Axonal transport of rabies virus in the central nervous system of the rat.

Stereotaxic inoculation of rabies virus into specific nuclei in the central nervous system has been used for the investigation of the central neural transport mechanisms of viral information. The infection was monitored by specific fluorescence and peroxidase studies and the titration of viral infectivity in dissected brain areas. Twenty-four hours after inoculation into the striatum, cortex, or substantia nigra, infected neurons were detected only in cells from areas and nuclei which were related to the site of inoculation. The distribution of infected neurons showed that retrograde axoplasmic flow plays a determining role in the transport of rabies virus 24 hours after delivery of virus to specific target nuclei. Local destruction of neurons by kainic acid at the site of viral inoculation did not prevent the uptake and subsequent retrograde axonal transport of virus. There was an overall correlation between the major neural connections of the inoculated areas (e.g. the striatum) and the infected areas 24 hours later (e.g. the substantia nigra).

Cajal-Retzius neurons in human cerebral cortex at midgestation show immunoreactivity for neurofilament and calcium-binding proteins.

Along with subplate neurons, Cajal-Retzius cells (CRc) are the first neurons to be generated in the cortical anlage. Studies of their chemical content, such as neurofilament and calcium-binding proteins, might give indications on their role in cortical development at midgestation in human fetuses (20-24 gestation weeks), when the CRc are morphologically mature. Cajal-Retzius cells were immunolabeled with antibodies to subunits of neurofilament proteins SMI31 and SMI32. The SMI32 antibodies (directed against the nonphosphorylated epitope) specifically labeled the CR cell bodies, dendrites, and proximal axons in a Golgilike fashion. Specific acetylcholinesterase activity is known to be typical of all the CRc, and double labeling for SMI32 immunoreactivity and acetylcholinesterase histochemistry demonstrated that all the CRc exhibited SMI32 immunoreactivity. The SMI31 antibodies (directed against the phosphorylated epitope) exclusively labeled the CRc axons, forming a dense positive network in the deep one-half of layer 1. This plexus was much denser than the one described with the Golgi method (Marin-Padilla, 1990: J. Comp Neurol 239:89-105). Calbindin D28k, parvalbumin, and calretinin immunoreactivities were visualized in the CRc. Double-labeling experiments showed that most of the CRc contained both calbindin and calretinin and sometimes parvalbumin. These colocalizations revealed a chemical heterogeneity within the CRc population even though they were described as morphologically homogeneous. These colocalizations of calcium-binding proteins in the CRc differed from the other nonpyramidal cortical neurons where calbindin, calretinin, and parvalbumin are contained in different (mutually exclusive) neuronal populations. Based on the morphological features and differential chemical contents described for the CRc, different hypotheses on their possible role and fate are discussed.

Axonal secretion of Reelin by Cajal-Retzius cells: evidence from comparison of normal and Reln(Orl) mutant mice.

A novel secretory pathway has been identified in the study of mice homozygous for the Reln(Orl) mutation, a line characterised by the defective secretion of the large extracellular matrix glycoprotein Reelin. By using both light and electron microscopy, immunohistochemical studies for Reelin in these mutants identified morphological changes in their Cajal-Retzius cells (CR cells). The CR cells of the mutant displayed the characteristic features of bipolar, tangentially elongated neurons with a dendritic proximal pole and an axonal cone at the opposite end of the soma. At either pole, cisterns of prominent rough endoplasmic reticulum (RER) were found to be rich in Reelin. However, the Reelin-positive RER cisterns of the axonal cones were hugely dilated in homozygous Reln(Orl) mice as compared with their wild type counterparts. CR cell axons displayed beads throughout their length, each contained a smooth spheroidal cistern filled with Reelin-immunoreactive fibrillar material, and were increased in number and size in Reln(Orl) mice. RER phenotype was rescued in the Reln(Alb2) mice, a mutation in which no Reelin protein is produced. We propose that the RER dilations viewed in the Reln(Orl) mutation are due to the accumulation of the defective Reelin protein, and the large axonal beads in Reln(Orl) mice reflect the accumulation of truncated Reelin as the result of defects in its secretion. These observations point to an original, hitherto unrecognised, mechanism of secretion by bulk transport in smooth cisterns from the axonal cone into the axon, followed by secretion in the cortical marginal zone from the axonal cisterns that we have named axonal reelin reservoirs.

