Klf9 is necessary and sufficient for Purkinje cell survival in organotypic culture.

During their phase of developmental programmed cell death (PCD), neurons depend on target-released trophic factors for survival. After this period, however, they critically change as their survival becomes target-independent. The molecular mechanisms underlying this major transition remain poorly understood. Here, we investigated, which transcription factors (TFs) might be responsible for the closure of PCD. We used Purkinje cells as a model since their PCD is restricted to the first postnatal week in the mouse cerebellum. Transcriptome analysis of Purkinje cells during or after PCD allowed the identification of Krüppel like factor 9 (Klf9) as a candidate for PCD closure, given its high increase of expression at the end of the 1st postnatal week. Klf9 function was tested in organotypic cultures, through lentiviral vector-mediated manipulation of Klf9 expression. In absence of trophic factors, the Purkinje cell survival rate is of 40%. Overexpression of Klf9 during PCD dramatically increases the Purkinje cell survival rate from 40% to 88%, whereas its down-regulation decreases it to 14%. Accordingly, in organotypic cultures of Klf9 knockout animals, Purkinje cell survival rate is reduced by half as compared to wild-type mice. Furthermore, the absence of Klf9 could be rescued by Purkinje cell trophic factors, Insulin growth factor-1 and Neurotrophin3. Altogether, our results ascribe a clear role of Klf9 in Purkinje cell survival. Thus, we propose that Klf9 might be a key molecule involved in turning off the phase of Purkinje PCD.

Expression of X-chromosome linked inhibitor of apoptosis protein in mature Purkinje cells and in retinal bipolar cells in transgenic mice induces neurodegeneration.

Transgenic mice with overexpression of the caspase-inhibitor, X-chromosome-linked inhibitor of apoptosis protein (XIAP) in Purkinje cell (PC) and in retinal bipolar cells (RBCs) were produced to study the regulation of cell death. Unexpectedly, an increased neurodegeneration was observed in the PCs in these L7-XIAP mice after the third postnatal week with the mice exhibiting severe ataxia. The loss of PCs was independent of Bax as shown by crossing the L7-XIAP mice with Bax gene-deleted mice. Electron microscopy revealed intact organelles in PCs but with the stacking of ER cisterns indicative of cell stress. Immunostaining for cell death proteins showed an increased phosphorylation of c-Jun in the PCs, suggesting an involvement in cell degeneration. Apart from PCs, the number of RBCs was decreased in adult retina in line with the expression pattern for the L7 promoter. The data show that overexpression of the anti-apoptotic protein XIAP in vulnerable neurons leads to enhanced cell death. The mechanisms underlying this neurodegeneration can be related to the effects of XIAP on cell stress and altered cell signaling.

Neuroprotective effect of mifepristone involves neuron depolarization.

In several regions of the developing nervous system, neurons undergo programmed cell death. In the rat cerebellum, Purkinje cell apoptosis is exacerbated when cerebellar slices are cultured during the first postnatal week. To understand the mechanism of this developmental apoptosis, we took advantage of its inhibition by the steroid analog mifepristone. This effect did not involve the classical steroid nuclear receptors. Microarray analysis revealed that mifepristone down-regulated mRNA levels of the Na+/K+-ATPase alpha3 subunit more than three times. Consistent with the down-regulation of the Na+/K+-ATPase, mifepristone caused Purkinje cell membrane depolarization. Depolarizing agents like ouabain (1 microM), tetraethylammonium (2 mM), and veratridine (2 microM) protected Purkinje cells from apoptosis. These results suggest a role of excitatory inputs in Purkinje cell survival during early postnatal development. Indeed, coculturing cerebellar slices with glutamatergic inferior olivary neuron preparations allowed rescue of Purkinje cells. These findings reveal a new neuroprotective mechanism of mifepristone and support a pivotal role for excitatory inputs in the survival of Purkinje neurons. Mifepristone may be a useful lead compound in the development of novel therapeutic approaches for maintaining the resting potential of neurons at values favorable for their survival under neuropathological conditions.

Lack of Purkinje cell loss in adult rat cerebellum following protracted axotomy: degenerative changes and regenerative attempts of the severed axons.

