GABA uptake and phenotypic characteristics of the subcommissural ependymocytes of the semi-desertic rodent, Meriones shawi: correlation with serotoninergic innervation.

Many studies have emphasized species differences in the serotoninergic innervation and phenotypic characteristics of the subcommissural organ in mammals. The post-natal distribution patterns of serotonin-containing fibers, the onset of gamma-aminobutyric acid uptake, and glial markers have been studied in the subcommissural organ of the semi-desertic rodent, Meriones shawi, by using immunohistochemical and autoradiographic techniques. Abundant serotoninergic fibers can be observed in the subcommissural organ of the newborn Meriones, some of them running among the ependymocytes and reaching the apical part of this organ. During the first 2 post-natal weeks of development, the subcommissural organ displays a progressive increase of serotonin fiber density throughout the organ, including the apical part. The existence of a dense serotonin-containing basal plexus concomitantly with a high apical innervation in this organ is a specific characteristic of Meriones. Ependymocytes of this organ have the ability to take up gamma-aminobutyric acid at birth. This uptake decreases and completely disappears from the 2nd week. The reappearance of gamma-aminobutyric acid accumulation in ependymocytes of the adult subcommissural organ after destruction of the serotonin innervation by a neurotoxin (5-7 dihydroxytryptamine) suggests an inhibitory effect of the serotonin innervation on this accumulation. Immunohistochemical studies of the phenotype of the ependymocytes with respect to glial markers during ontogeny show the transitory expression of glial fibrillary acidic protein, the presence of vimentin and the absence of S100 protein expression. No correlation has been found between the serotonin innervation and the expression of the glial markers.

Developmental expression of tryptophan hydroxylase mRNAs in the rat pineal gland: an in situ hybridization study.

The expression of messenger RNAs encoding for tryptophan hydroxylase (TPOH), the first enzyme involved in serotonin and melatonin synthesis, has been investigated by in situ hybridization during the development of the rat pineal gland. TPOH mRNAs were detected as early as the twentieth day of gestation (E20) in the rat embryo before any nerve ending was observed in the pineal gland. After birth, their expression increased strongly, and attained a plateau during the second week. This coincides with the setting up of sympathetic innervation. From day 17 (D17), the TPOH mRNA expression diminished. These results indicate that noradrenergic innervation is not involved in the initiation of rat pinealocyte differentiation, but might modulate cell maturation. This study showed the existence of three types of cells arranged in patches in the young rat pineal gland (D6): regions in which cells expressed TPOH mRNAs, regions in which cells expressed vimentin, an intermediate filament protein present in the cytoskeleton of immature cells, and regions in which both TPOH mRNAs and vimentin are expressed. In older rat pineal gland (D20), almost all cells express TPOH mRNAs, and some cells still express vimentin. This suggests that all cells do not reach the same level of differentiation at the same time in the rat pineal gland.

Tropism of serotonergic neurons towards glial targets in the rat ependyma.

During development, recognition mechanisms between neurons and their targets are necessary for the formation of the neuronal network. Neural connections are synaptic or non-junctional. Both types of communication can be found between neurons and glial elements in the periventricular walls. Serotonergic fibers form synaptic contacts on the specialized ependymocytes of the subcommissural organ, a structure which forms the roof of the third ventricle at its junction with the aqueduct. A network of non-junctional fibers containing both GABA and serotonin spread between the cilia of the classical ependymocytes in the ventricles. These anatomical, morphological and biochemical features suggest a tropism and specific recognition mechanisms between glial elements and serotonergic neurons. This hypothesis can be tested by the study of the innervation of the subcommissural organ and the classical ependyma by grafted embryonic neurons after a chemical destruction of the serotonergic endogenous innervation. Solid implants or cell suspensions prepared from embryonic metencephalon were transplanted to either the third ventricle or the periventricular gray matter in 5,7-dihydroxytryptamine denervated rats. Grafted serotonergic neurons were able to reinnervate the classical ependyma and the subcommissural organ. The fibers forming the supraependymal plexus were non-junctional and contained both serotonin and GABA while those innervating the subcommissural organ formed synaptic contacts and contained only serotonin. The signals capable of inducing the ependymal innervation were specific for serotonergic neurons since catecholaminergic neurons present in the grafts were unable to innervate either classical or specialized ependymocytes. These results demonstrate that glial cells are targets for serotonergic neurons and that the morphological and biochemical characteristics of the serotonergic innervation are closely related to the target cell phenotype.

Developmental neuron-glia interactions: role of serotonin innervation upon the differentiation of the ependymocytes of the rat subcommissural organ.

The rat subcommissural organ (SCO), which forms the roof of the third ventricle is an adequate model to study certain mechanisms of neuron-glia interactions in vivo. The ependymocytes, the main component of the SCO, have a glial origin. They possess particular phenotypic characteristics: they accumulate [3H]GABA by a specific uptake mechanism, contain transitory GFAP during ontogenesis and do not express PS100; on the other hand they receive a 5HT input which forms typical synaptic contacts. This innervation is of particular interest to approach neuron-glia interactions during the differentiation. Studies of GABA uptake carriers during ontogenesis in SCO ependymocytes show a correlation between the onset of the 5HT innervation and the advent of the GABA uptake. Moreover, destruction of the 5HT innervation by a neurotoxin (5-7-dihydroxytryptamine), before its arrival at the SCO in newborn rat, inhibits the formation of the GABA uptake system and causes the expression of PS100 in adult SCO cells. On the other hand, the SCO of newborn rats transplanted to the fourth ventricle of an adult host rat had no capacity to take up GABA and expressed PS100 3 months after its transplantation. Finally, the SCO ependymocytes of species devoid of 5HT innervation (rabbit, mice) were unable to take up GABA and contain PS100. These data suggest that neuron-glia interactions are necessary for the advent of GABA uptake carriers and can control the expression of glial markers during ontogenesis in SCO ependymocytes.

