J. M. Pérez-Fígares

Osmoregulatory responses to abrupt salinity changes in the euryhaline gilthead sea bream (Sparus aurata L.)

Abstract 1. 1. Gilthead sea breams ( Sparus aurata L.) adapted to sea water (SW, 39‰ salinity) and brackish water (BW, 7‰) were submitted to abrupt osmotic stress by transferring the specimens to 7‰ and 39‰, respectively. 2. 2. Plasma osmolality, Na, + Cl, − K, + Ca, 2+ cortisol and glucose were measured before and after the transfers. 3. 3. The transfer from SW to BW led to transitory hypomineralization and hyperglycemia. In long-term adapted fish cortisol level increased, and osmolality slightly decreased. 4. 4. Conversely, the transfer from BW to SW provoked transitory hypermineralization. In adapted fish, cortisol levels strongly decreased, and osmolality slightly increased.

Spontaneous congenital hydrocephalus in the mutant mouse hyh. Changes in the ventricular system and the subcommissural organ

The subcommissural organ is an ependymal gland located at the entrance of the cerebral aqueduct. It secretes glycoproteins into the cerebrospinal fluid, where they aggregate to form Reissners fiber. This fiber grows along the aqueduct, fourth ventricle, and central canal. There is evidence that the subcommissural organ is involved in the pathogenesis of congenital hydrocephalus. This organ was investigated in the mutant mouse hyh developing a congenital hydrocephalus. The central nervous system of normal and hydrocephalic hyh mice, 1 to 40 days old, was investigated using antibodies recognizing the subcommissural organ secretory glycoproteins, and by transmission and scanning electron microscopy. At birth, the affected mice displayed open communications between all ventricles, absence of a central canal in the spinal cord, ependymal denudation of the ventricles, stenosis of the rostral end of the aqueduct, and hydrocephalus of the lateral and third ventricles and of the caudal end of the aqueduct. Around the 5th postnatal day, the communication between the caudal aqueduct and fourth ventricle sealed, and hydrocephalus became severe. It is postulated that the hyh mice carry a genetic defect affecting the ependymal cell lineage. The subcommissural organ showed signs of increased secretory activity; it released to the stenosed aqueduct a material that aggregated, but it did not form a Reissners fiber. A large area of the third ventricular wall differentiated into a secretory ependyma synthesizing a material similar to that secreted by the subcommissural organ. It is concluded that the subcommissural organ changes during hydrocephalus; whether these changes preceed hydrocephalus needs to be investigated.

Ependymal Denudation, Aqueductal Obliteration and Hydrocephalus after a Single Injection of Neuraminidase into the Lateral Ventricle of Adult Rats

To investigate the role of sialic acid in the ependyma of the rat brain, we injected neuraminidase from Clostriditum perfingens into the lateral ventricle of 86 adult rats that were sacrificed at various time intervals. After administration of 10 µg neuraminidase, ciliated cuboidal ependymal cells of the lateral ventricles, third ventricle, cerebral aqueduct, and the rostral half of the fourth ventricle died and detached. The ependymal regions sealed by tight juntions such as the choroid plexus and the subcommissural organ were not affected. Debris was removed by infiltrating neutrophils and macrophagic cells. At the same time, after ependymal disappearance, the aqueduct was obliterated. In this region, mitoses were evident and cystic ependymal cells were frequent. Hydrocephalus of the lateral and third ventricles was evident 4 days after neuraminidase injection. Gliosis was restricted to the dorsal telencephalic wall of the injected lateral ventricle. It is thought that cleavage of sialic acid from ependymal surface glycoproteins or glycolipids, likely involved in cell adhesion, led to the detaching and death of the ependymal cells. Thereafter, ependymal loss, together with edema, led to fusion of the lateral walls of the cerebral aqueduct and this in turn provoked hydrocephalus of the third and lateral ventricles. This model of experimental hydrocephalus is compared with other models, in particular those of hydrocephalus after viral invasion of the cerebral ventricles.

