Early increase of apoptosis-linked gene-2 interacting protein X in areas of kainate-induced neurodegeneration.

Apoptosis-linked gene-2 interacting protein X (Alix) is thought to be involved in both cell death and vesicular trafficking. We examined Alix expression 2 h, 6 h and 24 h after triggering seizure-dependent neuronal death by i.p. kainic acid injection. In the hippocampus, intense, transient immunolabelling was observed in the strata lucidum, oriens and radiatum, areas of high synaptic activity. The similarity of this distribution to those of synaptophysin and endophilin suggests a presynaptic localisation. Alix labelling was increased in neuronal cell bodies in kainate-sensitive regions before or concomitant with the first signs of oedema and/or neuronal eosinophilia. The increase persisted 24 h after kainate-injection in CA3 and the piriform cortex which are areas with massive swelling and numerous pyknotic neurons. This suggests that Alix may play an early role in the mechanisms leading to cell death. Taken together, our results suggest that Alix may be a molecular link between synaptic functioning and neuronal death.

Chondroitin and keratan sulfates have opposing effects on attachment and outgrowth of ventral mesencephalic explants in culture.

During rat brain development, striatal proteoglycan (PG) expression shows specific spatio-temporal modifications suggesting a possible role in the guidance of its dopaminergic afferents. The effects of individual glycosaminoglycans (GAGs) on dopaminergic (DA) neuronal adhesion and outgrowth were therefore studied. We tested the behavior of dissociated embryonic rat mesencephalic cells cultivated on substrate-bound GAGs. Neuronal attachment was very limited and quantitative morphometry revealed variations in DA fiber outgrowth depending on the type and the concentration of GAG used. Next, we developed a cryoculture system to examine how neurons react toward GAGs expressed in situ. Rat brain slices from different developmental stages were used as substrates for embryonic mesencephalic explants. Preferential regions of adherence and outgrowth were observed: the striatum was found to be the most permissive, whereas the cortex was inhibitory. Western blotting experiments confirmed quantitative and qualitative changes in chondroitin sulfate (neurocan, phosphacan) and keratan sulfate (KS) containing PGs in these substrates and enzymatic digestion of GAGs before cryoculture revealed a substantial involvement of PGs in DA neuron adhesion and outgrowth. In particular, CSPGs seemed to mediate the permissive effect of the striatum, whereas KS confers an inhibitory effect to the cortex. PGs may thus be important for limiting midbrain projections to the striatum during development and for maintaining topography in the adult.

Mosaic distribution of chondroitin and keratan sulphate in the developing rat striatum: possible involvement of proteoglycans in the organization of the nigrostriatal system.

The striatum of the mammalian basal ganglia is composed of two neurochemically distinct compartments termed patches and matrix that contribute overall to a mosaic organization. Glycosaminoglycans (GAGs), the sugar moieties of proteoglycans, provide specific spatio-temporal guidance cues during the development of several functional neural systems. However, their distribution within the nigrostriatal system has not been investigated yet. Here, the immunohistochemical distributions of unsulphated (C0S), 4-sulphated (C4S) and 6-sulphated chondroitin (C6S) and keratan sulphate (KS) were examined in the developing neostriatum of rat and compared with the distribution of dopaminergic terminals. All the chondroitin sulphate (CS) isomers are homogeneously expressed in the embryonic striatum. After birth, C0S and C6S reveal the striatal mosaic in being preferentially expressed within the matrix compartment and in boundaries around patches whereas the C4S epitope is present in both compartments, with a slight patchy distribution. KS expression is detected first in the patches during the early postnatal period and subsequently only in the matrix compartment. All these GAG expressions disappear as the brain matures except for C4S which remains high throughout adult life. Furthermore, studies within the developing medial forebrain bundle reveal that CS isomers, but not KS, are expressed in and around the dopamine axonal tract but show similar developmental patterns of distribution which do not appear to be specifically associated with the nigrostriatal pathway. These results suggest a possible implication of proteoglycans during the development of the striatum and may be useful for understanding the complex cellular and molecular interactions in degeneration and plasticity of the nigrostriatal circuit in Parkinson's disease.

Regulated expression of keratan sulphate and peanut agglutinin binding sites during organogenesis in the developing chick.