The cerebellar Purkinje cells, due to their geometrical disposition and their high calbindin content, offer an optimal system in which to test the adequacy of current opinions on axotomy effects. We have, therefore, analyzed with calbindin immunostaining the morphological changes of Purkinje cells from 1 day to 6 months after axonal section in the cerebellar white matter. This method allows us to study the morphological changes in their dendrites, cell bodies, and axons. We have also searched for simultaneous changes in glial cells and vascularization by using cell type-specific markers. In addition, an ultrastructural study of Purkinje cells, 7 days after large electrolytic lesions affecting the white matter and the overlying granular layer, was carried out to determine whether amputation of the recurrent collateral system provokes a fast neuronal death. Neither the Purkinje cells axotomized close to their cell bodies (electrolytic lesions) nor those axotomized in the white matter (cerebellar transection) degenerated. Thus, this study demonstrates that Purkinje cells are extremely resistant to axotomy; those severed in the white matter at distances varying from 100 microns to 3 mm remain alive for as long as 6 months. At all survival times studied, axotomized Purkinje cells exhibited few changes in their somata and dendrites, as well as in their glial microenvironment. The major changes occurred in the axonal compartment. Axonal alterations, namely the presence of torpedoes and hypertrophy of the recurrent collateral system, were early events already noticeable 24 hours after the lesion, although they later differed in their time course and spatial distribution. It is remarkable that the distal segments of the central stumps of the cut axons survived in large numbers without any apparent retraction, with their terminal varicosities apposed to the wall of the wound cavity even 6 months after the lesion. Nevertheless, these segments were thinner than normal Purkinje cell axons (axonal atrophy). Despite this apparent immutability, some regenerative attempts did occur in the severed axons, such as axonal sprouts penetrating the deeper region of the granular layer in zones close to the lesion, presence of arciform axons, and hypertrophy of the recurrent collateral system. However, the Purkinje cell axons did not regenerate, and these neurons remained separated from their targets by a cavity in virtually all cases.

Late axonal sprouting of injured Purkinje cells and its temporal correlation with permissive changes in the glial scar.

Purkinje cells can survive axotomy for as long as 18 months without retracting their severed axons. During this period of time, the fate of the terminal bulbs of axotomized Purkinje cell axons and their relationship with the glial scar were determined. Terminal axonal sprouting begins three months after the lesion and continuously increases up to 18 months (the longest survival time studied), when the sprouts establish synaptic contacts, mainly on granule cell dendrites at the glomeruli. Cellular changes in the glial scar were analyzed to determine whether the late onset and continuous increase of axonal sprouting could be correlated with an increase of permissive factors and/or a decrease of inhibitory factors for axonal growth. Activated macrophages disappeared much earlier than did the initiation of sprouting. Myelin and its associated neurite growth inhibitory molecules began to decrease from three months after the lesion. This decrease was uneven and not correlated spatially with the sprouting. Reactive astrogliosis was heterogeneous: only some of the reactive astrocytes expressed PSA-NCAM, the embryonic form of the neural cell adhesion molecule, a permissive substratum for neurite outgrowth. The expression of PSA-NCAM occurred concurrently with sprouting in the area of gliosis containing Purkinje cell sprouts. Moreover, the ultrastructural study showed that the majority of sprouts (75%) were totally ensheathed by astrocytic processes. Thus, long-term glial scars are permissive to axonal sprouting, suggesting that reactive astrocytes, either through the expression of permissive molecules or by preventing direct contact between axonal elements and myelin inhibitory molecules, regulate the sprouting.

Stathmin: cellular localization of a major phosphoprotein in the adult rat and human CNS.