Astrocytes acquire morphological and functional characteristics of ependymal cells following disruption of ependyma in hydrocephalus

Hydrocephalic hyh mutant mice undergo a programmed loss of the neuroepithelium/ependyma followed by a reaction of periventricular astrocytes, which form a new cell layer covering the denuded ventricular surface. We present a comparative morphological and functional study of the newly formed layer of astrocytes and the multiciliated ependyma of hyh mice. Transmission electron microscopy, immunocytochemistry for junction proteins (N-cadherin, connexin 43) and proteins involved in permeability (aquaporin 4) and endocytosis (caveolin-1, EEA1) were used. Horseradish peroxidase (HRP) and lanthanum nitrate were used to trace the intracellular and paracellular transport routes. The astrocyte layer shares several cytological features with the normal multiciliated ependyma, such as numerous microvilli projected into the ventricle, extensive cell–cell interdigitations and connexin 43-based gap junctions, suggesting that these astrocytes are coupled to play an unknown function as a cell layer. The ependyma and the astrocyte layers also share transport properties: (1) high expression of aquaporin 4, caveolin-1 and the endosome marker EEA1; (2) internalization into endocytic vesicles and early endosomes of HRP injected into the ventricle; (3) and a similar paracellular route of molecules moving between CSF, the subependymal neuropile and the pericapillary space, as shown by lanthanum nitrate and HRP. A parallel analysis performed in human hydrocephalic foetuses indicated that a similar phenomenon would occur in humans. We suggest that in foetal-onset hydrocephalus, the astrocyte assembly at the denuded ventricular walls functions as a CSF–brain barrier involved in water and solute transport, thus contributing to re-establish lost functions at the brain parenchyma–CSF interphase.

Structure and function of the ependymal barrier and diseases associated with ependyma disruption.

The neuroepithelium is a germinal epithelium containing progenitor cells that produce almost all of the central nervous system cells, including the ependyma. The neuroepithelium and ependyma constitute barriers containing polarized cells covering the embryonic or mature brain ventricles, respectively; therefore, they separate the cerebrospinal fluid that fills cavities from the developing or mature brain parenchyma. As barriers, the neuroepithelium and ependyma play key roles in the central nervous system development processes and physiology. These roles depend on mechanisms related to cell polarity, sensory primary cilia, motile cilia, tight junctions, adherens junctions and gap junctions, machinery for endocytosis and molecule secretion, and water channels. Here, the role of both barriers related to the development of diseases, such as neural tube defects, ciliary dyskinesia, and hydrocephalus, is reviewed.

Hydrocephalus induced by immunological blockage of the subcommissural organ-Reissner's fiber (RF) complex by maternal transfer of anti-RF antibodies

Abstract. Stenosis of the cerebral aqueduct seems to be a key event for the development of congenital hydrocephalus. The causes of such a stenosis are not well known. Overholser et al. in 1954 (Anat Rec 120:917–933) proposed the hypothesis that a dysfunction of the subcommissural organ (SCO) leads to aqueductal stenosis and congenital hydrocephalus. The SCO is a brain gland, located at the entrance of the cerebral aqueduct, that secretes glycoproteins into the cerebrospinal fluid that, upon release, assemble into a fibrous structure known as Reissners fiber (RF). By the permanent addition of new molecules to its rostral end, RF grows and extends along the aqueduct, fourth ventricle, and central canal of the spinal cord. The immunological blockage of the SCO-RF complex has been used to test Overholsers hypothesis. The following was the sequence of events occurring in pregnant rats that had been immunized with RF glycoproteins: the mother produced anti-RF antibodies and transferred them to the fetus through the placenta and to the pup through the milk, and the antibodies reached the brain of the fetus and pup and blocked the SCO-RF complex. This resulted in a permanent absence of RF that was followed by stenosis of the cerebral aqueduct, and then by the appearance of hydrocephalus. The latter was patent until the end of the 6-month observation period. The chronic hydrocephalic state appeared, in turn, to induce new alterations of the SCO. It is concluded that a selective immunological knock out of the SCO-RF complex leads to hydrocephalus.

Effect of cortisol on brackish water adaptation in the euryhaline gilthead sea bream (Sparus aurata L.)

Abstract Gilthead sea breams (Sparus aurata L.) adapted to sea water (SW, 39%o salinity) were injected with either saline or cortisol (10 μg/g) 3 hours before being abruptly transferred to brackish water (BW, 7% salinity). Plasma osmolality, Na+, Cl−, Ca2+ and cortisol were measured before and after the transference. The transference led to a transitory hypoosmolality and hypomineralization in both groups. The Na+ and Cl− levels showed a lower decrease in the cortisol-injected group. Osmolality and Ca2+ were similar in both groups. In the control group plasma cortisol reached a peak soon after the transference. Long term BW-adapted specimens showed a stable 2.5-fold increase in cortisol levels with respect to SW-adapted. Cortisol injected group showed an increased cortisolemia, in relation to control, for 9 hr after the injection, after this time, cortisol levels were similar to the control group.

