Quantitation of dopaminergic neurons in 3-dimensional reaggregate tissue culture by computer-assisted image analysis

Vidal, Luis; Heller, Bárbara; Won, Lisa; Heller, Alfred

doi:10.1016/0165-0270(94)00110-3

Cited by 7 publications

(9 citation statements)

References 21 publications

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“…In these aggregates, the number of dopaminergic neurons per flask increased in linear fashion with increasing numbers of mesencephalic cells added, i.e., there was a linear relationship (r 2 5 0.927) between the number of mesencephalic cells initially added and the number of tyrosine hydroxylase-positive cells observed as we have previously reported (Vidal et al, 1995). Similarly the amount of dopamine per flask increased linearly with the number of mesencephalic cells added (r 2 5 0.895).…”

Section: Resultssupporting

confidence: 60%

“…Given the finding that dopaminergic axonal fiber density did not vary with dopaminergic cell numbers per individual aggregate, it was possible to use the volume of aggregate tissue containing patches of dopaminergic axonal processes as a measure of total axonal arbor size per aggregate. The volume of aggregate tissue containing dopaminergic axonal arbors was determined using a computer-assisted image analysis program as previously described (Vidal et al, 1995). Areas containing axonal processes in aggregate sections were determined without operator knowledge of the number of dopaminergic neurons within a given aggregate.…”

Section: Resultsmentioning

confidence: 99%

“…Cell counts obtained by random selection of 30 sections, without the application of any correction for possible double counting of neurons, were found to be in excellent agreement with those obtained by serial reconstruction. The utility of this method of assessing and comparing numbers of dopaminergic cells was demonstrated by the finding that the number of dopaminergic cells observed in the aggregates was directly proportional to the number of mesencephalic cells initially added, as long as there was an excess of target cells (Vidal et al, 1995), a finding replicated in this study over a more extended ratio of striatum to mesencephalon. …”

Section: Quantitation Of Dopaminergic Cell Numbers Per Flaskmentioning

confidence: 92%

“…The total number of dopaminergic neurons in aggregates within a flask was quantitated by counting THpositive cells in a random sample of aggregate sections and determining section and aggregate volumes by a computerassisted image analysis system developed for this purpose and described in detail in previous publications (Heller et al, 1988;Vidal et al, 1995). The number of TH-positive cells in a given aggregate flask was estimated by counting cells from a random selection of 30 sections.…”

Section: Quantitation Of Dopaminergic Cell Numbers Per Flaskmentioning

confidence: 99%

“…The areas containing dopaminergic axonal arbors were determined by comparison of the photographs projected on the computer screen with the drawings made from direct microscopic observation of the sections. The boundaries of histofluorescent patches were delineated on the monitor screen by placing dots around their perimeter with a ''mouse'' pointing device (Vidal et al, 1995). The area of each patch outlined was computed by using an image analysis program, and the volumes of patches containing dopamine axonal histofluorescence were then calculated from the section thickness (10 µm).…”

Section: Determination Of Cell Number and Volume Of Dopaminergic Axonmentioning

confidence: 99%

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

1997

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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.

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Section: Resultssupporting

confidence: 60%

Section: Resultsmentioning

confidence: 99%

Section: Quantitation Of Dopaminergic Cell Numbers Per Flaskmentioning

confidence: 92%

Section: Quantitation Of Dopaminergic Cell Numbers Per Flaskmentioning

confidence: 99%

Section: Determination Of Cell Number and Volume Of Dopaminergic Axonmentioning

confidence: 99%

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

1997

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

et al. 1997

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The murine weaver (wv) mutation is characterized by a genetically determined loss of several neuronal populations, which include the nigrostriatal dopaminergic neurons. Animals homozygous for the wv gene exhibit marked deficits in dopaminergic morphological and neurochemical parameters. The wv gene shows incomplete dominance in that heterozygous (wv/+) mice exhibit moderate reductions in midbrain dopaminergic neuron number. It is unclear whether the dopaminergic neuronal loss in homozygous and heterozygous animals results from an effect of the wv gene solely on the dopaminergic neurons or is due to a failure of interaction of dopaminergic neurons with target cells of the striatum. This issue has been addressed utilizing three-dimensional reaggregate tissue cultures to determine whether the wv gene acts directly on the mesencephalic dopaminergic neurons. Embryonic mesencephalon and striatum from wv/+ and wild-type (+/+) brains were dissociated and the cells recombined into four mesencephalic-striatal aggregate combinations: (1) mesencephalic(+/+)-striatal(+/+)aggregates; (2) mesencephalic(wv/+)-striatal(wv/+)aggregates; (3) mesencephalic(wv/+)-striatal(+/+)aggregates; and (4) mesencephalic(+/+)-striatal(wv/+)aggregates. At 29 days and 57 days of culture, the number of dopaminergic neurons and dopamine content from mesencephalic-striatal aggregates consisting of mixed genotype or from only wv/+ tissue were quantitated and compared with that from mesencephalic-striatal cultures prepared from +/+ tissue alone. At both culture time points, aggregates containing wv/+ mesencephalon coaggregated with either wv/+ or +/+ striatum contained fewer dopaminergic neurons than mesencephalic-striatal cultures composed of only +/+ cells. Coaggregation of +/+ mesencephalon with wv/+ striatum did not have a detrimental effect on dopaminergic cell number. The findings demonstrate that the difference in the number of mesencephalic dopaminergic neurons between wv/+ and +/+ animals seen in vivo can be reproduced in three-dimensional reaggregate culture. Since the coculture of +/+ striatum with wv/+ mesencephalon did not appear to rescue wv/+ dopaminergic neurons in the aggregates as compared to wv/+ striatum and, wv/+ striatum proved to be a perfectly adequate target for +/+ mesencephalic dopaminergic neurons, it appears that the effect of the wv gene is on the dopaminergic neurons themselves.

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Preferential resistance of dopaminergic neurons to glutathione depletion in a reconstituted nigrostriatal system

et al. 2000

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Quantitation of dopaminergic neurons in 3-dimensional reaggregate tissue culture by computer-assisted image analysis

Cited by 7 publications

References 21 publications

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

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

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

Preferential resistance of dopaminergic neurons to glutathione depletion in a reconstituted nigrostriatal system

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