Stathmin is a ubiquitous, 19 kDa cytoplasmic protein the phosphorylation of which is associated with many cellular signaling pathways. It is particularly abundant in neurons and reaches a peak of expression in the neonatal period, although it remains highly expressed in the adult brain. In order to determine whether this abundant expression is associated with discrete cellular populations that are still at an immature stage during adulthood, as suggested by others, the cellular localization of stathmin was investigated in the adult rat and human central nervous system. Western blotting with a specific antiserum indicated that stathmin was ubiquitous in the brain and spinal cord but that its relative concentration varied up to 2.6 times between regions. To characterize the distribution of stathmin within the brain, its cellular localization was analyzed by immunocytochemistry. Highly immunoreactive neurons and oligodendrocytes were observed, and stathmin immunoreactivity was localized to the perikaryon and all processes, but not the nucleus. Most brain and spinal cord cell groups showed stathmin immunoreactivity, although the extent and intensity of labeling differed largely from one place to another. Particularly numerous stathmin-immunoreactive neuronal cell bodies were found in the pyriform, cingulate, and neocortex, as well as in many cholinergic nuclei of the basal forebrain and brainstem, in the medial thalamus, in various brainstem nuclei, in the dorsalmost layers of the spinal cord, and in brain areas lacking a blood-brain barrier to macromolecules. In addition to neuronal populations, stathmin-antibodies intensely labeled choroid plexuses. Many other brain regions exhibited moderate neuronal immunostaining. The distribution of stathmin-immunoreactive processes was in some areas relatively heterogeneous. Intense immunoreactivity was observed in some fiber tracts (corpus callosum, anterior commissure, inferior cerebellar peduncle, etc.) but was missing in others (internal capsule, posterior commissure, etc.). Some brain areas rich in immunoreactive neurons also displayed an intense immunoreactivity of the neuropile, whereas others contained either immunoreactive cells or fibers. In the human brain, stathmin immunostaining occurred in many areas, corresponding to those identified in the rat, with the exception of the cerebral cortex, the hippocampal fascia dentata, and the substantia nigra. The present results support our suggestion that, in addition to its involvement in cell proliferation and differentiation, stathmin may also be related to regulation of differentiated cell functions, as it appears to be a major signaling protein in widespread areas of the adult brain in both rat and human.

Glial changes following an excitotoxic lesion in the CNS--I. Microglia/macrophages.

When an area of the adult rat CNS is depleted of neurons by an in situ excitotoxic injection, afferent axons to the area exhibit morphological alterations reminiscent of growth cones. These morphological changes are likely to be related to the deprivation of target cells. In addition, however, the area of neuronal loss is itself the site of profound changes in glial cell content, and altered axon-glial interactions may play a role in the axonal changes. In an attempt to define these interactions, we have undertaken a systemic study of glial populations in excitotoxically lesioned CNS over time. The microglial/macrophagic response is analysed in this paper; the astrocytic response is described in the companion paper [Dusart et al. (1991) Neuroscience 45, 541-549]. The microglial/macrophagic response was studied following kainic acid-induced neuronal loss in the thalamus of the adult rat. These microglial/macrophagic cells were labeled with the B4 isolectin from Griffonia simplicifolia, and the time-course of their response was studied between one day and one year post-lesion. This time-course study revealed different stages in the evolution of the response. At one day post-lesion, cell counts indicated that there was no increase in the number of non-neuronal cells in the neuron-depleted area. However, activated labeled cells were present in the entire thalamus on the side of the lesion, neuron-depleted or not. They were characterized by both increased lectin-binding and altered morphology when compared to quiescent microglia. In the absence of recruitment and/or proliferation, this result indicates that the early response consisted solely of the activation of resident microglia. By contrast, we observed a progressive increase in the number of non-neuronal cells in the lesion from four to 15 days post-lesion. A recruitment of blood-borne monocytes was apparent, and the observation of mitotic labeled cells indicated a proliferation of microglial/macrophagic cells in situ. There was a progressive decrease in the microglial/macrophagic reaction that began one month after lesion. In a thin band of parenchyma surrounding the neuron-depleted area, activated microglial/macrophagic cells were seen contacting neurons, and clusters of glial cells were observed around neurons up to one year post-lesion. These results suggest that neurons around the lesion site itself may be injured, secondarily, from a long term deleterious effect of the inflammatory process. This study allows us to conclude that activated microglia/macrophages are the predominant glial cell type in the excitotoxically lesioned CNS over the first weeks.(ABSTRACT TRUNCATED AT 400 WORDS)

Glial changes following an excitotoxic lesion in the CNS--II. Astrocytes.

