M. Santacana

Formation of an olfactory glomerulus: morphological aspects of development and organization.

We have studied the development of olfactory nerves in the rat from their first contact with the telencephalic vesicle until the formation of glomerular structures in the olfactory bulb at early postnatal period. The study is based on serial semithin and ultrathin sections of material prepared for electron microscopy and antibodies to label radial glial cells, glial fibrillary acidic protein and Rat-401. Beginning on embryonic day 12, developing olfactory axons from the olfactory placode are accompanied by migratory cells, also derived from the olfactory placode, that reach the prospective olfactory bulb by embryonic day 13. The mass of migratory cells accumulate superficial to the telencephalic vesicle. The cells increase in number by mitotic divisions. The majority of these cells represent precursor elements that will later develop into the ensheathing cells of the olfactory nerves and olfactory nerve layer of the adult. Some migratory cells penetrate into the prospective olfactory bulb early during development. The first synaptic contacts of olfactory axons with dendritic processes in the olfactory bulb were observed at embryonic day 18. Glomerular formation is initiated by penetration of cells from the migratory mass into the prospective glomerular layer by embryonic day 20 to postnatal day 0. These cells form walls surrounding zones of high synaptic density forming protoglomeruli. Postnatally, the peripheral processes of radial glial cells branch profusely delimiting glomerular formations and transform into periglomerular astrocytes. Rat-401 stains radial glial cells from embryonic day 14. Immunoreactivity becomes restricted to the olfactory glomeruli during the first postnatal weeks and it virtually disappears by the end of the first postnatal month. We conclude that the early penetration of cells from the migratory mass into the prospective olfactory bulb, observed immediately after the first synaptic contacts were established, initiates the formation of olfactory glomeruli which becomes completed by the transformation of radial glial cells into periglomerular astrocytes.

Distribution of adrenomedullin-like immunoreactivity in the rat central nervous system by light and electron microscopy

Adrenomedullin is a peptide of marked vasodilator activity first isolated from human pheochromocytoma and subsequently demonstrated in other mammalian tissues. Using a polyclonal antiserum against human adrenomedullin-(22-52) amide and the avidin-biotin peroxidase complex technique, we have demonstrated by light and electron microscopy that adrenomedullin-like immunoreactivity is widely distributed in the rat central nervous system. Western blotting of extracts of different brain regions demonstrated the fully processed peptide as the major form in the cerebellum, whereas a 14-kDa molecular species and a small amount of the 18-kDa propeptide were present in other brain regions. Immunoreactive neurons and processes were found in multipolar neurons and pyramidal cells of layers IV-VI of the cerebral cortex and their apical processes, as well as in a large number of telencephalic, diencephalic, mesencephalic, pontine and medullary nuclei. Cerebellar Purkinje cells and mossy terminal nerve fibers as well as neurons of the cerebellar nuclei were immunostained, as were neurons in area 9 of the anterior horn of the spinal cord. Immunoreactivity was also found in some vascular endothelial cells and surrounding processes that probably originated from perivascular glial cells. Electron microscopy confirmed the light microscopy findings and showed the reaction product in relation to neurofilaments and the external membrane of small mitochondria. Immunoreactive terminal boutons were occasionally seen. The distribution of adrenomedullin-like immunoreactivity in the central nervous system suggests that it has a significant role in neuronal function as well as in the regulation of regional blood flow.

Neuronal and inducible nitric oxide synthase expression and protein nitration in rat cerebellum after oxygen and glucose deprivation

A perfusion model of global cerebral ischemia was used for the immunohistochemical study of changes in the glutamate-nitric oxide (NO) system in the rat cerebellum and cerebellar nuclei during a 0-14 h reperfusion period after 30 min of oxygen and glucose deprivation, with and without administration of 1.5 mM N(omega)-nitro-L-arginine methyl ester (L-NAME). While immunostaining for N-methyl-D-aspartate receptor subunit 1 (NMDAR1) showed no marked changes during the reperfusion period, neuronal NO synthase (nNOS) immunostaining increased in stellate and basket cells, granule cells and neurons of the cerebellar nuclei. However, global cerebellar nNOS concentrations determined by Western blotting remained largely unchanged in comparison with actin expression. Inducible NOS (iNOS) immunostaining appeared in Purkinje cells and neurons of the cerebellar nuclei after 2-4 h of reperfusion and intensified during the 6-14 h period. This was reflected by an increase in global cerebellar iNOS expression determined by Western blotting. Immunostaining for protein nitrotyrosine was seen in Purkinje cells, stellate and basket cells, neurons of the cerebellar nuclei and glial cells in controls, and showed a progressive translocation in Purkinje cells and neurons of the cerebellar nuclei from an initial perinuclear or nuclear location towards the periphery. At the end of the reperfusion period the Purkinje cell apical dendrites were notably retracted and tortuous. Prior and concurrent L-NAME administration eliminated nitrotyrosine immunostaining in controls and blocked or reduced most of the postischemic changes observed. The results suggest that while nNOS expression may be modified in certain cells, iNOS is induced after a 2-4 h period, and that changes in protein nitration may be associated with changes in cell morphology.