A normally laminated afferent projection to an abnormally laminated cortex: some olfactory connections in the reeler mouse.

The relative positions of pyramidal and polymorphic cell classes are inverted in the central olfactory cortical structures of the reeler mutant mouse. Each cell class is generated at the normal embryonic time. The polymorphic cells of the mutant, like those of the normal, are generated between E11-E13. The pyramidal cells are formed between E11-E16 in both. Despite the anomalous positions of their somata deep in the cortex the apical dendrites of many pyramidal cells reach and ramify at a superficial cortical level subjacent to the lateral olfactory tract. The main and accessory olfactory bulbs are cytoarchitectonically normal in the mutant and project normally upon the anterior olfactory nucleus, the olfactory tubercle, the hippocampal rudiment, the piriform cortex, the amygdaloid region and the entorhinal cortex. As in the normal animal the axons transverse layer Ialpha, and their terminals are concentrated in the immediately subjacent laminar zone. The rostrally directed cortic-cortical association system of the piriform cortex projects upon the anterior olfactory nucleus in the mutant just as in the normal with a relative concentration of terminals in a lamina subjacent and complementary to the zone of termination afferent systems in the abnormally laminated olfactory cortex of the mutant syggests that, in this system at least, the developmental mechanisms which determine relative position of neuron somata and those which govern axon trajectories and the distribution of axon terminals are largely independent.

Projections from the hypothalamus to the posterior lobe in rats during ontogenesis: 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate tracing study.

The objective of this study was to determine the schedule of the arrival of the axons from the hypothalamus to the posterior lobe of the pituitary (PL) in rats during ontogenesis by using the fluorescent lipophilic carbocyanine dye 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI) as a retrograde tracer. After preliminary fixation of the brain, DiI crystals were implanted in the PL on embryonic day 15 (E15), E16, E17, and E19 as well as on postnatal day 2 (P2) and P9. This was followed by a DiI retrograde diffusion along the plasma membrane and subsequent staining of hypothalamic neuronal cell bodies. The supraoptic nucleus (SO) contained an accumulation of fluorescent cells that extended toward the diamond-like swelling of the third ventricle as early as E15. These data suggest that the magnocellular neurons of the SO send their axons to the PL at the very beginning of differentiation, perhaps even before reaching their final position. The initial axons of the neurons of the paraventricular nucleus proper (PV) appeared to reach the PL significantly later, at E17. In addition to the SO and the PV, accessory magnocellular nuclei contributed to the innervation of the PL in perinatal rats. The neurons of the retrochiasmatic accessory nucleus first sent their axons to the PL on E16-E17. Axons that originated from other accessory hypothalamic nuclei reached the PL after birth, suggesting a delay in their involvement in the regulation of visceral functions compared with other magnocellular nuclei. Thus, the axons of magnocellular neurons reach the PL unexpectedly early in embryogenesis, raising the possibility of the functional significance of vasopressin and oxytocin as fetal neurohormones.

Evidence that mouse astrocytes may be derived from the radial glia. An immunohistochemical study of the cerebellum in the normal and reeler mouse.

Astrocytic cells of unusual aspect can be detected in the cerebellum of normal mice during the first 4 weeks of life. They are visualized with anti-GFAP (glial fibrillary acidic protein), anti-S100 and anti-vimentin immune sera. Their perikaryons, located in the white matter or in the granular layer, extend long processes which are inserted onto the pial surface. These cells may be transitional forms between the radial glial cells and some of the differentiated astroglial elements. These unusual astrocytes are more numerous and heavily stained in the reeler mutant than in the normal mouse and it is suggested that our observations signify some degree of glial immaturity in the cerebellum of the mutant.