Astrocytes are involved, as are microglia/macrophages [Marty et al. (1991) Neuroscience 45, 529-539], in the formation of a glial scar after CNS lesions. This study was undertaken to follow the time-course of changes in the morphology and distribution of astrocytes that takes place during the formation of a glial scar after kainic acid injection in the rat thalamus. The astrocytes were identified using an antibody raised against glial fibrillary acidic protein (GFAP) and the progression of their reaction to the lesion was followed from 24 h to one year after the kainate injection. Three periods could be distinguished during the evolution of the astrocytic response in the neuron-depleted area. There was an initial appearance of a large number of GFAP+ cells. These cells displayed profound morphological differences from the normal. They were enlarged, round and devoid of processes. These GFAP+ astrocytes disappeared four days after the lesion. This increase of the GFAP+ cells in the neuron-depleted area may be due to cytoskeletal changes and thus an increased exposure of antigenic sites. In a second period between four and 14 days, the only GFAP+ elements present in the neuron-depleted area were long and straight processes. These processes entered the lesioned area from the periphery and seemed to follow axon bundles. Additionally, during the first weeks, the number of reactive astrocytes increased in a small band just around the area of neuronal loss. The third period began after two weeks. The lesioned area became gradually occupied by GFAP+ astrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Demyelination, and remyelination by Schwann cells and oligodendrocytes after kainate-induced neuronal depletion in the central nervous system.

Excitotoxins are thought to kill neurons while sparing afferent fibers and axons of passage. The validity of this classical conclusion has recently been questioned by the demonstration of axonal demyelination. In addition, axons are submitted to a profound alteration of their glial environment. This work was, therefore, undertaken to reassess axonoglial interactions over time after an excitotoxic lesion in the rat. Ultrastructural studies were carried out in the ventrobasal thalamus two days to 18 months after neuronal depletion by in situ injections of kainic acid. In some cases, lemniscal afferents were identified by using anterograde transport of wheatgerm agglutinin conjugated to horseradish peroxidase from the dorsal column nuclei. Two and four days after kainate injection, numerous dying axons displaying typical signs of Wallerian degeneration were observed in a neuropile characterized by the loss of neuronal somata and dendrites, an increase in number of microglia/macrophages and the disappearance of astrocytes. Ten and 12 days after kainate injection, degenerating axons were no longer observed although myelin degeneration of otherwise unaltered axons was ongoing with an accumulation of myelin remnants in the neuropile. At 16 and 20 days, the demyelination process was apparently complete and axons of different diameters were sometimes packed together. One and two months after kainate injection, the axonal environment changed again: remyelination of large-caliber axons occurred at the same time as reactive astrocytes, oligodendrocytes and numerous Schwann cells appeared in the tissue. Schwann cell processes surrounded aggregates of axons of diverse calibers, ensheathed small ones and myelinated larger ones. Axons were also remyelinated by oligodendrocytes. Horseradish peroxidase-labeled lemniscal afferents could be myelinated by either of the two cell types. After three months, the neuropile exhibited an increase in number of hypertrophied astrocytes and the progressive loss of any other cellular or axonal element. At this stage, remaining Schwann cells were surrounded by a glia limitans formed by astrocytic processes. These data indicate that although excitotoxins are sparing the axons, they are having a profound and complex effect on the axonal environment. Demyelination occurs over the first weeks, accompanying the loss of astrocytes and oligodendrocytes. Axonal ensheathment and remyelination takes place in a second period, associated with the reappearance of oligodendrocytes and recruitment of numerous Schwann cells, while reactive astrocytes appear in the tissue at a slightly later time. Over the following months, astrocytes occupy a greater proportion of the neuron-depleted territory and other elements decrease in number.(ABSTRACT TRUNCATED AT 400 WORDS)

First month of development of fetal neurons transplanted as a cell suspension into the adult CNS.