Keratan sulphate proteoglycans are potentially important during development and are possible binding molecules for the lectin, peanut agglutinin, a marker for areas that are inhibitory for axonal growth in early embryos. The present study describes the spatiotemporal distributions of keratan sulphate epitopes and peanut agglutinin binding sites during organogenesis in the developing chick from E5 to hatching. The widespread distributions of these molecules did not often overlap but clearly delimited different carbohydrate compartments demonstrating that peanut agglutinin does not necessarily bind to keratan sulphate proteoglycans. These markers were mostly extracellular but keratan sulphate, in particular, was found within certain specific cells in cartilage, gonad, heart and pancreas, at certain ages. The presence of keratan sulphate in putative germ cells during their migrations and in the gonads may be of particular importance. Their distributions generally evoke modulation of adhesion allowing cell migrations or morphogenetic movements related to epitheliomesenchymal interactions, but may also suggest an involvement in axonal guidance in skin, cartilage, gut and possibly heart. Furthermore, in the kidney, peanut agglutinin binding sites seem to be related to the functional differentiation of the nephrons.

Transient compartmental expression of neurocan in the developing striatum of the rat.

The expression of the chondroitin sulfate proteoglycan neurocan was examined in the developing striatum of the rat and compared with the distribution of dopaminergic terminals. Neurocan immunoreactivity shows a homogeneous pattern in the embryonic striatum. In the postnatal striatum, neurocan was first expressed within the matrix but not the patch compartments, and subsequently within both. These results suggest that chondroitin sulfate proteoglycans are involved in formation of connections between the substantia nigra and striatum.

Regulation of the chick cutaneous innervation pattern in retinoic acid-induced ectopic feathers and in the naked neck mutant.

In chick skin, nerve fibers develop in a typical network formed by arcades around the base of feathers. In this study, we tried to dissociate the morphogenesis of nerve arcades and feathers, and to clarify the implication of several matricial molecules in these two developmental events. For this purpose, cutaneous nerve pattern and distribution of fibronectin, tenascin, and three epitopes of chondroitin sulfate proteoglycans (CSPGs) have been immunohistologically studied in the skin of the specific apteria of naked neck chick mutants, which lack feathers in the neck area, and in the tarso-metatarsal zone of retinoic acid-treated embryos where ectopic feathers grow. The presence of feathers was always associated with nerve arcades; no arcades were present in featherless areas. Specific immunofluorescence for tenascin and two epitopes of CSPGs revealed different distributions in the naked-neck neo-apteria as compared to control apteria. Moreover, the only difference in matricial composition in ectopic feathers concerned a CSPG isoform, bringing additional evidence that extracellular matrix molecules, and especially some (but not all) CSPGs, are involved both directly and indirectly in the cutaneous nerve pattern development.

Close link between cutaneous nerve pattern development and feather morphogenesis demonstrated by experimental production of neo-apteria and ectopic feathers: implication of chondroitin sulphate proteoglycans and other matrix molecules.

In chick skin, nerve arcades develop around the base of feathers. In order to understand the mechanisms of their formation, we have tried to dissociate arcade formation from feather morphogenesis in various ways. Nerve patterns were analysed (1) in hydrocortisone-treated embryos that are partially devoid of feathers, (2) after retinoic acid treatment that produces ectopic feathers, (3) in dorsal root ganglia-skin co-cultures. Whenever tested, immunochemistry revealed that nerve arcades form around chondroitin sulphate proteoglycan-rich areas. Hydrocortisone treatment modifies the distribution of two out of three chondroitin sulphate proteoglycan epitopes tested, as well as the shapes of the feathers and nerve arcades, but not fibronectin, tenascin or laminin localizations. Chondroitinase digestion in co-cultures eliminated the nerve arcade formation and produced abnormally thin feathers, but nevertheless with a normal spatial distribution. Thus, chondroitin sulphate proteoglycans are probably not involved in the overall arrangement of feathers, but appear to play a fundamental role in both the formation of nerve arcades and the morphogenesis of the feather.

Keratan sulphate is present in developing chick skin in vivo where it could constitute a barrier to advancing neurites as observed in vitro.

Proteoglycans play an important role in axonal guidance. The glycosaminoglycan chondroitin sulphate is known to be inhibitory. Keratan sulphate is structurally similar and may play a similar role. The lectin peanut agglutinin potentially labels chondroitin or keratan sulphate rich areas. In order to elucidate the mechanisms by which the regular nerve pattern develops in chick skin, keratan sulphate epitopes and peanut agglutinin binding sites were localized in chick embryos and compared to previous studies of chondroitin sulphate. The markers all display specific, developmentally regulated staining patterns, thus reflecting their individuality. Their maximal expression coincides with the formation of feathers and their nerve pattern in both time and space. Furthermore, culture experiments using dorsal root ganglia demonstrate the avoidance of keratan sulphate by the growing sensory neurites, although outgrowth and elongation occur if they grow directly on the keratan sulphate substrate. Thus, keratan sulphate proteoglycans in developing chick skin are potentially involved in guidance of sensory neurites or maintenance of the new nerve pattern, but are clearly different to the chondroitin sulphate proteoglycans which have a complementary distribution. Furthermore, peanut agglutinin binding sites are independent of both proteoglycans.

