M.D. López-Avalos

IGF‐I stimulates neurogenesis in the hypothalamus of adult rats

In the brain of adult rats neurogenesis persists in the subventricular zone of the lateral ventricles and in the dentate gyrus of the hippocampus. By contrast, low proliferative activity was observed in the hypothalamus. We report here that, after intracerebroventricular treatment with insulin‐like growth factor I (IGF‐I), cell proliferation significantly increased in both the periventricular and the parenchymal zones of the whole hypothalamus. Neurons, astrocytes, tanycytes, microglia and endothelial cells of the local vessels were stained with the proliferative marker 5‐bromo‐2′‐deoxyuridine (BrdU) in response to IGF‐I. Conversely, we never observed BrdU‐positive ciliated cubic ependymal cells. Proliferation was intense in the subventricular area of a distinct zone of the mid third ventricle wall limited dorsally by ciliated cubic ependyma and ventrally by tanycytic ependyma. In this area, we saw a characteristic cluster of proliferating cells. This zone of the ventricular wall displayed three cell layers: ciliated ependyma, subependyma and underlying tanycytes. After IGF‐I treatment, proliferating cells were seen in the subependyma and in the layer of tanycytes. In the subependyma, proliferating glial fibrillary acidic protein‐positive astrocytes contacted the ventricle by an apical process bearing a single cilium and there were many labyrinthine extensions of the periventricular basement membranes. Both features are typical of neurogenic niches in other brain zones, suggesting that the central overlapping zone of the rat hypothalamic wall could be considered a neurogenic niche in response to IGF‐I.

Obesity-dependent cannabinoid modulation of proliferation in adult neurogenic regions

Endocannabinoid signalling participates in the control of neurogenesis, especially after brain insults. Obesity may explain alterations in physiology affecting neurogenesis, although it is unclear whether cannabinoid signalling may modulate neural proliferation in obese animals. Here we analyse the impact of obesity by using two approaches, a high‐fat diet (HFD, 60% fat) and a standard/low‐fat diet (STD, 10% fat), and the response to a subchronic treatment with the cannabinoid receptor type 1 (CB1) inverse agonist AM251 (3 mg/kg) on cell proliferation of two relevant neurogenic regions, namely the subventricular zone in the striatal wall of the lateral ventricle (SVZ) and the subgranular zone of the dentate gyrus (SGZ), and also in the hypothalamus given its role in energy metabolism. We found evidence of an interaction between diet‐induced obesity and CB1 signalling in the regulation of cell proliferation. AM251 reduced caloric intake and body weight in obese rats, as well as corrected plasma levels of cholesterol and triglycerides. AM251 is shown, for the first time, to modulate cell proliferation in HFD‐obese rats only. We observed an increase in the number of 5‐bromo‐2‐deoxyuridine‐labelled (BrdU+) cells in the SGZ, but a decrease in the number of BrdU+ cells in the SVZ and the hypothalamus of AM251‐treated HFD rats. These BrdU+ cells expressed the neuron‐specific βIII‐tubulin. These results suggest that obesity may impact cell proliferation in the brain selectively, and provide support for a role of CB1 signalling regulation of neurogenesis in response to obesity.

Bovine Reissner’s fiber (RF) and the central canal of the spinal cord: an immunocytochemical study using a set of monoclonal antibodies against the RF-glycoproteins

