[Development of the central nervous system in mammals]. / Développement du système nerveux central chez les mammifères.

In humans, the nervous system is induced during the third gestational week by molecular signals coming from the mesoderm, which modulate the temporal and spatial expression of specific genes in the cells of the dorsal ectoderm. The induced neural plate closes to form the neural tube where the cells actively proliferate in the germinal zone. The neuroblasts which have completed their last division migrate along the fibers of the radial glia to which they adhere, and this movement is essential to establish the normal cerebral organization. The regional identity of the developing brain is governed by the expression of homeobox genes, and the main central structures are clearly delineated by the end of the fifth week. The cerebral cortex begins to form on the seventh week, and the early specification of the cortical areas, which is under genetic control, would be modulated later on by environmental influences. When the neurons have reached their final position, they extend an axon, using surface molecules or diffusible molecules present along its pathway as cues to reach the appropriate target and form a synapse, and this process is a critical step for the establishment of neuronal relationships. The maturation and stabilization of neural networks is characterized by the apoptotic death of roughly 50% of the neurons, due to insufficient neurotrophic support, and by the remodeling of the initial synaptic connections in the surviving neurons. These regressive events occur late in development and depend on both the interactions with the environment and the resulting neuronal activity.

A comparative study of Purkinje cells in two RORalpha gene mutant mice: staggerer and RORalpha(-/-).

The staggerer (Rora(sg/sg)) mutation is a deletion in the RORalpha gene, one member of a family of nuclear receptor genes related to the retinoic acid receptor. Recently Steinmayr et al. (Proc. Natl. Acad. Sci. USA 95 (1998) 3960) generated a RORalpha null-mutant mouse (Rora(-/-)) by using a targeting vector in which a beta-Gal gene replaces the second finger of the DNA-binding domain of RORalpha. The Rora(-/-) cerebellum is qualitatively a phenocopy of the Rora(sg/sg) one, but the two strains differ slightly in their motor skills. To address the question whether the morphological defects in the Rora(-/-) cerebellum are identical to the Rora(sg/sg) one, we compared number and size of Purkinje cells in both staggerer and RORalpha null-mutant mice, using calbindin (CaBP) immunohistochemistry and revelation of beta-Gal activity. Compared to control cerebella the Rora(sg/sg) cerebellum has 82% fewer CaBP-positive cells. In Rora(-/-) mouse, all the the beta-Gal-positive Purkinje cells also expressed CaBP, but the cerebellum contained 78% less CaBP-positive cells than control, a deficit not different from the one observed in Rora(sg/sg). We show similar mediolateral compartments in Purkinje cell number and cytological abnormality in Rora(sg/sg) and Rora(-/-) mice. These results provide quantitative support for the hypothesis that the cerebellar phenotype in the homozygous Rora(sg/sg) is due to the lack of function of the RORalpha gene.

Progressive atrophy of cerebellar Purkinje cell dendrites during aging of the heterozygous staggerer mouse (Rora(+/sg)).

Staggerer (Rora(sg/sg)) is an autosomal mutation in an orphan nuclear hormone receptor gene, RORalpha, that acts intrinsically within the Purkinje cells and causes dysgenesis of the cerebellar cortex. Purkinje cell number is severely reduced, and the surviving cells are small with poorly developed dendrites. In contrast, the cytoarchitecture of the cerebellar cortex of the heterozygous staggerer (Rora(+/sg)) appears to be normal. However, quantitative studies have revealed a premature loss of Purkinje cells with advancing age. Most of the loss (25--30%) is complete by 13 months with little change thereafter. To address the question of whether all Purkinje cells, even the surviving ones, are affected by aging even though their cell bodies remain intact, we studied the evolution with age of the dendritic arbor through a semi-quantitative analysis of Golgi-impregnated Purkinje cells. A total of ten different morphological parameters were measured in 4-, 12- and 22-month-old wild type and heterozygous Rora(+/sg) mice. While the effects of the aging process are apparent in the wild type cerebellum, they are considerably accelerated in the Rora(+/sg) mouse. By 12 months the Rora(+/sg) Purkinje cell dendrite is as atrophic as a wild type dendrite from a 22-month-old and the dendritic regression continues well beyond the period of cell death in the heterozygous Rora(+/sg) mouse.

Target-related and intrinsic neuronal death in Lurcher mutant mice are both mediated by caspase-3 activation.