[Development of the cutaneous nervous system]. / Développement du système nerveux cutané.

Skin of vertebrates is richly innervated, mainly by sensory nerve fibres which form a well organized pattern, particularly around phaners. This innervation develops segmentally (dermatomes) from cutaneous branches provided by spinal nerves. The innervation begins at 13 days (E 13) in the mouse embryo and, although hair buds form at E 16, follicles are only innervated from 5 days postnatally being complete at about 20 days. In the chick skin, innervation forms a regular and characteristic pattern around feathers, and can be visualized on whole mounts. Its development can be traced from 6 days of development in relation to feather morphogenesis. Experiments producing non formation of spinal ganglia (X-ray irradiation or neural tube ablation) or production of neoapteria (hydrocortisone treatment) or ectopic feathers on scales (retinoic acid treatment) show there is a close link between feather development and nerve pattern formation. In vitro co-cultures of dorsal root ganglia and epidermis combined with the use of synthesis inhibitors and antibodies, showed that epidermis has a repulsive effect on nerve fibres mediated, at least in part, by chondroitin sulphate proteoglycans. These compounds have been localized, using antibodies mainly at the base of the feather buds and seem to play a key role in the construction of the fine nerve pattern around feather follicles. In conclusion, the specific nerve patterns are the final result of selective responses of growing nerve endings to unique combinations of local cues and conflicting interactions which are developmentally regulated in parallel with the morphogenesis of phaners.

Development of sensory innervation in chick skin: comparison of nerve fibre and chondroitin sulphate distributions in vivo and in vitro.

In bird skin, nerve fibres develop in the dermis but do not enter the epidermis. In co-cultures of 7-day-old chick embryo dorsal root ganglia and epidermis, the neurites also avoid the epidermis. Previous studies have shown that chondroitin sulphate proteoglycans may be involved. Chondroitin sulphate has therefore been visualized by immunocytochemistry, using the monoclonal antibody CS-56, both in vivo and in vitro using light and electron microscopy. Its distribution was compared to those of 2 other chondroitin sulphate epitopes and to that of the growing nerve fibres. In cultures of epidermis from 7-day-old embryonic chicks, immunoreactivity is found uniformly around the epidermal cells while at 7.5 days the distribution in dermis is heterogeneous, and particularly marked in feather buds. In vivo, chondroitin sulphate immunoreactivity is detected in the epidermis, on the basal lamina, on the surfaces of fibroblasts and along collagen fibrils. This localization is complementary to the distribution of cutaneous nerves. Chondroitin sulphate in the basal lamina could prevent innervation of the epidermis and the dermal heterogeneities could partly explain the nerve fibres surrounding the base of the feathers. Chondroitin sulphate could therefore be important for neural guidance in developing chick skin.

Fetal development and regulation of pituitary cell types.

The ontogenesis of the pituitary gland is considered from anatomical and functional points of view. Embryogenesis of the hypothalamo-pituitary unit involving development of the hypothalamo-hypophyseal portal system is complete during early life as shown in several mammalian species. The ultrastructural characteristics of the different cell types during development are described according to observations made by using immunochemical techniques. The patterns of differentiation of the cell types are reviewed according to studies of pituitary glands from human anencephalic fetuses and encephalectomized rat fetuses as well as in vitro studies of cultured pituitary primordia in synthetic media. The maturation of the neuroendocrine mechanisms controlling the secretion of fetal hormones is also analyzed. During fetal life, the factors implicated in the regulation of pituitary hormone secretion are generally the same as in adults, but the intensity of the response of pituitary cells to their action is variable according to the species, thus reflecting an immaturity in the functioning of certain cell types.

Differentiation of fetal rat somatotropes in vitro: effects of cortisol, 3,5,3'-triiodothyronine, and glucagon, a light microscopic and radioimmunological study.