Cajal-Retzius cell ontogenesis and death in mouse brain visualized with horseradish peroxidase and electron microscopy.

The ontogenetic development of Cajal-Retzius cells was studied in mouse by local application of horseradish peroxidase over the developing neocortex, revelation with 3,3'-diaminobenzidene and examination from horizontal thick sections. Cajal-Retzius cells were completely stained in Golgi-like fashion. The Cajal-Retzius cells were seen to be elongated spindle-shaped bipolar neurons with their main processes horizontally oriented. They were exclusively located in the first cortical layer and were connected to the cortex surface by the numerous vertical appendages. Except for these appendages, the Cajal-Retzius cells were two-dimensional, with an immature structure at their tips resembling a growth cone. Cajal-Retzius cell dendrites were up to 400-microns-long and reached their maximal length prenatally. Their axon and its collaterals were very fine and sometimes measured several millimetres. It followed a random but planar trajectory confined to the first layer. Healthy Cajal-Retzius bearing growth cones were seen until one week after birth when signs of Cajal-Retzius cell degeneration began to occur and intensified in the days that followed. Rough endoplasmic reticulum and Golgi complex swelling along with a progressive darkening of the Cajal-Retzius cells were revealed by electron microscopy, strongly suggesting that most Cajal-Retzius cells disappear from the first cortical layer. Usually neuronal death is the result of cell deafferentation following synapse retraction; however, this effect does not seem to apply to Cajal-Retzius cells engaged in the process of death since normal synaptic junctions were seen on them. No signs of the morphological transformation of Cajal-Retzius cells into persisting horizontal first layer cells were observed. The concept of dual origin of neocortex is discussed in light of the similar fate of Cajal-Retzius cells and subplate neurons which both are transient neurons.

The inferior colliculus of the mouse. A Nissl and Golgi study.

Serial sections of cell- and fiber-stained and Golgi-impregnated material from adult mice were used to study the cytoarchitectonics, fiber and neuronal architecture of the inferior colliculus. The size of the cells, the pattern of dendritic branching, and the appearance of the neuropil were the features used to delineate the three main regions of the auditory tectum: the central mass of cells or central nucleus, the cortex, and the paracentral nuclei. The central nucleus contains two major cell types: the bipolar cells, which are the most abundant, and the multipolar cells. The dendrites of the bipolar cells are oriented in the same direction and the afferent axons of the lateral lemniscus run along them, contributing to form fibrodendritic strips: the laminae of the central nucleus. The orientation of these laminae differs in the various parts of the central nucleus and delineates four subdivisions. In these four subdivisions, the laminae maintain the same relative position throughout the anteroposterior axis of the central nucleus, but they stop abruptly at the periphery of the nucleus. The cortex surrounds the central nucleus dorsally and caudally. The lamination in four layers concentric to the surface, the increasing gradient of size from the periphery to the deep tissue, the existence of two major types of cells, stellate and pyramidal, permit this structure to be considered as a true cortex. The paracentral nuclei are scattered around the central nucleus. The commissural nucleus is composed of cells with a simple dendritic branching pattern perpendicular or parallel to the fibers of the intercollicular commissure. The dorsomedial and ventrolateral nuclei are characterized by the presence of large multipolar cells. The nucleus of the rostral pole, distinct from the anterior pole of the central nucleus, is composed of small and medium-sized multipolar cells. The lateral nucleus appears as an extension of the dorsal cortex with only two or three layers of cells. The neuronal organization in the central nucleus appears similar in the mouse and in the cat, suggesting an identical processing of auditory information in the two species. Our results seem to establish definitely the cortical nature of the sheet of cells covering the central nucleus.

A calcium/calmodulin kinase pathway connects brain-derived neurotrophic factor to the cyclic AMP-responsive transcription factor in the rat hippocampus.