Abstract.The subcommissural organ secretes N-linked complex-type glycoproteins into the cerebrospinal fluid. These glycoproteins condense to form Reissner’s fiber (RF), which extends along the fourth ventricle and central canal of the spinal cord. A set of three monoclonal antibodies (Mabs 3E6, 3B1, and 2A5) has been obtained using these glycoproteins as immunogens. Competitive and sandwich enzyme-linked immunoassay methods have demonstrated that the three monoclonal antibodies are directed against different epitopes, and that there is no competition among them for their binding to glycoproteins of RF. Mab 3E6 displays immunoblotting properties that are similar to those of a polyclonal antibody against the pool of glycoproteins from RF, but that are different from those of Mabs 3B1 and 2A5. All three antibodies immunostain the bovine subcommissural organ and RF. A population of ependymal cells is stained by the polyclonal antibody, and Mabs 2A5 and 3E6, but not by Mab 3B1. The material present in a population of ependymal cells of the central canal, and the glycoproteins secreted by the subcommissural organ thus probably have partial chemical identity. Some evidence suggests that the immunoreactive ependymal cells are secretory cells. The luminal surface of the central canal is coated by a thin layer of material with immunocytochemical characteristics different from those of the ependymal cells; such a coat may correspond to material released from RF.

Pharmacological administration of the isoflavone daidzein enhances cell proliferation and reduces high fat diet-induced apoptosis and gliosis in the rat hippocampus.

Soy extracts have been claimed to be neuroprotective against brain insults, an effect related to the estrogenic properties of isoflavones. However, the effects of individual isoflavones on obesity-induced disruption of adult neurogenesis have not yet been analyzed. In the present study we explore the effects of pharmacological administration of daidzein, a main soy isoflavone, in cell proliferation, cell apoptosis and gliosis in the adult hippocampus of animals exposed to a very high-fat diet. Rats made obese after 12-week exposure to a standard or high-fat (HFD, 60%) diets were treated with daidzein (50 mg kg−1) for 13 days. Then, plasma levels of metabolites and metabolic hormones, cell proliferation in the subgranular zone of the dentate gyrus (SGZ), and immunohistochemical markers of hippocampal cell apoptosis (caspase-3), gliosis (GFAP and Iba-1), food reward factor FosB and estrogen receptor alpha (ERα) were analyzed. Treatment with daidzein reduced food/caloric intake and body weight gain in obese rats. This was associated with glucose tolerance, low levels of HDL-cholesterol, insulin, adiponectin and testosterone, and high levels of leptin and 17β-estradiol. Daidzein increased the number of phospho-histone H3 and 5-bromo-2-deoxyuridine (BrdU)-ir cells detected in the SGZ of standard diet and HFD-fed rats. Daidzein reversed the HFD-associated enhanced immunohistochemical expression of caspase-3, FosB, GFAP, Iba-1 and ERα in the hippocampus, being more prominent in the dentate gyrus. These results suggest that pharmacological treatment with isoflavones regulates metabolic alterations associated with enhancement of cell proliferation and reduction of apoptosis and gliosis in response to high-fat diet.

Analysis of the secretory glycoproteins of the subcommissural organ of the dogfish (Scyliorhinus canicula)

The subcomissural organ (SCO) is an ancient and conserved brain gland secreting glycoproteins into the cerebrospinal fluid which condense to form Reissners fiber (RF). The SCO of an elasmobranch species, the dogfish Scyliorhinus canicula, was investigated applying morphological and biochemical methods. The SCO of 34 dogfishes were processed for the following techniques: (1) conventional transmission electron microscopy; (2) light and electron microscopy lectin histochemistry (Concanavalin A, Con A; wheat germ agglutinin, WGA; Limax flavus agglutinin, LFA); (3) light and electron microscopy immunocytochemistry using antisera raised against the glycoproteins of the bovine RF (anti-bovine RF), and the secretory material of the dogfish SCO (anti-dogfish SCO). The former reacts with the SCO of virtually all vertebrate species [19] (conserved epitopes); the latter reacts only with the SCO of elasmobranchs [Cell Tissue Res., 276 (1994) 515-522] (class-specific epitopes). At the light microscopic level both antisera immunoreacted selectively with the SCO and RF; no other structure of the central nervous system was reactive. Within the SCO the binding sites for WGA (affinity = glucosamine, sialic acid) and LFA (affinity = sialic acid) displayed the same density and intracellular distribution. At the ultrastructural level two types of granules were distinguished. Type I granules (200-400 nm) were numerous, reacted with both antisera, bound WGA but not Con A. Type II granules (0.8-1.8 microns) reacted with the anti-bovine RF serum but not with the anti-dogfish SCO serum, bound Con A and WGA. The content of dilated cisternae of the rough endoplasmic reticulum reacted with both antisera and bound Con A; it did not bind WGA. The SCOs of 4500 dogfishes were extracted in ammonium bicarbonate. This extract was used for SDS-PAGE and blotting. Blots were processed for immunolabeling using anti-bovine RF and anti-dogfish SCO sera, and for lectin binding (Con A, WGA and LFA). The anti-bovine RF revealed four compounds with apparent molecular weights of 750, 380, 145 and 35 kDa. The two former also reacted with the anti-dogfish SCO serum and bound Con A. Only the 380 kDa compound bound WGA and LFA. The findings indicate that both the conserved and the class-specific epitopes are part of the same compounds (780, 380 kDa), which would be stored in type I granules. The lectin binding properties of these compounds point to the 780 kDa compound as a precursor form and the 380 kDa polypeptide as a processed form.

