Rosine Wehrlé

Postnatal maturation of Na+, K+, 2Cl– cotransporter expression and inhibitory synaptogenesis in the rat hippocampus: an immunocytochemical analysis

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.

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. J. Comp. Neurol. 408:399–418, 1999.

Implication of Bcl-2 and Caspase-3 in age-related Purkinje cell death in murine organotypic culture: an in vitro model to study apoptosis

Neuronal cell death is an essential feature of nervous system development and neurodegenerative diseases. Most Purkinje cells in murine cerebellar organotypic culture die when taken from 1–5‐day‐old mice (P1–P5), whereas they survive when taken before or after these ages. Using DNA gel electrophoresis, terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labelling (TUNEL) and electron microscopic analyses, we were able to show that this massive Purkinje cell death is apoptotic in nature and reaches a peak at P3. From the several endogenous genes known to be involved in the apoptotic process, we have focused on two: the bcl‐2 and the caspase‐3 that encode for anti‐apoptotic and pro‐apoptotic proteins, respectively. Immunostaining for activated Caspase‐3 correlated with Purkinje cell death. A better survival of Purkinje cells was observed in P3 slices taken from hu‐bcl‐2 transgenic mice, and in slices treated with z‐DEVD.fmk (an inhibitor of numerous caspases). Thus, these two genes are implicated in the age‐related Purkinje cell apoptosis in organotypic culture. As Purkinje cell death in vitro takes place at the same age as Purkinje cells engaged in intense synaptogenesis and dendritic remodeling in vivo, we propose that this apoptosis reflects a naturally occurring Purkinje cell death during this critical period.

Gene expression signature of cerebellar hypoplasia in a mouse model of Down syndrome during postnatal development

BackgroundDown syndrome is a chromosomal disorder caused by the presence of three copies of chromosome 21. The mechanisms by which this aneuploidy produces the complex and variable phenotype observed in people with Down syndrome are still under discussion. Recent studies have demonstrated an increased transcript level of the three-copy genes with some dosage compensation or amplification for a subset of them. The impact of this gene dosage effect on the whole transcriptome is still debated and longitudinal studies assessing the variability among samples, tissues and developmental stages are needed.ResultsWe thus designed a large scale gene expression study in mice (the Ts1Cje Down syndrome mouse model) in which we could measure the effects of trisomy 21 on a large number of samples (74 in total) in a tissue that is affected in Down syndrome (the cerebellum) and where we could quantify the defect during postnatal development in order to correlate gene expression changes to the phenotype observed. Statistical analysis of microarray data revealed a major gene dosage effect: for the three-copy genes as well as for a 2 Mb segment from mouse chromosome 12 that we show for the first time as being deleted in the Ts1Cje mice. This gene dosage effect impacts moderately on the expression of euploid genes (2.4 to 7.5% differentially expressed). Only 13 genes were significantly dysregulated in Ts1Cje mice at all four postnatal development stages studied from birth to 10 days after birth, and among them are 6 three-copy genes. The decrease in granule cell proliferation demonstrated in newborn Ts1Cje cerebellum was correlated with a major gene dosage effect on the transcriptome in dissected cerebellar external granule cell layer.ConclusionHigh throughput gene expression analysis in the cerebellum of a large number of samples of Ts1Cje and euploid mice has revealed a prevailing gene dosage effect on triplicated genes. Moreover using an enriched cell population that is thought responsible for the cerebellar hypoplasia in Down syndrome, a global destabilization of gene expression was not detected. Altogether these results strongly suggest that the three-copy genes are directly responsible for the phenotype present in cerebellum. We provide here a short list of candidate genes.