Brain-derived neurotrophic factor (BDNF) plays fundamental roles in synaptic plasticity in rat hippocampus. Recently, using rat hippocampal slices, we found that BDNF induces activation of calcium/calmodulin-dependent protein kinase 2 (CaMKII), a critical mediator of synaptic plasticity. CaMKII in turn activates the p38 subfamily of mitogen-activated protein kinases (MAPK) and its downstream effector, MAPK-activated protein kinase 2 (MAPKAPK-2). Herein, we determined whether some kinases of this pathway connect BDNF to the cyclic AMP response element -binding protein (CREB), a transcription factor also involved in plasticity and survival. Crude cytosolic and nuclear fractions were prepared from hippocampal slices of adult rat, and then kinase involvement in CREB phosphorylation was studied with a combination of pharmacologic inhibition and antibody depletion. In addition, the regional localization of this signaling pathway was immunohistochemically investigated. We show that: (i). the BDNF-stimulated CaMKII cascade phosphorylates the key positive regulatory site of CREB via its end MAPKAPK-2 component; (ii). this process appears to be highly localized in the outermost cell layer of the dentate gyrus. The present findings suggest that CaMKII is involved in neurotrophic-dependent activation of CREB in the dentate gyrus. Such a signaling process could be important for controlling synaptic plasticity in this major area for the afferent inputs to the hippocampal formation.

Early cortical histogenesis in the primary olfactory cortex of the mouse.

The primary olfactory cortex of mouse embryos varying in age from the 13th to the 15th day of gestation is investigated with the aid of several techniques: Nissl and Golgi preparations, autoradiography and electron microscopy. It is observed that the earliest cells to complete their migrations are diffusely scattered at all levels of the emerging cortical plate. The dendrites of these cells are predominantly tangentially aligned. Subsequently these early formed cells are subdivided into two groups. One group remains superficial in close relation to the lateral olfactory tract while the second is displaced to the deepest level of the cortex. The dendrites of many cells which remain at superficial cortical levels become realigned in a radially upward direction. "Maculae adherens diminutae" are abundant at points of apposition of membranes of cells of all classes at superficial cortical levels. It is suggested that these serve to stabilize cell position and that the upward dendritic realignment is an expression of radially directed stresses. Synapses appear relatively late and probably play no role as determinants of cell position.

Evidence for the occurrence of early modifications in the 'glia limitans' layer of the neocortex of the reeler mutant mouse.

A comparative EM study of the marginal zone of the neocortex in E15 foetuses of normal and 'reeler' mice was carried out. In the mutant a direct contact between 'proneurons' and the superficial basal lamina bordering the 'glia limitans' layer was observed. Since this phenomenon occurs with a much higher frequency in the mutant than in the normal animal, it is thought to be important in the genesis of the 'reeler' anomaly. In the discussion, different hypotheses concerning the possible nature, mechanism and importance of the interaction described are suggested.

Comparative localization of Cajal-Retzius cells in the neocortex of normal and reeler mutant mice fetuses.

The Cajal-Retzius cells (CRc) appear as an histochemically advanced subpopulation, since they are the only neocortical neurons able to express an acetylcholinesterase activity at an early stage of corticogenesis. Thus a comparative study of the CRc population was carried out on normal and reeler mice fetuses. The use of an appropriate cytochemical technique to reveal this endogenous cell marker, in conjunction with coronal and tangential sections show that: the Cajal-Retzius cells form a sheet in the more superficial layer of the embryonic cortex, with a relative cell density giving the same value in both phenotypes; a difference is observed in the relative depth position of these cells with regard to the pial basal lamina and in the pattern of cell grouping between the heterozygous and homozygous mutant littermates. This technique appears of great interest for a further analysis of the CRc population during cortical ontogenesis.

Ontogenesis of NADPH-diaphorase neurons in the mouse forebrain.