Rostral floor plate flexural organ secretes glycoproteins immunologically similar to subcommissural organ glycoproteins in / dogfish Scyliorhinus canicula embryos

The subcommissural organ of vertebrates secretes glycoproteins into the cerebrospinal fluid of the third cerebral ventricle. This material polymerizes in Reissners fiber. During ontogenetic development, besides the subcommissural organ, the ependyma lining the pontine flexure constitutes an additional Reissners fiber-secreting gland named flexural organ. We have studied the secretion of the flexural organ and the subcommissural organ in dogfish (Scyliorhinus canicula) embryos using three different antisera and the lectins concanavalin A and wheat germ agglutinin. AFRU is an antiserum against the bovine Reissners fiber, Ab-600 is an antiserum against 600 kDa dogfish subcommissural organ glycoproteins; and APSO is an antiserum against immunoaffinity purified bovine subcommissural organ secretory glycoproteins. These three antisera immunostained the flexural organ indicating that it contains epitopes similar to those present in bovine and dogfish subcommissural organ glycoproteins. It seems highly probable that the flexural organ and the subcommissural organ of dogfish embryos secrete similar compound(s). Other ependymal regions were also immunostained with Ab-600 and APSO antisera. Then, Reissners fiber-like glycoproteins were transiently expressed by most embryonary ependymal cells. These glycoproteins might play a role in the development of the central nervous system of vertebrates.

A comparative analysis of intraperitoneal versus intracerebroventricular administration of bromodeoxyuridine for the study of cell proliferation in the adult rat brain

Bromodeoxyuridine (BrdU) is the most widely used marker to detect proliferative cells in the adult brain. Here we analyse whether the route of administration of the tracer influences the number of labelled cells. For the intraperitoneal (ip) administration of BrdU, we performed two daily injections during 7 days, and for an intracerebroventricular (icv) delivery, it was continuously infused into one lateral ventricle for a 7 days period as well. After ip administration, cells labelled with BrdU were seen in the subventricular zone of the striatal wall of the lateral ventricle, the hippocampus and the neurohemal circumventricular organs. Also, the habenula and large myelinated tracts, such as the fornix and the corpus callosum, showed many BrdU-positive nuclei. Labelled nuclei were scarce in the parenchymal regions of the rest of the brain. In contrast, a significant increase in the number of BrdU-positive nuclei was observed in the parenchyma of the periventricular zones after icv administration of the marker, thus showing a greater availability of the tracer when it was administered directly into the ventricular cerebrospinal fluid. We suggest that the availability of BrdU in the vicinity of proliferating cells may depend on the permeability of the brain vessels to nucleosides in each location. By using double immunocytochemistry we found that neurons, astrocytes, oligodendrocytes, tanycytes and microglia had incorporated the tracer, demonstrating their proliferation capacity.

