Regulation of developing dopaminergic axonal arbor size in three-dimensional reaggregate tissue culture

Abstract

The role of target availability in determining axonal size of the developing dopaminergic neuron was examined in three-dimensional reaggregate culture in which these neurons undergo normal neurochemical and morphological development. The ratio of striatal target cells to dopaminergic neurons was systematically increased from 2:1 to 50:1 (striatal to mesencephalic cells). These ratios provide sufficient target for quantitative survival of dopaminergic neurons similar to that seen in vivo. After 3 weeks in culture, dopamine levels increased linearly and proportionally to dopaminergic cell number over a 25-fold relative increase in target cell availability. The amount of neurotransmitter per dopaminergic cell remained constant, suggesting that the axonal arbor did not increase as a function of target availability. This conclusion was directly tested by morphological analysis of the dopaminergic axonal arbor in single aggregates containing between 0 and 45 dopaminergic cells under conditions in which sufficient target was not a factor in dopaminergic cell survival. As was the case with respect to dopamine concentration, axonal arbor size per cell remained constant in the face of increasing target availability. Thus, availability of target cells under these conditions, is not, per se, the limiting factor on the rate of growth of the developing dopaminergic axonal arbor.

In vitro evidence that the reduction in mesencepalic dopaminergic neurons in the weaver heterozygote is not due to a failure in target cell interaction

“…These results, showing that the decreases in dopaminergic cell number are directly proportional to reductions in dopamine content, are consistent with recent investigations using reaggregate tissue cultures which demonstrate that the size of the dopaminergic axonal arbor as well as dopamine content per cell is constant and, as a result, dopamine content is directly proportional to dopaminergic cell number (Heller et al 1997).…”

Computer experiments to determine whether over- or under-counting necessarily affects the determination of difference in cell number between experimental groups

“…Within hours after the formation of the aggregates, the cells within them form clusters of 'like' cells by migration. The cells subsequently put out axons which form selective specific synaptic connections with appropriate target cells resulting in the construction of projection pathways analogous to those observed in the intact brain (Won et al, 1989;Heller et al, 1992Heller et al, , 1993Heller et al, , 1997. Experiments involving this 'three-dimensional reaggregate' system typically yield estimates of cell number or cell density for the various experimental groups.…”

“…By using computer-assisted image analysis, the volumes of the sampled aggregate sections are estimated and the total aggregate volume for the flask is measured (Vidal et al, 1995;Heller et al, 1997). The sum of the cell counts for the 30 sampled aggregate sections from a flask divided by the sum of the corresponding section volumes provides an estimate of cell density for the flask.…”

“…In this paper, computer experiments are performed in order to examine the consequences of over-and undercounting of cells by comparing with the ideal method of counting cell centers and with the optical disector method. Since cell counting must take place in a specific laboratory environment, the computer experiments here are modeled on the counting of dopaminergic neurons in three-dimensional reaggregate cultures (Won et al, 1989;Heller et al, 1992Heller et al, , 1993Heller et al, , 1997. For modeling purposes, this culture system has the advantage of being more easily mimicked than in vivo systems in which it is difficult to model the spatial arrangement of cells realistically by use of spatial stochastic processes (Schmitz, 1998;Schmitz et al, 1999).…”

Reassessment of Caspase Inhibition to Augment Grafted Dopamine Neuron Survival

“…encephalic cells dissociated and grafted in the presence of 100 µM Ac-YVAD-CMK; groups 3 and 4 were grafted Mesencephalic Reaggregates with mesencephalic reaggregates representing 300,000 Preparation of VM reaggregates followed a protocol mesencephalic cells that were exposed to either control modified from Heller and colleagues (15) and recently aggregate medium or to aggregate medium plus 100 µM described by Sortwell et al (36). Employment of re-Ac-YVAD-CMK.…”