It has been demonstrated elsewhere that fetal thalamic tissue, when transplanted as a cell suspension into the excitotoxically neuron-depleted adult somatosensory thalamus, can grow, differentiate, and receive projections from host afferents. In the present study, we used the same paradigm to analyse the transplanted neurons during their morphogenesis, i.e. during the first month after transplantation. Using various anatomical criteria, at the light and electron microscope levels, we compared the development of transplanted neurons with the normal ontogeny of homologous neuronal populations. Confined solely to the mechanically lesioned area during implantation at seven days post-grafting, the transplant increased in size to occupy most of the previously neuron-depleted area by the third week after grafting. The final size of the transplant thus depended upon the size of the lesion. At seven days post-grafting, the neurons were small in size and the cellular density was high. At this immature stage few synaptic contacts were visible and the ultrastructure was characterized by large extracellular spaces. At 10 days post-grafting, the size of the neurons had increased and the cellular density had decreased. Both an extensive dendritic proliferation and a simultaneous active synaptogenesis could also be observed. All these events continued to evolve and during the third week the neuropil progressively acquired more mature ultrastructural characteristics. Synaptic contacts exhibiting characteristics comparable to those observed in the intact thalamus also became more numerous. At 20 days post-grafting, axonal myelination had started, the development of the graft apparently stopped and the various criteria had stabilized. Until that developmental stage, growth of grafted neurons compared to that of normal thalamic ones. At later stages, however, grafted neurons failed to grow larger and did not reach the size of the homologous population in the adult animal. It seems, therefore, that transplants of thalamic fetal neurons can be used as a tool with which to study thalamic neuronal development, within definable limits.

Parasagittal compartmentation of adult rat Purkinje cells expressing the low-affinity nerve growth factor receptor: changes of pattern expression after a traumatic lesion.

The pattern of expression of p75, the low affinity nerve growth factor receptor, in the adult rat cerebellum and its fate after a traumatic lesion were analysed using immunohistochemical localization of this receptor. A subset of Purkinje cells was immunoreactive for low affinity nerve growth factor receptor in the intact adult cerebellum. These cells were arranged in alternating positive and negative parasagittal compartments along the cerebellar cortex. This pattern of expression had 90% homology with zebrin I. After a traumatic lesion, the specific pattern of expression of zebrin I remained unchanged, whereas the low affinity nerve growth factor receptor pattern changed as early as one day: Purkinje cells near the lesion site, independent of zebrin I staining, became immunoreactive. During the first week, the increase in immunoreactivity remained high. Thereafter, there was a short, fast decrease followed by a long period in which a faint immunostaining on lesioned Purkinje cells is maintained for up to one year. The increase in the expression of the low affinity nerve growth factor receptor by all traumatically affected Purkinje cells suggests a correlation between this specific up-regulation and the high resistance of these neurons to axotomy or other traumatic injuries.

Axotomy does not up-regulate expression of sodium channel Na(v)1.8 in Purkinje cells.

Aberrant expression of the sensory neuron specific (SNS) sodium channel Na(v)1.8 has been demonstrated in cerebellar Purkinje cells in experimental models of multiple sclerosis (MS) and in human MS. The aberrant expression of Na(v)1.8, which is normally present in primary sensory neurons but not in the CNS, may perturb cerebellar function, but the mechanisms that trigger it are not understood. Because axotomy can provoke changes in Na(v)1.8 expression in dorsal root ganglion (DRG) neurons, we tested the hypothesis that axotomy can provoke an up-regulation of Na(v)1.8 expression in Purkinje cells, using a surgical model that transects axons of Purkinje cells in lobules IIIb-VII in the rat. In situ hybridization and immunocytochemistry did not reveal an up-regulation of Na(v)1.8 mRNA or protein in axotomized Purkinje cells. Hybridization and immunostaining signals for the sodium channel Na(v)1.6 were clearly present, demonstrating that sodium channel transcripts and protein were present in experimental cerebella. These results demonstrate that axotomy does not trigger the expression of Na(v)1.8 in Purkinje cells.

Fetal neural transplants into an area of neurodegeneration in the spinal cord of the adult rat.