Secretory glycoproteins of the subcommissural organ of the dogfish (Scyliorhinus canicula): evidence for the existence of precursor and processed forms

Abstract.The subcommissural organ of the dogfish, Scyliorhinus canicula (L), has been investigated by use of antibodies and lectins applied to blots and tissue sections processed for light and electron microscopy. Antibodies have been raised against each of the bands that have previously been identified in immunoblots by the use of antisera raised against secretory glycoproteins extracted from the dogfish subcommissural organ, viz., the 600-kDa band and two gel regions including the 475 to 400-kDa and the 145-kDa bands obtained from preparative gels; they are referred to as Ab-600, Ab-475/400, and Ab-145. These antisera and the lectins concanavalin A and wheat germ agglutinin have been used for the staining of: (1) blots of extracts of the dogfish subcommissural organ and optic tectum; (2) tissue sections of the dogfish brain. The findings indicate that the bands of 600, 475 and 400 kDa contain compounds that should be regarded as secretory glycoproteins of the dogfish subcommissural organ. The 600-kDa and 400-kDa bands are labeled by concanavalin A; wheat germ agglutinin labels the 475-kDa band strongly and the other two weakly. Ab-600 reacts with the bands at 600, 475 and 400 kDa and stains materials stored in the rough endoplasmic reticulum and secretory granules of 200–600 nm in diameter. The 600-kDa compound is probably a precursor form. Ab-475/400 stains the same three bands revealed by Ab-600; immunocytochemically, it reacts with two types of secretory granules (200–600 and 800–1200 nm in diameter) but it does not label the rough endoplasmic reticulum. Ab-145 reveals the bands at 600, 475 and 400 kDa and a diffuse zone in the region of 145 kDa; in light-microscopic immunocytochemistry, it behaves as Ab-475/400. The 475-kDa and 400-kDa glycoproteins, and a compound of approximately 145 kDa thus probably correspond to processed forms. Ab-475/400 stains granules present in cell processes ending on local blood vessels and at the leptomeninges. Since this antiserum selectively labels secretory granules, this finding may be taken as evidence for a basal route of secretion.

Identification of a high molecular weight polypeptide in the subcommissural organ of the chick embryo

Abstract.The subcommissural organ is an ependymal brain gland that secretes, into the ventricular cerebrospinal fluid, high molecular weight glycoproteins that form Reissner’s fiber. Precursor and processed forms of secretion have been demonstrated by immunoblotting in the subcommissural organ of mammals and fish. In the chicken only a processed form has as yet been identified. In the present report, we have studied the subcommissural organ of 13-day-old chick embryos using (1) an antiserum against bovine Reissner’s fiber, and (2) the lectins, concanavalin A and Limax flavus agglutinin. Paraffin sections of the subcommissural organ and blots of subcommissural organ extracts have been analyzed. The ependymal cells of sectioned subcommissural organ are strongly stained with the antiserum. Concanavalin A binds to materials in all cytoplasmatic regions, whereas Limax flavus agglutinin identifies materials confined to the apex of the ependymal cells. In the blots, a band of 540 kDa is immunostained. This band is positive for concanavalin A positive but negative for Limax flavus agglutinin and is thereby regarded as representing a precursor form of the secretion.

A sensitive method to analyse the effect of putative regulatory ligands on the release of glycoprotein from primary cultures of dispersed bovine subcommissural organ cells

The subcommissural organ (SCO) releases into the cerebrospinal fluid (CSF) large glycoproteins that polymerize forming the Reissners fibre (RF), which is involved in CSF circulation and homeostasis. We obtained high purity primary cultures of bovine secretory SCO cells and measured glycoprotein release by a reliable and sensitive ELISA method. We also analysed the effect of regulatory ligands known to control the secretory activity of the SCO. Cells cultured for short time (4h) released a high amount of glycoproteins that decreased with time. In young cultures, ATP increased and serotonin inhibited secretion rate. By contrast the acetylcholine agonist carbachol and high potassium did not evoke any detectable change in SCO glycoprotein release. These results support not only the suitability of the methodological approach but an important role of both ATP and serotonin in regulating SCO secretory activity as well.