the presence of pargyline and nomifensine (PN), and was highly dependent on calcium and sensitive to TTX. Basal DA release increased 2.4-fold between week 3 (1st DA release experiment) and week 4 in vitro (3rd DA release experiment), DA tissue levels increased 1.6-fold and DA release expressed as a percentage of total DA (medium + tissue contents) increased from 20% to 34% during this growth period in vitro. Co-treatments with high K(+) or veratridine did not cause major changes in percentages of DA release. Tyrosine hydroxylase activity was increased by high K(+), but not by the other drug treatments. The acute (single or multiple) treatments with depolarizing agents did not affect the survival of dopaminergic neurons, but chronic low-level veratridine treatments were toxic.

Properties of dopaminergic neurons in organotypic mesencephalic-striatal co-cultures - evidence for a facilitatory effect of dopamine on the glutamatergic input mediated by α-1 adrenergic receptors

“…, 1997), also express VGluT2. These results suggest that also in OC dopaminergic neurons release glutamate, in addition to release of DA (Larsen et al. , 2008), and are in agreement with the recent data obtained with optogenetic techniques (Tecuapetla et al.…”

Intermittent, low dose carbon monoxide exposure enhances survival and dopaminergic differentiation of human neural stem cells

“…Sample preparation; medium : Cells were washed twice in Hank’s balanced salt solution (Life Technologies), followed by incubation (2 hrs/36°C) in 200 μl of Hank’s balanced salt solution containing 10μM nomifensine (Research Biochemicals International). A 100 μl sample was transferred to HPLC vials containing 50 μl of mobile phase (10% methanol (v/v), 20 g/l citric acid monohydrate, 100 mg/l octane-1-sulfonic acid sodium salt, 40 mg/l EDTA dissolved in Milli-Q water and pH adjusted to 4.0; all from Merck/VWR Chemicals) and stored at -20°C until HPLC analysis with electrochemical detection [ 60 , 61 ].…”

Effects of ciliary neurotrophic factor and leukemia inhibiting factor on oxytocin and vasopressin magnocellular neuron survival in rat and mouse hypothalamic organotypic cultures

“…Nevertheless, despite the latter drawback, organotypic cultures provide the best experimental models to study the physiological and molecular properties of a wide variety of specific neuronal phenotypes. Among these phenotypes are neurons found in the hippocampus (Bergold and Casaccia-Bonnefil, 1997; Bruce et al, 1995; Gahwiler and Hefti, 1985), cortex (Baratta et al, 1996; Dammerman et al, 2000; Snyder-Keller, 2004), cerebellum (Birgbauer et al, 2004; Davids et al, 2002; Dupont et al, 2006), spinal cord (Bonnici and Kapfhammer, 2008; Eustache and Gueritaud, 1995; Li et al, 2008), brain stem (Rusnak and Gainer, 2005), hypothalamus (House et al, 1998; Israel et al, 2008; Shimizu et al, 2008; Wray et al, 1988), Mesencephalon-(Franke et al, 2003; Holmes et al, 1995; Larsen et al, 2008; Lyng et al, 2007; Plenz and Kitai, 1998), retina (Kaempf et al, 2008) and cochlear nucleus (Kesser et al, 2007; Khan et al, 2007; Qi et al, 2008). One important technical advance that has made the organotypic culture method accessible to many laboratories has been the development of the stationary explant (Stoppini et al, 1991)as an alternative to the roller-tube approach which was originally developed by Gahwiler and co-workers (Gahwiler, 1981; Gahwiler et al, 1997).…”