The Lurcher (Lc) mutation in the delta2 glutamate receptor gene leads to the presence of a constitutive inward current in the cerebellar Purkinje cells of Lurcher heterozygous mice and to the postnatal degeneration of these neurons. In addition, cerebellar granule cells and olivary neurons of Lc/+ mice die as an indirect effect of the mutation after the loss of their target Purkinje cells. The apoptotic nature of Lc/+ Purkinje cell death remains controversial. To address this question, we studied the involvement of caspase-3, a key effector of apoptosis, in the neurodegenerative processes occurring in Lc/+ cerebellum. Several antibodies recognizing different regions of caspase-3 were used in immunoblotting and immunohistochemical experiments. We demonstrate that pro-caspase-3 is specifically upregulated in the dying Lc/+ Purkinje cells, but not in granule cells and olivary neurons, suggesting that different death-inducing signals trigger variant apoptotic pathways in the CNS. The subcellular localization of pro-caspase-3 was shown to be cytoplasmic and mitochondrial. Active caspase-3 as well as DNA fragmentation was found in numerous granule cells and some Purkinje cells of the Lc/+ cerebellum. Thus, caspase-3 activation is involved in both the direct and indirect neuronal death induced by the Lurcher mutation, strongly supporting the idea that the Lc/+ Purkinje cell dies by apoptosis.

Hemisynaptic distribution patterns of presenilins and beta-APP isoforms in the rodent cerebellum and hippocampus.

Healthy brain neurons co-express Alzheimer's disease (AD) related proteins presenilins (PS) and beta-amyloid precursor protein (beta-APP). Deposition of beta-amyloid and PS in the senile plaques of AD brain and their ability to interact in vitro suggest that AD pathology could arise from a defect in the physiological interactions between beta-APP and PS within and/or between neurons. The present study compares the immunocytochemical distribution of PS (1 and 2) and beta-APP major isoforms (695 and 751/770) in the synapses of the cerebellum and hippocampus of the adult rat and mouse. In the cerebellar cortex of both species, the four molecules are immunodetected in the presynaptic or the postsynaptic compartments of synapses, suggesting that they are involved in interneuronal relationships. In contrast, PS and beta-APP are postsynaptic in almost all the immunoreactive synapses of the hippocampus. The different distribution patterns of these proteins in cerebellar and hippocampal synapses may reflect specific physiological differences, responsible for differential vulnerability of neurons to AD synaptic pathology. Defective interactions between beta-APP and PS at the synapses could impede the synaptic functions of beta-APP, inducing the selective loss of synapses that accounts for cognitive impairment in AD.

Role of an enriched environment on the restoration of behavioral deficits in Lurcher mutant mice.

Lurcher mutant mice, characterized by massive degeneration of the cerebellar cortex, and normal littermate controls were reared from birth either in standard conditions or in an enriched environment. The effects of this manipulation on motor functions, landmark water maze learning, exploration, and anxiety were evaluated at 3 months of age. Under standard conditions, Lurcher mutants were impaired in comparison to controls on tests of sensorimotor function and had altered exploratory tendencies. The enriched housing improved the motor coordination of Lurcher mutants and decreased the number of trials before reaching criterion in the landmark water maze. In addition to its effects in Lurcher mutants, enriched rearing also increased some behavioral abilities in normal mice. It is hypothesized that enriched housing altered brain morphology or neurochemistry in both normal and cerebellar-damaged animals.

Cerebellar Purkinje cell loss during life span of the heterozygous staggerer mouse (Rora(+)/Rora(sg)) is gender-related.

The staggerer mutation causes dysgenesis of the cerebellar cortex in the homozygous mutant (Rora(sg)/Rora(sg)). The mutation acts intrinsically within the Purkinje cells (PCs), leading to cytological abnormalities and a severe deficit in the number of these cells. In contrast, in the heterozygous staggerer (Rora(+)/Rora(sg)), the cytoarchitecture of the cerebellar cortex appears to be normal, but quantitative studies have revealed a significant loss of cerebellar neurons with advancing age. In the heterozygous reeler (+/rl), another mutant presenting a PC loss with age, we have found that only males were affected (Hadj-Sahraoui et al., 1996). In the present study, we have investigated whether a similar gender effect exists in the heterozygous staggerer during life span. PCs were counted on cerebellar sagittal sections in male and female Rora(+)/Rora(sg) and in their Rora(+)/Rora(+) littermates at 1, 3, 9, 13, 18, and 24 months of age. In the Rora(+)/Rora(+), the number of PCs remained stable until 18 months, but there was a 25% significant loss in 24- month-old mice of both genders. During life span, Rora(+)/Rora(+) males had slightly more PC than females. In the Rora(+)/Rora(sg) of both genders, the deficit in PC number was similar at 13 months but it appeared earlier in males, beginning between 1 and 3 months, and was aggravated regularly up to 13 months. By contrast, the decline was delayed and more abrupt in Rora(+)/Rora(sg) females, from a value still normal at 9 months to its maximal extent at 13 months. In view of these results, the heterozygous (Rora(+)/Rora(sg)) mouse offers an interesting model to test the interaction between sex, age, and genetic background on the development and maintenance of cerebellar neuronal populations.

Hu-Bcl-2 transgenic mice with supernumerary neurons exhibit timing impairment in a complex motor task.