Distribution of intraventricularly injected horseradish peroxidase in cerebrospinal fluid compartments of the rat spinal cord

SummaryThe circulation of the cerebrospinal fluid along the central canal and its access to the parenchyma of the spinal cord of the rat have been analyzed by injection of horseradish peroxidase (HRP) into the lateral ventricle. Peroxidase was found throughout the central canal 13 min after injection, suggesting a rapid circulation of cerebrospinal fluid along the central canal of the rat spinal cord. It was cleared from the central canal within 2 h, in contrast with the situation in the brain tissue, where it remained in the periventricular areas for 4 h. In the central canal, HRP bound to Reissners fiber and the luminal surface of the ependymal cells; it penetrated through the intercellular space of the ependymal lining, reached the subependymal neuropil, the basement membrane of local capillaries, and appeared in the lumen of endothelial pinocytotic vesicles. Furthermore, it accumulated in the labyrinths of the basement membrane contacting the basolateral aspect of the ependymal cells. In ependymocytes, HRP was found in single pinocytotic vesicles. The blood vessels supplying the spinal cord were classified into two types. Type-A vessels penetrated the spinal cord laterally and dorsally and displayed the tracer along their external wall as far as the gray matter. Type-B vessels intruded into the spinal cord from the medial ventral sulcus and occupied the anterior commissure of the gray matter, approaching the central canal. They represented the only vessels marked by HRP along their course through the gray matter. HRP spread from the wall of type-B vessels, labeling the labyrinths, the intercellular space of the ependymal lining, and the lumen of the central canal. This suggests a communication between the central canal and the outer cerebrospinal fluid space, at the level of the medial ventral sulcus, via the intercellular spaces, the perivascular basement membrane and its labyrinthine extensions.

Neurogenesis in explants from the walls of the lateral ventricle of adult bovine brain: role of endogenous IGF-1 as a survival factor

Previous studies have shown the existence of proliferating cells in explants from bovine (Bos Taurus) lateral ventricle walls that were maintained for several days in vitro in the absence of serum and growth factors. In this study we have characterized the nature of new cells and have assessed whether the insulin‐like growth factor‐1 (IGF‐1) receptor regulates their survival and/or proliferation. The explants were composed of the ependymal layer and attached subependymal cells. Ependymal cells in culture were labelled with glial markers (S‐100, vimentin, GFAP, BLBP, 3A7 and 3CB2) and did not incorporate bromodeoxiuridine when this molecule was added to the culture media. Most subependymal cells were immunoreactive for βIII‐tubulin, a neuronal marker, and did incorporate bromodeoxiuridine. Subependymal neurons displayed immunoreactivity for IGF‐1 and its receptor and expressed IGF‐1 mRNA, indicating that IGF‐1 is produced in the explants and may act on new neurons. Addition to the culture media of an IGF‐1 receptor antagonist, the peptide JB1, did not affect the incorporation of bromodeoxiuridine to proliferating subependymal cells. However, JB1 significantly increased the number of TUNEL positive cells in the subependymal zone, suggesting that IGF‐1 receptor is involved in the survival of subependymal neurons. In conclusion, these findings indicate that neurogenesis is maintained in explants from the lateral cerebral ventricle of adult bovine brains and that IGF‐1 is locally produced in the explants and may regulate the survival of the proliferating neurons.

The distribution of corticotropin-releasing factor-immunoreactive neurons and nerve fibers in the brain of the snake, Natrix maura

SummaryThe anatomical distribution of neurons and nerve fibers containing corticotropin-releasing factor (CRF) has been studied in the brain of the snake, Natrix maura, by means of immunocytochemistry using an antiserum against rat CRF. To test the possible coexistence of CRF with the neurohypophysial peptides arginine vasotocin (AVT) and mesotocin (MST) adjacent sections were stained with antisera against the two latter peptides. CRF-immunoreactive (CRF-IR) neurons exist in the paraventricular nucleus (PVN). In some neurons of the PVN, coexistence of CRF with MST or of CRF with AVT has been shown. Numerous CRF-IR fibers run along the hypothalamo-hypophysial tract and end in the outer layer of the median eminence. In addition, some fibers reach the neural lobe of the hypophysis. CRF-IR perikarya have also been identified in the following locations: dorsal cortex, nucleus accumbens, amygdala, subfornical organ, lamina terminalis, nucleus of the paraventricular organ, nucleus of the oculomotor nerve, nucleus of the trigeminal nerve, and reticular formation. In addition to all these locations CRF-IR fibers were also observed in the lateral septum, supraoptic nucleus, habenula, lateral forebrain bundle, paraventricular organ, hypothalamic ventromedial nucleus, raphe and interpeduncular nuclei.