Lesioning the spinal cord with an excitotoxic agent provides a model of neuronal degeneration while sparing afferent axons. The present study has been undertaken to determine whether homotypic fetal neurons transplanted as a cell suspension were able to rebuild a neural circuitry in the neuron-depleted adult cord. Fetal spinal cords, taken from rat embryos (gestational day E12-13), were transplanted as cell suspensions into an area of the lumbar cord previously depleted of neurons using kainic acid. The excitotoxic lesion extended over ventral and intermediate horns, implying the death of all motoneurons with consequent paralysis and muscular atrophy of corresponding hindlimb. During the first month after injection, the damaged cord was characterized by proliferation and recruitment of various glial cell and Schwann cell populations. First to appear were activated microglia/macrophages and next reactive astrocytes which entered the lesion from its borders with the intact tissue. Schwann cells also ensheathed central axons. Differential sensitivity of various afferents to loss of postsynaptic target neurons was observed: rubrospinal and corticospinal afferents decreased in density while no conspicuous changes were observed for immunostained CGRP-containing or monoaminergic fibers. Two to fourteen months after surgery, transplants occupied most of the neuron-depleted area. The grafts did not display a laminar organization. Monoaminergic afferents grew for a long distance and formed a network within transplants. Similarly, primary sensory CGRP-immunoreactive fibers entering in the dorsal roots penetrated deeply into transplants. In contrast, cortico- and rubrospinal afferents entered only the most peripheral portion of transplants. Our results indicate that fetal spinal neurons can be successfully transplanted into the adult neuron-depleted spinal cord. Host-to-graft connections can be formed, although their spatial extent in the transplants may depend upon features of the afferent fiber systems.

Somatic activation of thalamic neurons transplanted into lesioned somatosensory thalamus.

Homotypic fetal neurons were transplanted into previously lesioned ventrobasal complex of rats. After 1-3 months of survival the animals received injections of 2-deoxy-[14C]glucose to reveal metabolic activity of the transplanted cells in response to somatic stimuli. These experiments indicated that stimulus-evoked activity in the transplants of animals receiving a somatic stimulus was significantly greater than in the transplants of animals that were not stimulated. Control studies using cell counts, cytochrome oxidase and acetylcholinesterase histochemistry established that the differences in activity values were not due to the number of surviving cells or the metabolic health of the individual grafts.

Postnatal development of cholecystokinin (CCK) binding sites in the rat forebrain and midbrain: an autoradiographic study.

The postnatal development of cholecystokinin (CCK) binding sites in the rat forebrain and midbrain was studied by in vitro receptor autoradiography. In the majority of structures, the densities of sites were low over the first week after birth, increased until the third week, and decreased over the fourth week to reach adult levels. However, both the rate of increase and the extent of the decrease varied in large proportions among structures. For instance, labeling in the neocortex underwent its largest increase from postnatal day 10, while this increase was already begun at day 7 in the paleocortex. On the other hand, over the fourth postnatal week, the densities could either remain roughly constant (cingulate cortex), slightly decrease (thalamic reticular nucleus), or even return to background levels (pyramidal layer of hippocampus). These different timetables may depend mostly on the differential growth of cells expressing the CCK receptor gene within the developing CNS. The absence of CCK binding sites in most of the regions during the early postnatal period precludes a major role of this peptide in the embryonic development of the rat brain. However, in some regions as the ventromedial hypothalamic nucleus, the endopyriform cortex or the medial nucleus of amygdala, 30-50% of the adult levels were already present at birth. Whether this observation reflects an earlier functional maturation of these structures or a direct participation of the corresponding CCK systems in their development remains to be established.

Vascularization of fetal cell suspension grafts in the excitotoxically lesioned adult rat thalamus.

Several studies have considered the establishment of vascularization in intracerebral solid transplants of neural tissue. The widely supported interpretation of the results is that the vascular network of the solid grafts is already present before implantation into the host brain. The situation is different when dissociated fetal tissue is transplanted as a cell suspension because in these conditions the fetal vascular network is disrupted. The present study has, therefore, been undertaken to follow the angiogenesis in a transplant of dissociated fetal cells implanted into the excitotoxically neuron-depleted thalamus. The vascular network is compared to that observed in the intact and in the lesioned thalamus both in terms of morphology of the capillaries and of the function of the blood-brain barrier (BBB). In the transplant, capillaries, stained by Indian ink, are very few in number and have very fine calibers during the first 20 days after grafting. Some structures can be identified as immature blood vessels at the electron microscopic level. The blood vessels are progressively more numerous in the graft and they demonstrate mature ultrastructural features 2 months after grafting. Last, there is no leakage of the BBB for peroxidase. The vascularization seems to follow a pattern of maturation comparable to that described during development in the literature. In contrast, in the lesioned area, there is a reactive angiogenesis: 10 days after the excitotoxic injection (shortest time studied), there are many wide caliber vessels with expanded perivascular spaces engorged with mesodermal cells. A microvascularization also develops transiently during the first two months. Capillaries are abnormal from the functional point of view, since there is a leakage of the BBB to macromolecules. The use of an experimental model in which transplant had to grow in a lesioned area permits to determine two types of vascularization: an apparently normal developmental timetable, normal morphological and functional characteristics, in the transplant; a reactive angiogenesis, in the lesioned area.

