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Notes: Summary The axonal transport of horseradish peroxidase (HRP) has been investigated in the nigro-neostriato-nigral loop. The HRP moves bidirectionally, i.e. from cell bodies in the caudoputamen to their terminal arborization, mainly in pars reticulate, and from axon terminals in the neostriatum to their nigral cell bodies. Eighteen hours after the neostriatal injection of HRP both axon terminals and cell bodies in the substantia nigra are already labeled, indicating that the transport of HRP is associated with the fast phase of the axoplasmic flow. The ultrastructural study of unstained thick (0.15–0.3 μm) plastic sections allows a precise intraneuronal localization of the HRP reaction product. In labeled axon terminals of the pars reticulata, the reaction product is confined to vesicular and tubular profiles of the smooth endoplasmic reticulum (SER). The synaptic vesicles are always free of labeling. In labeled nigral neurons, numerous HRP-containing SER tubules are present in the cell bodies as well as in their proximal dendrites. These SER tubules are in contiguity and/or in continuity with dense bodies of an average diameter of about 0.5 μm and filled with reaction product. In addition, in the pars compacta numerous thin unmyelinated axons, of a calibre similar to that of dopaminergic axons, are labeled. In these instances, the HRP reaction product is exclusively confined to SER cisterns, and it can be followed for several microns within the section. All these results provide morphological evidence that the retrograde transport of HRP also takes place through the endoplasmic reticulum pathway.

Notes: Summary Correlation of morphological and electrophysiological data strongly suggest that in rat, the giant cells of the lateral vestibular nucleus (L.V.N.) are electrotonically coupled. 1. in addition to “active zones” large terminals synapsing on the perikaryon and/or the main dendritic trunk of the cells bear “gap” junctions which are interpreted as low electrical resistance pathways between neurons. 2. electrical activity of the giant cells was recorded intracellularly as the vestibulo-spinal tract was stimulated. Graded antidromic stimulation produced graded antidromic depolarizations (G.A.Ds) in 69% of cells with high threshold axons. 3. the latency of the G.A.Ds was too short to allow for chemical transmission through afferents or recurrent collaterals. 4. collision experiments demonstrated that directly evoked spikes blocked the antidromic spikes but did not block the G.A.Ds which thus were accounted for by activation of cells others than the impaled ones. 5. lesion experiments indicated that afferent fibers from the spinal cord terminate exclusively in the dorsal part of the L.V.N. Since G.A.Ds were recorded all throughout the nucleus, they were not excitatory post synaptic potentials (EPSPs) from spinal afferents. 6. when the strength of the spinal cord stimulation was increased EPSPs were also generated but they were distinct from the G.A.Ds by their latencies, time course and maximum amplitude. 7. since no direct contact is observed between neurons it is inferred that, as in other documented cases, coupling between giant cells is mediated by way of presynaptic fibers.

Notes: The expression of calcitonin gene-related peptide (CGRP) immunoreactivity in certain inferior olivary neurons is transient and developmentally regulated. Labelled neurons begin to appear at embryonic day 16 (E16), and reach their maximal extent by postnatal day 2 (P2). The extinction of the labelling occurs between P13 and P16. Expression of CGRP immunoreactivity is also observed in a few cerebellar fibres from E17, when axons in the restiform bundle begin to enter medially the cerebellar parenchyma. Their maximal extent is reached by P6, and thereafter they slowly disappear following a precise pattern, although fibre extinction is not complete. The spatio-temporal changes in the olivary distribution of the labelled neurons and the changes in the cerebellar labelled fibres follow the known pattern of topographic arrangement of the olivocerebellar system in adult rats. Moreover, the developmental phases of the CGRP-labelled fibres in postnatal rats correspond to those known for climbing fibre phenotypic acquisition. Thus, CGRP immunocytochemistry identifies in the fetal rat a subset of inferior olivary neurons and their corresponding cerebellar climbing fibres. Using this approach, we have analysed some of the initial events leading to the formation of the olivocerebellar projection, and obtained the following information: (i) Olivocerebellar axons are not randomly distributed in the restiform bundle before they enter the cerebellum. (ii) In the presence of a large spectrum of choices at the surface of the rostral half of the cerebellar plate the labelled olivary axons begin to enter the cerebellum at a precise medial point to abut a region composed solely of migrating Purkinje cells, and establish contacts with their targets before these neurons reach their final cortical location. (iii) From E18 to E19, the bundle of labelled fibres loses its superficial location, being bypassed by migrating Purkinje cells, to occupy a region corresponding to the prospective white matter. This translocation is coincident with the occurrence of a second axonal entry point, somewhat more lateral than the previous one, and with the appearance of a new lateral stripe of labelled fibres. (iv) Both the early and the late appearing labelled stripes remain confined from the time of their formation in precise cerebellar territories, indicating that only some clusters of Purkinje cells are contacted by the CGRP fibres. The results obtained imply that there is neither a waiting period nor an initial phase of randomness in the formation of the olivocerebellar projection map. This absence of chaotic cerebellar invasion, and the high selectivity of the entry points, suggest that the orientation of CGRP-positive olivocerebellar fibres towards their targets is regulated by positional information shared between subsets of olivary neurons and clusters of Purkinje cells. The result of this process would be the formation of a precocious coarse topography that would need further refinement.