Cortisol stimulates somatotrope differentiation in vitro. T3 and/or glucagon may also be involved. Fetal rat pituitary primordia were explanted at 14 days gestation and cultured for 7 days in medium supplemented with cortisol (50-500 nM), and either T3 (0.67 nM) or glucagon (0.5 nM). Also, to determine the time of first appearance of the somatotropes, explants were cultured 4, 5, or 6 days with cortisol alone. Immunoreactive somatotropes were detected by immunohistochemistry, and their size and number were estimated for each medium. GH was measured by RIA in explants and media. Immunoreactive somatotropes first appear at 18-19 days gestation. Their size and number depend on cortisol concentration: no cells at 50 nM, a few small ones at 100 nM, and many large ones at 250-500 nM. This progression was reflected by RIA of GH in explants and media, although small quantities were detected with 50 nM. The effect of T3 was only visible with a low dose of cortisol. With 100 nM cortisol, it increased the size and number of cells. Differentiation was also triggered with 50 nM cortisol plus T3. RIA detected significantly higher GH content and secretion after T3 stimulation. The decreases in number, size, and GH secretion and content elicited by glucagon were not significant, probably due to the high variability. Both techniques used provide similar information on somatotrope differentiation: stimulation by cortisol alone, or alternatively by a synergistic action between cortisol and T3.

[The ontogeny of hormones of the somatotrophic axis]. / Ontogenèse des hormones de l'axe somatotrope.

The ontogenesis of the somatotropic function involving its regulatory hormones is considered from an anatomical and functional point of view. Embryogenesis of the hypothalamo-pituitary unit involving development of the hypothalamo-hypophyseal portal system is complete during early fetal life as shown in a certain number of mammalian species. The maturation of the neuroendocrine mechanisms controlling the secretion of fetal pituitary growth hormone is analyzed in the present paper. Corticosteroids may act directly on the differentiation of the somatotropic cells. During fetal life, the plasma level of GH is always higher than after birth. Despite large gaps in our knowledge, SRIF may play a prominent part in the regulation of the somatotropic function, sometimes mediating the effect of other factors, and GRF may also be active during late gestation. However, maturation is not complete until after birth especially that of somatomedins as well as the pulsatile release of GH.

[Differentiation of pituitary cells in culture]. / Différenciation des cellules antéhypophysaires en culture.

Gonadotrophs were first detected at 18 days of gestation in normal rat fetus. Encephalectomy performed at 16 days of gestation did not modify the normal aspect of cells at term. In adenohypophysial primordia explanted from 13 days of gestation differentiated gonadotrophs were detected after culture (8 days) in medium containing insulin (minimal dose required: 0.5 microgram/ml) and transferrin (5 micrograms/ml). In contrast, in primordia explanted at 11 and 12 days of gestation, GnRH 10(-9) to 10(-12) M was required for the first 24 hours of culture to induce differentiation of cells which was obtained in synergy with insulin and transferrin. On the other hand, fetal hypothalamic GnRH and pituitary GnRH receptors were observed from 12 days of gestation which can explain the observations made on primordia explanted at 13 days. Lactotrophs first appeared at term in normal rat fetus. In vitro, differentiation of lactotrophs was not observed in primordia explanted from 13 days in the presence of insulin and transferrin alone, but it was induced by GnRH (10(-9) M) for the 24 hours of culture in the same medium. The action of GnRH was mediated through glycoproteins and specifically isolated alpha subunit. Indeed, purified LH alpha-subunit added in medium instead of GnRH induced differentiation of lactotrophs from 10(-9) M with an increase in the number of cells related to the dose of hormone. Differentiation of the two cell types is very linked in these culture conditions. Gonadotroph differentiation is regulated by an hypothalamic endocrine secretion whereas lactotroph differentiation is more dependent on a paracrine secretion.

Somatotrophs and lactotrophs in the anterior pituitary of fetal and neonatal rats. Electron-microscopic immunocytochemical identification.

The ultrastructure of immunoreactive somatotrophs and lactotrophs in pituitaries of fetal rats at 19, 20 and 21 days of gestation and on the day of birth was studied. Somatotrophs, first detectable at 19 days of gestation, undergo only minor modifications before reaching the structure described for adults. In particular there is an increase in the endoplasmic reticulum and Golgi apparatus. Lactotrophs, first identifiable in newborn rats, are very different in ultrastructure from adult cells, because the secretory granules are generally small, but variable in shape and size and the Golgi complex is prominent.

Induction of pituitary lactotrope differentiation by luteinizing hormone alpha subunit.

Addition of gonadotropin releasing hormone to cultures of fetal rat pituitary induced differentiation of lactotropes as revealed by immunocytochemistry. Antiserum to luteinizing hormone (LH) (recognizing native LH), but not antiserum to LH-beta (recognizing both native LH and its beta subunit), inhibited this induction. Further addition of highly purified LH-alpha subunit in culture medium also induced lactotrope differentiation. Thus, the alpha subunit may have a specific biological activity of its own with probable practical use in clinical investigations.