Developmental expression of glial markers in ependymocytes of the rat subcommissural organ: role of the environment.

The rat subcommissural organ (SCO), principally composed of modified ependymocytes (a type of glial cell), is a suitable model for the in vivo study of glial differentiation. An immunohistochemical study of the ontogenesis of rat SCO-ependymocytes from embryonic day 13 to postnatal day 10 shows that these cells express transitory glial fibrillary acidic protein (GFAP) from embryonic day 19 until postnatal day 3. However, S100 protein (S100) is never expressed in the SCO-cells, contrasting with the ventricle-lining cells of the third ventricle, which contain S100 as early as embryonic day 17. Environmental factors could be responsible for the repression of GFAP and S100 in adult rats, because GFAP and S100 are observed in ependymocytes of SCO 3 months after being grafted from newborn rat into the fourth ventricle of an adult rat. Neuronal factors might be involved in the control of the expression of S100, since after the destruction of serotonin innervation by neurotoxin at birth, S100 can be observed in some SCO-ependymocytes of adult rats. On the other hand, GFAP expression is apparently not affected by serotonin denervation, suggesting the existence of several factors involved in the differentiation of SCO-cells.

Developmental neuron-glia interaction: role of the serotonin innervation upon the onset of GABA uptake into the ependymocytes of the rat subcommissural organ.

The subcommissural organ (SCO) of the rat allows the analysis of neuron-glia interactions, in vivo, during the maturation of the brain. The SCO contains a single glial cell type which receives a homogeneous serotonin (5-HT) innervation. The onset of gamma-aminobutyric acid (GABA) uptake transport into the SCO ependymocytes is dependent on the 5-HT innervation since destruction of this innervation, at birth, or transplantation of newborn rat SCO ependymocytes to the fourth ventricle of adult host rats prevented the appearance of [3H]GABA uptake as visualized by autoradiography.

Comparative marker analysis of the ependymocytes of the subcommissural organ in four different mammalian species.

The subcommissural organ (SCO), classified as one of the circumventricular organs, is composed mainly of modified ependymal cells, attributable to a glial lineage. Nevertheless, in the rat, these cells do not possess glial markers such as glial fibrillary acidic protein (GFAP), protein S100, or the enzyme glutamine synthetase (GS). They receive a synaptic 5-HT input and show pharmacological properties for uptake of GABA resembling the uptake mechanism of neurons. In this study, we examine the phenotype of several mammalian SCO (cat, mouse, rabbit) and compare them with the corresponding features of the rat SCO. In all these species, the SCO ependymocytes possess vimentin as an intermediate filament, but never express GFAP or neurofilament proteins. They do not contain GS as do glial cells involved in GABA metabolism, and when they contain protein S100 (rabbit, mouse), its rate is low in comparison to classical glial or ependymal cells. Thus, these ependymocytes display characteristics that differentiate them from other types of glial cells (astrocytes, epithelial ependymocytes and tanycytes). Striking interspecies differences in the capacity of SCO-ependymocytes for uptake of GABA might be related to their innervation and suggest a species-dependent plasticity in their function.

Neuronal control of [3H]GABA uptake in the ependymocytes of the subcommissural organ: an in vivo model of neuron-glia interaction.

The rat subcommissural organ (SCO) is a particular but adequate paradigm for the approach, in vivo, to some aspects of neuron-glia interaction in gamma-aminobutyric acid (GABA) uptake. The rat SCO ependymocytes (the main component of this structure lying at the junction of the aqueduct and the third ventricle) accumulate [3H]GABA by a highly specific uptake mechanism and receive a serotoninergic input forming typical synaptic contacts. It seems that there is a correlation between the capacity of the rat SCO ependymocytes to take up [3H]GABA and the presence of a serotonin (5-HT) innervation. Indeed, in the newborn rat, no uptake of [3H]GABA was observed before the onset of this innervation and the increased [3H]GABA accumulation in the SCO was correlated with the appearance of the 5-HT terminals in the SCO. Moreover, in the mouse, whose SCO is devoid of a 5-HT innervation, no accumulation of [3H]GABA was observed in the SCO ependymocytes. Thus, the 5-HT innervation could be involved directly or indirectly in the onset of the GABA uptake carriers. On the other hand, in adult rats parachlorophenylalanine (pCPA) treatment decreased the 5-HT content of the SCO, and increased [3H]GABA accumulation; such an augmentation was not observed when rats were treated with pCPA plus 5-hydroxytryptophan to restore the 5-HT content. However, an increase in 5-HT content of the SCO by pargyline treatment appeared to have no effect on [3H]GABA uptake. Control of GABA uptake activity by 5-HT in the SCO ependymocytes could be an interesting model for the study of a possible interaction between amino-acids and other neurotransmitters by terminating their action in the extracellular space.