Dopaminergic development of prenatal ventral mesencephalon and striatum in organotypic co-cultures

Abstract

Using organotypic co-cultures of rat embryonic day 14 (E14) ventral mesencephalon (VM and E21 striatum, we have described the developmental changes in (i) dopamine (DA) neurochemistry; (ii) numbers of DA neurons; and (iii) protein expression of tyrosine hydroxylase (TH), DA transporter (DAT), and glutamic acid decarboxylase (GAD 65/67), over 17 days in vitro (DIV). Co-cultures demonstrated changes in DA development similar to those observed in vivo. The numbers of VM DA neurons remained relatively constant, while levels of VM DA progressively increased through 10 DIV. After 3 DIV, the levels of striatal DA increased substantially, through 10 DIV. Tissue levels of DA metabolites homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) reflected changes in tissue DA concentrations, indicating that release and metabolism of DA are similar to these characteristics observed in vivo. Western blot analysis of TH protein expression revealed large increases in VM TH after only 3 DIV, followed by a decline in levels through 17 DIV; levels of striatal TH, in contrast, increased through this period. Additionally, DAT and GAD 65/67 expression increased, in both the VM and striatum, over 17 DIV. By 17 DIV, many measures of DA function had decreased from those assessed at 10 DIV, thus providing an approximate limit to the effective duration of use of this co-culture model. Our results provide a much-needed description of the neurochemical changes that occur during the maturation of VM and striatum in organotypic cocultures. Additionally, these results provide a foundation for future studies to assess toxic challenges of the developing nigrostriatal DA system, in vitro.

Polychlorinated biphenyl-induced oxidative stress in organotypic co-cultures: Experimental dopamine depletion prevents reductions in GABA

“…Organotypic co-cultures of VM and striatum were prepared based on the methods described by Lyng et al (2007a) and Snyder-Keller et al (2001). Under aseptic conditions, E14 and E21 fetuses (the presence of sperm after mating was considered embryonic day E0) were removed from timed-pregnant Sprague-Dawley rats (Taconic Farms, Germantown, NY) under isoflurane anesthesia of the dam.…”

“…Our studies of DA and GABA development and the neurotoxicological consequences of PCB exposure in organotypic co-cultures of developing rat striatum and ventral mesencephalon (VM) (Lyng et al, 2007a(Lyng et al, , 2007b have provided a framework to further study the mechanisms of PCB-induced neurotoxicity. There have been reports that PCB-induced inhibition of VMAT subsequently affects DA neurochemistry (Mariussen et al, 1999;Mariussen et al, 2001;Bemis & Seegal 2004;Seegal et al, 1997;Lyng et al, 2007b).…”

“…The organotypic co-culture system, originally developed by Stoppini et al (1991) and modified by Snyder-Keller et al (2001), provides an in vitro model of the nigrostriatal DA system that closely mimics that of the intact animal (Lyng et al, 2007a). Exposure of co-cultures to PCBs, for periods ranging from 1 to 14 days, resulted in depletion of tissue DA, DA associated proteins (TH and DAT), GABA, the proteins glutamic acid decarboxylase (GAD 65/67), as well as increased DA neuronal death (Lyng et al, 2007b).…”

Graft outcomes influenced by co‐expression of Pax7 in graft and host tissue

“…To date, the major approach has been to use stem/progenitor cells taken from differentiating regions of fetal brain (Ostenfeld et al 1999;Hitoshi et al 2002;Parmar et al 2002;Hurelbrink & Barker, 2005). Whereas even stem/progenitor cells can be coaxed into becoming the appropriate neurons in culture (Simon et al 2003;Lyng et al 2007), a similar outcome in vivo has been problematic (Svendsen et al 1996;Lundberg et al 1997;Barker & Dunnett, 1999;Mendez et al 2005).…”

Corticosterone enhances N-methyl-d-aspartate receptor signaling to promote isolated ventral tegmental area activity in a reconstituted mesolimbic dopamine pathway

“…In addition, though the effects of in vitro aging on receptor density have not yet been examined in co-cultures of the NAcc and VTA, Martens and Wree (2001) noted that NMDA receptor distribution was comparable in hippocampal slices aged in vitro and those taken from aged-matched rats and previous studies have found that a number of synaptic components remain at a steady level following a brief initial depression (Bahr et al, 1995). Additionally, numerous studies have investigated the use of long-term organotypic co-cultures of the VTA/NAcc and reported in tact morphology, re-innervation of dopaminergic tracts into the NAcc, and survival up to 3 months (Jaumotte and Zigmond, 2005; Lyng et al, 2007; Ostergaard et al, 1990). Thus, organotypic cell culture studies allow for long-term and detailed manipulations investigating mechanisms of brain activity while minimizing environmental influences and may provide a better understanding of CORT-NMDA interactions in vivo .…”