Presence of Schwann cells in neurodegenerative lesions of the central nervous system.

Ultrastructural analysis of neurodegenerative CNS lesions produced by an excitotoxic substance revealed that the majority of cells ensheathing axons were not oligodendrocytes. By their morphology and the presence of both a basal lamina and collagen fibers they were identified as Schwann cells. The presence of Schwann cells, whose growth-promoting role in the peripheral nervous system has been largely documented, may account for the development of regenerating growth cones which have been observed in the excitotoxically lesioned central nervous system. Further support for this hypothesis came from the analysis of fetal neural transplants implanted into the lesioned area. Schwann cells ensheathing axons were indeed numerous in the neuron-depleted area surrounding the transplants, where neurite outgrowth of graft origin occurred.

Lack of adenylate cyclase 1 (AC1): consequences on corticospinal tract development and on locomotor recovery after spinal cord injury.

Cyclic AMP (cAMP) signalling pathways are involved in axonal growth and regeneration. The calcium-calmodulin- stimulated adenylate cyclase 1 (AC1), a regulator of cAMP levels, is strongly expressed in the corticospinal motor neurons (CSMN) in cerebral cortex layer V during development, but its role in the development of the corticospinal tract (CST) is unknown. Here, we analyse the organization of the CST pathway using anterograde and retrograde tracers in the barrelless (brl) mouse that carries an inactivating mutation of the AC1 gene. We show that in brl mice the general organization of the CST is normal but there is an increase in the number of axons in the ipsilateral contingent in the dorsal and ventral medial funiculi of the cervical spinal cord. The density of CSMN in layer V of the motor cortex is increased in brl compared to wild-type mice. Thus, lack of AC1 likely perturbs late phases of CSMN and CST development. Next, we examine the motor recovery after a spinal cord injury (SCI). We find that brl mice show enhanced locomotor functions as assessed by the BMS (Basso mouse scale) as early as 6h and up to 6 weeks after SCI, indicating a smaller responsiveness of brl mice to SCI. It is therefore possible that developmental effects on motor systems might decrease the locomotor effects consecutive to a SCI. This point is particularly important with regards to the use of transgenic animals for testing SCI recovery.

Intrinsic versus extrinsic determinants during the development of Purkinje cell dendrites.

The peculiar shape and disposition of Purkinje cell (PC) dendrites, planar and highly branched, offers an optimal model to analyze cellular and molecular regulators for the acquisition of neuronal dendritic trees. During the first 2 weeks after the end of the proliferation period, PCs undergo a 2-phase remodeling process of their dendrites. The first phase consists in the complete retraction of the primitive but extensive dendritic tree, together with the formation of multiple filopodia-like processes arising from the cell body. In the second phase, there is a progressive disappearance of the somatic processes along with rapid growth and branching of the mature dendrite. Mature Purkinje cell dendrites bear two types of spiny protrusions, named spine and thorn. The spines are numerous, elongated, located at the distal dendritic compartment and form synapses with parallel fibers, whereas the thorns are shorter, rounded, emerge from the proximal compartment and synapse with climbing fibers. Different culture models and mutant mice analyses suggest the identification of intrinsic versus extrinsic determinants of the Purkinje cell dendritic development. The early phase of dendritic remodeling might be cell autonomous and regulated by specific transcription factors such as retinoid-related orphan receptor alpha (RORalpha). Afferent fibers, trophic factors and hormones regulate the orientation and growth of the mature dendritic tree contributing, with still unknown intrinsic factors, to sculpt its general architecture. The formation of spines appears as an intrinsic phenomenon independent of their presynaptic partner, the parallel fibers, and confined to the distal compartment by inhibitory influences of the climbing fibers along the proximal compartment.