Notes: Physiological, pharmacological and radioautographic binding studies have suggested the presence of the 5-HT1A autoreceptors on midbrain serotoninergic neurons. The recent production of specific anti-rat 5-HT1A receptor antibodies in rabbits injected with a synthetic peptide has provided a tool to examine this problem directly. Using the immunoperoxidase method to localize the receptor protein, neurons of all the sizes and forms characterizing the neuronal populations in the dorsal and median raphe nuclei were stained. Reaction product was distributed along the neuronal surface, outlining the contours of perikarya and dendrites in a continuous but uneven manner. Intracellular staining was scarce and confined to the perinuclear region. Double immunohistochemical staining using the anti-5-HT1A receptor antibodies and an anti-serotonin (5-HT) antiserum showed that all the 5-HT1A receptor immunoreactive neurons in the dorsal raphe, and the vast majority of them in the median raphe, are serotoninergic neurons. These data provide the first direct demonstration of the existence of 5-HT1A autoreceptors on the perikarya and dendrites of serotoninergic neurons in the anterior raphe nuclei.

Notes: This study reports the spatio-temporal pattern of BEN expression (a molecule of the immunoglobulin superfamily) during early stages of the first axonal tract formation, in the fore- and midbrain of chick embryos [Hamburger and Hamilton (HH) stages 12–22]. The expression of BEN has been analysed using immunohistochemistry and non-radioactive in situ hybridization. Furthermore, double labelling experiments (combining anti-class III β-tubulin, a pan-neuronal marker, and anti-BEN antibodies) have been carried out to determine whether BEN is expressed by all first axonal tracts. The first neurons expressing BEN appear around stage HH13–14, in the caudal diencephalon. They belong to the interstitial nucleus of Cajal, and their axons are the first components of the medial longitudinal fasciculus. By HH14, two other early axonal tracts appear: the tract of the postoptic commissure and the descending root of the mesencephalic nucleus of the trigeminal nerve. Only the latter expresses BEN. At later stages of development numerous new axonal tracts appear in the telencephalic, diencephalic and mesencephalic domains. Only a few of them (the fourth nerve, the lemniscus lateralis, the tectobulbar and habenulopeduncular tracts) express BEN. In all BEN positive systems, the cell bodies, axons and growth cones are uniformly labelled by the antibody. We have found that none of the early axonal tracts grows preferentially at interneuromeric boundaries. Moreover, each tract is formed by several thin fascicles rather than a single one. The expression of BEN is transient and disappears shortly before hatching. These results suggest that BEN may serve to promote axonal outgrowth of precise neuronal systems involved in ‘axonal scaffolding’.

Notes: Neural progenitors are thought to be multipotent cells whose adult phenotype is determined by extrinsic influences acting during and immediately after their last mitosis. To test this hypothesis, postnatal cerebellar precursor cells were placed in the heterochronic cellular environment of the embryonic mouse cerebellar anlage and the resulting phenotypes were determined. To identify the cells arising from postnatal precursors, tissue fragments taken from 3- to 8–day-old cerebellum of several transgenic mouse lines (each expressing the lacZ reporter gene in different sets of neuronal populations) were mixed with fragments taken from the wild-type cerebellar primordium of 12- or 13–day-old embryos. The fragments were dissociated and grafted into the cerebellum of adult mice. The phenotype acquired by postnatal precursors in the mixed grafts was determined by their morphology and ultrastructural features and by the expression of specific markers. Only two adult phenotypes were generated by these precursors: granule cells and molecular layer interneurons. Most granule cells were well integrated in the trilaminated cortex of the graft, being positioned in their proper layer both during development and after complete maturation. By contrast, basket and stellate cells were always ectopic, remaining outside the molecular layer. These results indicate that at least two distinct progenitor cells are present in the postnatal cerebellar cortex under the experimental conditions of this study. Both progenitors appear to be strictly specified at the time of grafting, and neither their identity nor the expression of their major distinctive features are significantly influenced by local signals emerging from the cellular environment of the embryonic cerebellar anlage.