Transient pattern of exuberant projections of olfactory axons during development in the rat

The purpose of our study was twofold: (1) to trace the development of the olfactory axons from early embryonic stages until the mature pattern of connectivity and (2) to determine whether a transient penetration of them exists beyond the olfactory glomeruli. Two techniques were employed: DiI applied in the olfactory epithelium after aldehyde fixation, and olfactory marker protein (OMP) immunostaining. At E13 and E14 olfactory axons were observed spreading over the telencephalic vesicle and entering deeply into the prospective olfactory bulb, extending near the ventricular zone. Growth cones were seen at the end of these axons. At E15, the bundles of olfactory axons form a network, in which axons, growth cones and cells were seen. Some of these axons entered the olfactory bulb. Using OMP immunostaining olfactory axons were observed along the external plexiform layer, the mitral cell layer and in the granular layer from E19 to P6. At P9 some OMP immunoreactive axons were observed in the external plexiform layer. No OMP immunostained axons could be observed outside the glomeruli at P10. Our conclusions are that a transient immature pattern of early invasion over the telencephalic vesicle and of the olfactory bulb by olfactory axons occurs in the olfactory system. By the second postnatal week the glomerular layer reaches its mature configuration, and no olfactory fibers are seen outside the glomerular layer.

Characterization of neuronal cell varieties migrating from the olfactory epithelium during prenatal development in the rat. Immunocytochemical study using antibodies against olfactory marker protein (OMP) and luteinizing hormone-releasing hormone (LH-RH)

The development of neurons located outside the olfactory epithelium was studied by using antisera against olfactory marker protein (OMP) and luteinizing hormone-releasing hormone (LH-RH) in the rat. The study was restricted to the localization of these cells in the nasal cavity and in the region of the olfactory bulb during development. We describe groups of cells that stain positively for OMP located principally on the ventro-lateral aspect of the olfactory bulbs. A comparison is made with the LH-RH-immunoreactive system of cells which predominate on the medial aspect following the known trajectory of the nervus terminalis. OMP-immunoreactive cells appeared along the course of the olfactory fibers when they were first detected at embryonic day 16. These cells became restricted to a small group above the cribriform plate, ventral to the olfactory bulbs that seemed to disappear shortly after birth. It is concluded that these cells, which like the LH-RH cells have most probably migrated from the olfactory placode, represent a group of intervening neurons between the olfactory receptor cells and the olfactory bulb, serving as hints for olfactory axons to reach their targets during prenatal development.

Effects of oxygen and glucose deprivation on the expression and distribution of neuronal and inducible nitric oxide synthases and on protein nitration in rat cerebral cortex.

Changes in the nitric oxide (NO) system of the rat cerebral cortex were investigated by immunohistochemistry, immunoblotting, NO synthase (NOS) activity assay, and magnetic resonance imaging (MRI) in an experimental model of global cerebral ischemia and reperfusion. Brains were perfused transcardially with an oxygenated plasma substitute and subjected to 30 minutes of oxygen and glucose deprivation, followed by reperfusion for up to 12 hours with oxygenated medium containing glucose. A sham group was perfused without oxygen or glucose deprivation, and a further group was treated with the NOS inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME) before and during perfusion. Global ischemia led to cerebrocortical injury as shown by diffusion MRI. This was accompanied by increasing morphologic changes in the large type I interneurons expressing neuronal NOS (nNOS) and the appearance of nNOS immunoreactivity in small type II neurons. The nNOS‐immunoreactive band and calcium‐dependent NOS activity showed an initial increase, followed by a fall after 6 hours of reperfusion. Inducible NOS immunoreactivity appeared in neurons, especially pyramidal cells of layers IV–V, after 4 hours of reperfusion, with corresponding changes on immunoblotting and in calcium‐independent NOS activity. Immunoreactive protein nitrotyrosine, present in the nuclear area of neurons in nonperfused controls and sham‐perfused animals, showed changes in intensity and distribution, appearing in the neuronal processes during the reperfusion period. Prior and concurrent L‐NAME administration blocked the changes on diffusion MRI and attenuated the morphologic changes, suggesting that NO and consequent peroxynitrite formation during ischemia–reperfusion contributes to cerebral injury. J. Comp. Neurol. 443:183–200, 2002.