Role of GAP‐43 in mediating the responsiveness of cerebellar and precerebellar neurons to axotomy

To determine whether the competence for axonal sprouting and/or regeneration in the cerebellar system correlates with GAP‐43 expression, we have studied GAP‐43 mRNA and protein expression in the postlesioned cerebellum and inferior olive. Purkinje cells transiently express GAP‐43 during their developmental phase (from E15 to P5 in the rat) which consists of fast axonal growth and the formation of the corticonuclear projection. Adult Purkinje cells, which in control adult rats do not express GAP‐43, are extremely resistant to the effects of axotomy but cannot regenerate axons. However, a late and protracted sprouting of axotomized Purkinje cells occurs spontaneously and correlates with a mild expression of GAP‐43 mRNA. In contrast, inferior olivary neurons, despite their high constitutive expression of GAP‐43, do not sprout but retract their axons and die after axotomy. Furthermore, mature Purkinje cells in cerebellar explants of transgenic mice that overexpress GAP‐43 do not regenerate after axotomy, even in the presence of a permissive substrate (cerebellar embryonic tissue) and, contrary to the case in wild‐type mice, they do not survive in the in vitro conditions and undergo massive cell death. These results show that the expression of GAP‐43 is not only associated with axonal growth, but also with neuronal death.

SCLIP Is Crucial for the Formation and Development of the Purkinje Cell Dendritic Arbor

Cerebellar Purkinje cells elaborate one of the most complex dendritic arbors among neurons to integrate the numerous signals they receive from the cerebellum circuitry. Their dendritic differentiation undergoes successive, tightly regulated phases of development involving both regressive and growth events. Although many players regulating the late phases of Purkinje cell dendritogenesis have been identified, intracellular factors controlling earlier phases of dendritic development remain mostly unknown. In this study, we explored the biological properties and functions of SCLIP, a protein of the stathmin family, in Purkinje cell dendritic differentiation and cerebellum development. Unlike the other stathmins, SCLIP is strongly expressed in Purkinje cells during cerebellar development and accumulates in their dendritic processes at a critical period of their formation and outgrowth. To reveal SCLIP functions, we developed a lentiviral-mediated approach on cerebellar organotypic cultures to inhibit or increase its expression in Purkinje cells in their tissue environment. Depletion of SCLIP promoted retraction of the Purkinje cell primitive process and then prevented the formation of new dendrites at early stages of postnatal development. It also prevented their elongation and branching at later phases of differentiation. Conversely, SCLIP overexpression promoted dendritic branching and development. Together, our results demonstrate for the first time that SCLIP is crucial for both the formation and proper development of Purkinje cell dendritic arbors. SCLIP appears thus as a novel and specific factor that controls the early phases of Purkinje cell dendritic differentiation during cerebellum development.

Neuronal promoter of human aromatic L-amino acid decarboxylase gene directs transgene expression to the adult floor plate and aminergic nuclei induced by the isthmus.

In order to analyze the regulatory sequences involved in the neuronal expression of aromatic L-amino acid decarboxylase (AADC), we have generated transgenic mice carrying the LacZ gene under the control of a 3.6-kb human aadc genomic fragment flanking the neuronal alternative first exon. A series of double labeling experiments were performed to compare the pattern of transgene expression to that of specific markers for catecholaminergic and serotonergic neurons. In the adult brain parenchyma, transgene expression was observed in the substantia nigra (SN), the ventral tegmental area (VTA) and the dorsal, medial and pontine raphe nuclei. A large degree of co-expression was observed with tyrosine-hydroxylase (TH) in the SN and VTA, and with serotonin (5-HT) in the dorsal raphe nucleus. Moreover, expression was observed in cells that were both TH- and 5-HT-negative, in particular in the ventral tegmental decussation and the dorsal tip of the VTA. Transgene expression was also observed in the walls of central cavities. Cells positive for both beta-gal and PSA-NCAM were localized in the ventral ependyma of the third and fourth ventricle, and of the central canal of the spinal cord, in what appears to be the adult floor plate. Transgene expressing, PSA-NCAM negative, cells located along the ventral midline of the spinal cord seemed to have migrated out of the ependyma. Our data thus reveal the complexity of aadc gene regulation. The present transgene provides a unique marker for monoaminergic nuclei induced by the isthmus and for the adult floor plate.