Notes: This paper examines the organization of host afferents within cerebellar grafts implanted into kainic acid lesioned cerebellum. Our selection of a cerebellum, a prime example of a ‘point-to-point’ system, permits precise determination of the degree and the specificity of host-graft interactions.One month after a cerebellar injection of kainic acid, the lesion produced can be divided into two concentric regions: (i) a central necrotic zone, totally depleted of neurons (zone 1), and (ii) a peripheral zone which lacks all Purkinje cells but preserves its cortical lamination (zone 2). Two months after the implantation of solid pieces of embryonic cerebellum, the graft has evolved into a minicerebellar structure, occupying most of zone 1. The grafted minicerebellum consists of a highly convoluted trilaminated cortex with a core containing deep nuclear neurons. Purkinje cells are positioned between the molecular and granular layer with their short and irregular dendrites branching within the former. Donor foetal Purkinje cells migrate into the contiguous portion of the molecular layer of the host zone 2. These embryonic neurons set up within the upper three-quarters of the host molecular layer, and develop monoplanar dendritic trees that span the whole width of the layer.The organization of host-graft interactions was studied by autoradiography of anterogradely transported tritiated leucine, injected in the host bulbar region containing the caudal half of the inferior olivary complex (origin of all vermal climbing fibres) and the dorsally adjacent paramedian reticular nucleus (origin of a few mossy fibres). Numerous labelled fibres cross the host-graft interface from the white matter of the host cerebellum, and provide innervation to the minicerebellar structure. The vast majority of these labelled axons terminate in the molecular layer, forming axonal arborizations that follow the shape of the Purkinje cell dendrites. The labelled climbing fibres are organized into uneven sagittally aligned strips, which mimic that of olivocerebellar projections in control rats. Only a small proportion of host labelled fibres end in the donor granular layer, forming typical mossy fibre rosettes. The latter are present in the region of the graft close to the host-graft interface. In addition, labelled axons are observed climbing over the dendritic trees of grafted Purkinje cells that have invaded a portion of the host molecular layer of zone 2. In all regions containing grafted Purkinje cells and labelled climbing fibres, the density of the innervation is close to normal with practically all Purkinje cells receiving a climbing fibre.The extensive integration of the grafted cells into the deficient neuronal networks of the host clearly illustrates the positive neurotropic effect exerted by immature cerebellar neurons on adult extracerebellar afferent fibres. The hodological integration, allowing a possible restoration of the impaired cerebellar circuitry, takes place respecting the specificity and topographic distribution which characterize the ‘point-to-point’ arrangement of normal cerebellar circuitry.