Demonstration of NADPH diaphorase (NADPH-d) activity in the nervous system has recently gained considerable interest since it has been shown that this enzyme is a nitric oxide synthase (NOS). Therefore, histochemical staining of NADPH-d activity provides a specific labelling of neurons that use nitric oxide (NO). In this work, spatiotemporal distribution of NADPH-d neurons has been determined during forebrain ontogenesis. NADPH-d neurons first appeared between embryonic days 15 and 16 and were confined to the fronto-lateral aspect of the incipient corpus striatum and cortical subplate but were not present in the cortical plate. Until birth, NADPH-d neurons differentiated progressively in cortical subplate and striatum in rostro-caudal and latero-medial directions. For both regions, the adult cortical pattern was established during the first postnatal week. The pattern of genesis of NADPH-d neurons might be related to the spatiotemporal ontogenesis of catecholaminergic afferents to the forebrain described previously in the literature.

[Demonstration of Cajal-Retzius cells and their processes in the neocortex of newborn mice using horseradish peroxidase]. / Mise en évidence des cellules de Cajal-Retzius et de leurs prolongements dans le néocortex de la souris nouveau-née à l'aide de la peroxydase de raifort.

In newborn mouse neocortex, the so-called Cajal-Retzius cells (CRc), which are usually considered as neurons due to their polarity, located exclusively in the first cortical layer, were visualized using local application of horseradish peroxidase (HRP) on the neocortex followed by its tangential sectioning. Close to the application site, the exogenous enzyme, taken up by some of the CRc, was revealed with 3,3' diaminobenzidin (DAB) according to the Graham and Karnovsky technique. The brown reaction product was seen to fill almost completely some of these cells giving a "Golgi like" picture. They had a fusiform bipolar pericaryon and processes extending only into the first cortical layer. A single thick process whose length reached 300 to 400 microns, almost rectilinear and tapering progressively at its end was a dendrite which bore thin expansions reaching the cortical surface where they sometimes ramified between the endfeet of the radial glia. The dendrite sometimes showed symmetrical synapses with an afferent axon of unknown origin. The CRc axon was very thin (0.5 microns in diameter) and gave off at random numerous collaterals whose number and trajectories varied greatly from one cell to the other. The axonal processes could often be followed over a millimeter. They ended either abruptly because HRP had not diffused far enough into the process or terminated with large growth cones bearing numerous digitiform filopodia. The presence of growth cones thus suggested that the processes were exploring the cellular environment of the first cortical layer. In the newborn, CRcs appeared as still immature neurons.

The supraependymal fibres (SEF) of the mouse brain. as visualized by the Golgi method.

The well-known Golgi method, which impregnates, among other elements, unmyelinated fibres, has been proved suitable to visualize a dense plexus of very winding and smooth axonal processes lying all over the ventricular surface of the mouse brain. Comparisons with other cytochemical studies correlate quite well the pattern of distribution of these supraependymal fibres (SEF), and allow the characterization of most of these axons as serotoninergic. Combining thick sections, selective impregnation and well-defined plane of section, a restricted exit zone of the SEF in the fourth ventricle has been found over the raphe dorsalis region, which is known from previous studies to represent the main site of origin of serotoninergic fibres in the brain. However, this fact does not exclude the possibility of other pathways, yet to be discovered.

Forebrain connections of the rat paraventricular thalamic nucleus as demonstrated using the carbocyanide dye DiI.

The anatomical connections of the rat paraventricular thalamic nucleus (PaVT) have been studied using the fluorescent dye DiI. The fact that this compound is able to dissolve and to diffuse in plasma membranes in formalin-fixed tissues (Godement et al., 1987) allowed us to depose precisely tiny quantities of DiI on the PaVT, in order to study its neuronal connections. Ventralward, the forebrain projections of the PaVT were directed to the reuniens nucleus of the thalamus, the dorso-medial hypothalamic region, the zona incerta and the lateral hypothalamic area. In the rostral direction, the PaVT appeared to innervate mainly the medial preoptic area, the bed nucleus of the stria terminalis (particularly the ventral and medial regions) and the medial septum. The forebrain afferent connections of the PaVT were found to originate mainly in the lateral and periventricular hypothalamic area, the medial preoptic area, the bed nucleus of the stria terminalis and the medial septum where the fluorescent nerve cell bodies appeared particularly numerous. From these results, it is concluded that DiI is a useful tool for demonstrating the neuroanatomical connections of small brain nuclei, as well as for post-mortem neuropathological studies.