Fetal rat somatotropes in vitro: effects of insulin, cortisol, and growth hormone-releasing factor on their differentiation: a light and electron microscopic study.

Somatotropes first appear in the fetal rat pituitary just before term. These cells have never been detected in cultured fetal pituitaries. A modified culture medium has, however, enabled their differentiation in vitro. Hypophysial primordia were explanted on days 13-18 of gestation and cultured in different media until the equivalent of term. Immunoreactive somatotropes could be detected, by light and electron microscopy, in cultured primordia explanted on day 14 of gestation or later. The size and numbers of immunoreactive cells depended on culture medium composition. The control medium, containing insulin, cortisol, T3, and glucagon, proved favorable to somatotrope differentiation and proliferation. Increased insulin concentration reduced somatotrope numbers. In the presence of only insulin and cortisol (or corticosterone) somatotropes were more numerous than in the control. Culture medium enriched with insulin alone, with insulin and T3, or with insulin and glucagon, was not suitable for development of this cell type. Addition of GH-releasing factor ( GHRF ) to the medium during the first culture day did not accelerate the first appearance of the somatotropes but did significantly increase their size. GHRF addition 1/2 h before the end of culture did not modify their morphology. The ultrastructure of somatotropes in vitro is very similar to that observed in vivo on day 21 of gestation. The cells were characterized by their lamellar endoplasmic reticulum and immunoreactive secretory granules (300-400 nm maximal section diameter). Fetal somatotropes can, therefore, be successfully caused to differentiate in vitro. Their appearance depends on insulin and glucocorticoid concentration. T3 and/or glucagon may be inhibitory. GHRF may increase storage in somatotropes. These factors may also regulate the development of somatotropes in vivo.

Cryoultramicrotomy versus plastic embedding: comparative immunocytochemistry of rat anterior pituitary cells.

The anterior pituitary of the rat is used as a model for the study of the effects of freezing or plastic embedding on the maintenance of antigenicity. Rat anterior pituitaries are fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.4. Some of the blocks are post-fixed before being divided into two lots. One batch is frozen, while the other is dehydrated and embedded. The indirect antibody enzyme method is applied to ultrathin sections obtained by cryoultramicrotomy after freezing or by sectioning after embedding. All six pituitary hormones are detected by both methods. Comparison shows that the morphological characteristics are identical for both techniques, though ultrastructural preservation is better after embedding. Immunoreactivity is found in secretory granules and sometimes in the endoplasmic reticulum. Osmium postfixation may reduce or even abolish antigenicity in plastic-embedded tissue. After cryoultramicrotomy, however, even after osmium fixation, antibody may be used 1000 times more diluted than after plastic embedding. Embedding preserves ultrastructure and limited antigenicity while the use of cryoultramicrotomy is a far more sensitive technique.

Gonadotropin releasing hormone (GnrH) stimulates immunoreactive lactotrope differentiation.

To study lactotrope differentiation in the fetal rat, immunocytochemistry was performed on pituitary primordia explanted from 13-day-old fetuses and cultured in different synthetic media until the equivalent of 21 days. Lactotropes were detected only by antirat prolactin antiserum when the synthetic medium was enriched with GnRH (10(-9)M). These results indicate that lactotrope differentiation may partly depend on stimulatory factors such as GnRH.

Simultaneous ultrastructural localization of serotonin and cholinesterases in Mytilus byssal retractor muscle (A.B.R.M.).

Ultrastructural localization techniques for cholinesterases (ChE) by the Karnovsky method, for monoamines by the technique of Wood, and Tranzer and Richards, and for 5HT by the technique of Wood, were performed on the anterior byssal retractor muscle of Mytilus. Simultaneous ultrastructural localization of 5HT and ChE was obtained by the use of the false neurotransmitter 5,6-DHT, followed by the method of Karnovsky for ChE. The glio-interstitial tissue presents a constant ChE activity, mainly localized on the plasma membrane, even in those processes which accompany tryptaminergic (5HT containing) neurites. Muscle cell membrane also reacts positively to the Karnovsky method. The tryptaminergic neurites themselves do not show any ChE activity; they contain dichromate reactive large (100 nm) dense cored vesicles. The presence of differentiated tryptaminergic neuromuscular junctions, suggested by other authors, is established. It was not possible to distinguish classes of nerve endings by the typology of their vesicular content. It is concluded that one can reasonably plan to study the effect of glial ChE inhibition on the physiology of the tryptaminergic (relaxing) response of the A.B.R.M. to nerve stimulation.