Notes: GABA, a major inhibitory neurotransmitter, depolarizes hippocampal pyramidal neurons during the first postnatal week. These depolarizations result from an efflux of Cl– through GABAA-gated anion channels. The outward Cl– gradient that provides the driving force for Cl– efflux might be generated and maintained by the Na+, K+, 2Cl– cotransporter (NKCC) that keeps intracellular Cl– concentration above electrochemical equilibrium. The developmental pattern of expression of the cotransporter in the hippocampus is not known. We studied the postnatal distribution pattern of NKCC in the hippocampus using a monoclonal antibody (T4) against a conserved epitope in the C-terminus of the cotransporter molecule. We also examined the temporal relationships between the developmental pattern of NKCC expression and the formation of perisomatic GABAergic synapses. This study was aimed at determining, with antivesicular inhibitory amino acid transporter (VIAAT) antibodies, whether perisomatic GABAergic synapses are formed preferentially at the time when GABA is depolarizing. During the first postnatal week, NKCC immunolabelling was restricted to cell bodies in the pyramidal cell layer and in the strata oriens and radiatum. In contrast, at postnatal day 21 (P21) and in adult animals little or no labelling occurred in cell bodies; instead, a prominent dendritic labelling appeared in both pyramidal and nonpyramidal neurons. The ultrastructural immunogold study in P21 rat hippocampi corroborated the light-microscopy results. In addition, this study revealed that a portion of the silver-intensified colloidal gold particles were located on neuronal plasmalemma, as expected for a functional cotransporter. The formation of inhibitory synapses on perikarya of the pyramidal cell layer was a late process. The density of VIAAT-immunoreactive puncta in the stratum pyramidale at P21 reached four times the P7 value in CA3, and six times the P7 value in CA1. Electron microscopy revealed that the number of synapses per neuronal perikaryal profile in the stratum pyramidale of the CA3 area at P21 was three times higher than at P7, even if a concomitant 20% increase in the area of these neuronal perikaryal profiles occurred. It is concluded that, in hippocampal pyramidal cells, there is a developmental shift in the NKCC localization from a predominantly somatic to a predominantly dendritic location. The presence of NKCC during the first postnatal week is consistent with the hypothesis that this transporter might be involved in the depolarizing effects of GABA. The depolarizing effects of GABA may not be required for the establishment of the majority of GABAergic synapses in the stratum pyramidale, because their number increases after the first postnatal week, when GABA action becomes hyperpolarizing.

Notes: The scarring process occurring after adult central nervous system injury and the subsequent increase in the expression of certain extracellular matrix molecules are known to contribute to the failure of axon regeneration. This study provides an immunohistochemical analysis of temporal changes (8 days to 1 year) in the cellular and molecular response of the Swiss mouse spinal cord to a dorsal hemisection and its correlation with the axonal growth properties of a descending pathway, the serotoninergic axons. In this lesion model, no cavity forms at the centre of the lesion. Instead, a dense fibronectin-positive tissue matrix occupies the centre of the lesion, surrounded by a glial scar mainly constituted by reactive astrocytes. NG2 proteoglycan and tenascin-C, potential axon growth inhibitors, are constantly associated with the central region. In the glial scar, tenascin-C is never observed and the expression of chondroitin sulphate proteoglycans (revealed with CS-56 and anti-NG2 antibodies) highly increases in the week following injury to progressively return to their control level. In parallel, there is an increasing expression of the polysialilated neural cell adhesion molecule by reactive astrocytes. These molecular changes are correlated with a sprouting process of serotoninergic axons in the glial scar, except in a small area in contact with the central region. All these observations suggest that while a part of the glial scar progressively becomes permissive to axon regeneration after mouse spinal cord injury, the border of the glial scar, in contact with the fibronectin-positive tissue matrix, is the real barrier to prevent axon regeneration.

Notes: To determine whether members of the Netrin-1 and Slit families and their receptors are expressed after central nervous system (CNS) injury, we performed in situ hybridization for netrin-1, slit-1, 2 and 3, and their receptors (dcc, unc5h-1, 2 and 3, robo-1, 2 and 3) 8 days, 2–3 months and 12–18 months after traumatic lesions of rat cerebellum. The expression pattern of these molecules was unchanged in axotomized Purkinje cells, whereas unc5h3 expression was upregulated in deafferented granule cells. Cells expressing slit-2 or dcc were never detected at the lesion site. By contrast, cells expressing netrin-1, slit-1 and slit-3, unc5h-1, 2 and 3, and robo-1, 2 and 3 (rig-1) could be detected at the cerebellar lesion site as soon as 8 days after injury. Expression of unc5h-2, robo-1, robo-2, slit-1 and slit-3 at the lesion site was maintained until 3 months, and up to 12–18 months for unc5h-1 and 3 and robo-3. Likewise, in the mouse spinal cord, netrin-1, slit-1 and slit-3 were also expressed at the lesion site 8 days after injury. Most of the cells expressing these mRNAs were located at the centre of the lesions, suggesting that they are macrophages/activated microglial cells (macrophagic cells) or meningeal fibroblastic cells. The macrophagic nature of most Netrin-1-positive cells and the macrophagic or fibroblastic nature of Robo-1-positive cells were corroborated by double staining. Thus, Netrin-1, Slits and their receptors may contribute to the regenerative failure of axons in the adult CNS by inhibiting axon outgrowth or by participating in the formation of the CNS scar.