[Identification of Cajal-Retzius cells during neocortical ontogenesis in mice with fluorescent carbocyanine]. / Identification des cellules de Cajal-Retzius durant l'ontogenèse du néocortex de la souris à l'aide d'une carbocyanine fluorescente.

Cajal-Retzius cells, which are present transiently in the first layer of the mammalian neocortex, have been revealed in the mouse by DiI. This lipophilic fluorescent dye, locally applied over the cortex after formaldehyde fixation, allowed the global view of cortical cells. During ontogenesis, Cajal-Retzius cells retained their initial characteristic bipolar shape and orientation parallel to the meningeal surface. The bright fluorescent light emitted by this dye allowed visualization of the labelled cells by "microtomoscopy" using a confocal scanning laser microscope and analysis of the detailed aspect of these neurons and of their connections.

Extracellular matrix distribution during neocortical wall ontogenesis in "normal" and "Reeler" mice.

The spatio-temporal distribution of extracellular spaces (ECS) was analysed during prenatal corticogenesis in the normal mouse or in the recessive mutant Reeler. Colloidal iron was used to visualize extracellular anionic substances (containing glycosaminoglycans) which constitute an extracellular matrix (ECM). The spatio temporal distribution of the ECM appeared closely related to ECS localization. Before the cortical plate formation, the ECM occurred only in the marginal zone. The plate appearance resulted in a redistribution of anionic sites in the subplate of the normal mouse in addition to the marginal zone. Although, in the Reeler, the ECM remained mainly located in a broad outer cortical zone. Until birth, the distribution of the ECM in the normal mouse, followed closely the inside-out gradient of cortical neuropile maturation, while in the Reeler and outside-in gradient was observed. With EM, following standard fixation and staining an extracellular matrix (formed of 5 to 10 nm fibrils) occurred in sites where the ECM was already detected with light microscopy. The ECM fibrils were seen mainly in the vicinity of young neurons showing characters of secretory functions, such as dilated RER cisternae filled with a fibrillar material and well developed golgi complexes. Young Cajal-Retzius cells appeared especially well suited to elaborate the ECM in the marginal zone of the normal mouse or in the outer cortical zone of the Reeler. The potential role of ECM for the differentiation and formshaping of young neurons is discussed.

Enhancement of passive avoidance learning through small doses of intra-amygdaloid physostigmine in the young rat. Its relation to the development of acetylcholinesterase.

Passive avoidance learning was studied in young rats 7-20 days old, in control conditions and after bilateral injections of physostigmine into the lateral amygdaloid nucleus. Acquisition in controls was possible from postnatal Day 8 on, progressed markedly after Day 11, and nearly reached maturity by Day 20. Physostigmine differentially altered acquisition depending on the dose: facilitation with low doses, no effect with moderate doses, and impairment with high doses. Enhanced learning through small doses of physostigmine was observed at all ages from Day 8 on, and was greater with 0.2 microgram than with 0.1 microgram. Maturation of the cholinergic innervation of the amygdaloid region was also studied between Days 9-20 using acetylcholine-esterase histochemistry. The results suggest that passive avoidance learning is dependent on amygdaloid cholinergic mechanisms early in life. In addition, very immature cholinergic systems, which are known to be uninfluenced by anticholinergic agents, react to anticholinesterases.

[Supra-ependymal "giant"" nerve terminals in rodent brains]. / Sur la présence de terminaisons sus-épendymaires "géantes" dans le cerveau des rongeurs

In the lateral ventricles and third ventricle of the Rat and Mouse brain a dense net of supraependymal amyelinic varicose axons is obvious. Moreover, orginating from this was found small quantities of "giant" supra-ependymal nerve endings evoking sometimes the aspect of sensory terminals. These two types of supra-ependymal structures seem to contain 5-hydroxytryptamine.