EXPRESSION OF NEURONAL NITRIC OXIDE SYNTHASE DURING EMBRYONIC DEVELOPMENT OF THE RAT CEREBRAL CORTEX

The expression of neuronal nitric oxide synthase (nNOS) during the development of the rat cerebral cortex from embryonic day (E) 13 to postnatal day (P) 0 was analyzed by immunocytochemical procedures using a specific antibody against rat brain nNOS. Expression of nNOS was first seen on E14 in cells of Cajal-Retzius morphology located in the marginal zone. Neuronal NOS immunoreactivity persisted in this layer throughout the embryonic period and only began to decrease on E20, when neuronal migration is coming to an end. From E17 onwards, migrating neurons expressing nNOS were observed in the intermediate zone with their leading processes directed towards the cortical plate. At the same time, efferent nNOS-immunoreactive axons originating from cortical plate cells entered the intermediate zone. From E19 onwards, cells expressing nNOS and with the morphological characteristics of migrating cells were observed in and near the subventricular zone. Confocal analysis of double immunostaining for nNOS and glial fibrillary acidic protein or nestin showed no coexpression of nNOS and glial markers in these cells, suggesting that nNOS-positive cells leaving the subventricular zone were not glial cells. Commissural, callosal and fimbrial fibers were seen to express nNOS on E18 and E19. This expression decreased from E20 and was very weak on E21 and P0. The observations suggest that nitric oxide is synthesized during embryonic life in relation to maturational processes such as the organization of cerebral lamination, and is involved in controlling migrational processes and fiber ingrowth.

Adrenomedullin expression is up-regulated by ischemia-reperfusion in the cerebral cortex of the adult rat.

Changes in the pattern of adrenomedullin expression in the rat cerebral cortex after ischemia-reperfusion were studied by light and electron microscopic immunohistochemistry using a specific antibody against human adrenomedullin (22-52). Animals were subjected to 30 min of oxygen and glucose deprivation in a perfusion model simulating global cerebral ischemia, and the cerebral cortex was studied after 0, 2, 4, 6, 8, 10 or 12 h of reperfusion. Adrenomedullin immunoreactivity was elevated in certain neuronal structures after 6-12 h of reperfusion as compared with controls. Under these conditions, numerous large pyramidal neurons and some small neurons were intensely stained in all cortical layers. The number of immunoreactive pre- and post-synaptic structures increased with the reperfusion time. Neurons immunoreactive for adrenomedullin presented a normal morphology whereas non-immunoreactive neurons were clearly damaged, suggesting a potential cell-specific protective role for adrenomedullin. The number and intensity of immunoreactive endothelial cells were also progressively elevated as the reperfusion time increased. In addition, the perivascular processes of glial cells and/or pericytes followed a similar pattern, suggesting that adrenomedullin may act as a vasodilator in the cerebrocortical circulation. In summary, adrenomedullin expression is elevated after the ischemic insult and seems to be part of CNS response mechanism to hypoxic injury.

Development and early postnatal maturation of the primary olfactory cortex

Tritiated thymidine autoradiography was used to study the origin and distribution of neurons in the primary olfactory cortex of the rat. The principal interest was devoted to animals injected at embryonic day 12 (E12) and sacrificed at different pre- and postnatal ages. The first generated neurons appearing at E12 were studied from E15 to P63. Animals sacrificed at E15 show a group of heavily labeled cells occupying a large area of the ventro lateral region of the telencephalic vesicle. At E16 this group differentiates into the principal cells of the accessory olfactory bulb and cells of the prospective primary olfactory cortex (POC). At E18-E20 the ventral tip of the cortical plate apparently divides this group into a superficial part corresponding to layer I and a deep part, corresponding to cells located in the adult in layer III. Labeled cells in layer I were found flanking the lateral olfactory tract (TOL), but rarely in the adult suggesting that they disappear or transform postnatally. Golgi observations were carried out from E15 to postnatal day 8. The morphology of different cells were studied. Layer I contains polymorphic cells resembling Cajal-Retzius cells. Among other cell types, layer II includes kinds of pyramidal cells lacking basal dendrites known as semilunar cells and intrinsic neurons. Layer III contains pyramidal cells having more than one apical dendrite ascending to the surface.

Coexistence of translocated cytochrome c and nitrated protein in neurons of the rat cerebral cortex after oxygen and glucose deprivation

Changes in the distribution of immunoreactive cytochrome c and protein nitration were studied in the rat cerebral cortex after oxygen and glucose deprivation by bright field, confocal and electron microscopy. In control cerebral cortex, nitrotyrosine immunoreactivity indicating protein nitration was found mostly in the neuronal nuclear region, with only a small amount distributed in the cytosol, whereas cytochrome c immunoreactivity was found at the inner membrane and in the intermembrane space of the mitochondria. During the recovery phase after oxygen and glucose deprivation, cytochrome c immunoreactivity was released from the intermembrane space of swollen mitochondria into the surrounding cytosol. The cytosol now also displayed nitrotyrosine immunoreactivity, which had diminished in the nuclear region. Both immunoreactivities were dispersed throughout the soma and processes of the cortical neurons. These changes were largely prevented by the administration of cyclosporin A, which inhibits both the mitochondrial permeability transition and the neuronal isoform of nitric oxide synthase while blocking the induction of the inducible isoform. Ischemia/reperfusion injury increases the production of nitric oxide, reactive oxygen species and intracellular factors that damage the mitochondria and liberate apoptotic factors. We suggest that translocation of cytochrome c from the mitochondria to the cytosol, which has been shown to precede the mitochondrial permeability transition, could result from peroxynitrite-mediated nitration. This phenomenon is attenuated by cyclosporin A administration, suggesting a neuroprotective role for this agent.