Comparison between normal developing striatum and developing striatal grafts using drug-induced fos expression and neuron-specific enolase immunohistochemistry

Abstract

The cell-level functional maturation of cell suspension grafts from embryonic day 14–15 rat striatal primordia implanted unilaterally into ibotenic acid lesioned striata of adult female rats was studied from two days to 10 weeks post-grafting. The functional and morphological characteristics of the grafts were compared with those of adult grafts (one year after implantation), normal adult striata and postnatal developing striata (up to four weeks after birth). Serial sections were stained with Cresyl Violet and investigated immunohistochemically with antibodies against dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein (DARPP-32, as a striatal marker), tyrosine hydroxylase (as a marker of dopaminergic fibres), Fos protein (as a cell-level marker of functional dopaminergic host-graft interactions), and neuron-specific enolase (correlated to differentiation and functional maturation of neuronal cells). Selected sections were double-stained for DARPP-32 and either tyrosine hydroxylase, Fos or neuron-specific enolase. The rats used to study dopamine receptor-activated expression of Fos were killed 2 h after administration of either the dopamine-releasing agent d-amphetamine (5mg/kg intraperitoneally) or the dopamine-receptor agonist apomorphine (0.25 mg/kg subcutaneously, at which dosage it is active only on supersensitive receptors of denervated neurons). In normally developing rats, amphetamine induced Fos expression in both the striatum and globus pallidus by two weeks after birth; by four weeks, the pattern of amphetamine-induced Fos immunoreactivity was similar to that observed in adults. In the globus pallidus of both two- and three-week-old rats, amphetamine induced greater expression of Fos than in adults. Apomorphine did-not induce appreciable Fos activation in either the striatum or the globus pallidus at any stage of development. In striatal grafts, amphetamine induced Fos expression from three weeks after implantation onwards, and by five to 10 weeks post-grafting the pattern of Fos immunore-activity was similar to that observed in adult grafts. However, apomorphine induced a considerable number of Fos-positive nuclei in striatal grafts at three and four weeks after grafting. Neuron-specific enolase immunoreactivity was moderate in normal adult striatum and very high in the adult globus pallidus, and mainly located in neuronal perikarya and processes. Before two weeks of age, most neuron-specific enolase immunoreactivity was observed in internal capsule fascicles and the striatal afferents. Between two and four weeks after birth, neuron-specific enolase immunoreactivity in striatal and globus pallidus neurons gradually increased, while that in afferent fibres decreased to adult levels. Neuron-specific enolase labelling of adult striatal grafts revealed striatum-like and non-striatum-like patches, the latter usually containing some strongly neuron-specific enolase positive neurons resembling cortical or pallidal neurons. During the first week after transplantation, neuron-specific enolase positivity in the graft was restricted to highly immunoreactive coarse fibres coming from the surrounding lesioned area. During the second and subsequent weeks after implantation, thin and less densely immunoreactive fibres sprouted from the coarse fibres and progressively innervated the grafts. Neuron-specific enolase immunoreactivity began to be found in grafted neurons at the end of the second week post-grafting; over the third and fourth weeks after transplantation neuron-specific enolase positive neurons became more frequent and more intensely stained in both striatum-like and non-striatum-like patches, and by week five to 10 the graft exhibited adult characteristics. This study shows that intrastriatal fetal striatal grafts develop rapidly not only in terms of morphology but also function, and with a time-course closely matching that of the developing striaturn in situ. Functional integration began at the end of the third week post-grafting, and adult characteristics were attained within five weeks of implantation.