Programmed neuronal cell death is common during development, and is thought to be important in the elimination of errors in axonal projection, cell position and sculpting of neuronal circuits. However, the potential importance of programmed cell death for complex behaviour in the adult animal has never been addressed. We studied motor abilities in a strain of transgenic mice with neuronal overexpression of the human Bcl-2 protein, which have supernumerary neurons due to reduced developmental cell death. Our results show that these mice have a clear deficiency in fine timing of motor coordination without impairment of basic motor functions. This is the first indication that altered developmental cell death and the consequent neuronal surplus can impair complex behaviour in the adult animal.

Afferent-target cell interactions in the cerebellum: negative effect of granule cells on Purkinje cell development in lurcher mice.

Lurcher (Lc) is a gain-of-function mutation in the delta2 glutamate receptor gene that results in a large, constitutive inward current in the cerebellar Purkinje cells of +/Lc mice. +/Lc Purkinje cells fail to differentiate fully and die during postnatal development. In normal mice, interactions with granule cells promote Purkinje cell dendritic differentiation. Partial destruction of the granule cell population in young +/Lc mice by x irradiation resulted in a significant increase in Purkinje cell dendritic growth and improved cytoplasmic structure but did not prevent Purkinje cell death. These results indicate two components to Purkinje cell abnormalities in +/Lc mice: a retardation/blockade of dendritic development that is mediated by interactions with granule cells and the death of the cell. Thus, the normal trophic effects of granule cell interaction on Purkinje cell development are absent in the +/Lc cerebellum, suggesting that granule cells are powerful regulators of Purkinje cell differentiation.

Dopamine turnover in the mediobasal hypothalamus in rat fetuses.

In this study, the dopamine turnover in the mediobasal hypothalamus, the key compartment of the neuroendocrine regulation of reproduction, was evaluated in fetal male and female rats. High-performance liquid chromatography with electrochemical detection was used to measure 3,4-dihydroxyphenylalanine, dopamine and 3,4-dihydroxyphenylacetic acid in the mediobasal hypothalamus of fetuses on the 21st day of intrauterine development and in primary cell culture (cell extracts and culture medium) of the same brain region, explanted at the 17th fetal day and maintained for seven days. The same technique was applied to determine dopamine release from fetal neurons of the mediobasal hypothalamus in response to an excess of K+ in the perifusion system or in culture. L-3,4-Dihydroxyphenylalanine, dopamine and 3,4-dihydroxyphenylacetic acid were detected both ex vivo and in culture. The ratios of the concentrations of L-3,4-dihydroxyphenylalanine/dopamine and 3,4-dihydroxyphenylacetic acid/dopamine were significantly higher in vitro than ex vivo, showing a lower rate of dopamine production and a higher rate of its degradation in the experiments in vitro. Moreover, it has been demonstrated that an excess of K+, i.e. a membrane depolarization, resulted in a highly increased release of dopamine in the perifusion system and in culture. The dopaminergic activity in the developing mediobasal hypothalamus showed sexual dimorphism that was manifested in a greater concentration of 3,4-dihydroxyphenylalanine and dopamine, at least in cell extracts of cultures, as well as in a higher rate of dopamine release, both in the perifusion system and in culture in males compared to females. Thus, dopamine is synthesized and released in response to a membrane depolarization in the mediobasal hypothalamus of rats as early as the end of intrauterine development, suggesting its contribution to the inhibitory control of pituitary prolactin secretion.

Severe atherosclerosis and hypoalphalipoproteinemia in the staggerer mouse, a mutant of the nuclear receptor RORalpha.

BACKGROUND: Hypoalphalipoproteinemia is the most common lipoprotein abnormality in patients with coronary artery disease, yet its causes are unknown. METHODS AND RESULTS: We show that the homozygous staggerer (sg/sg) mutant mouse, which carries a deletion within the nuclear receptor RORalpha gene, develops severe atherosclerosis when maintained on an atherogenic diet. In addition, sg/sg mice display a profound hypoalphalipoproteinemia, which is associated with decreased plasma levels of the major HDL proteins, apolipoprotein (apo) A-I and apoA-II. This decrease in HDL levels in sg/sg mice is due to lowered apoA-I gene expression in the intestine but not in the liver. ApoA-II gene expression is unaffected. CONCLUSIONS: These results suggest that the RORalpha gene contributes to the plasma HDL level and susceptibility to atherosclerosis.

Abnormal IL-1beta cytokine expression in the cerebellum of the ataxic mutant mice staggerer and lurcher.

To assess the extent to which interleukin-1beta (IL-1beta) may contribute to the development and/or progression of neurodegenerative processes, we have examined the levels of IL-1beta in the brain of two types of neurological mutant mice, staggerer and Lurcher. Using a quantitative immunological method (enzyme-linked immunosorbent assay, ELISA), we measured IL-1beta in the cerebellum, hippocampus and cerebral cortex of mutant mice at baseline and after peripheral LPS treatment. Two types of IL-1beta expression abnormalities were found in the mutant cerebella: higher basal level in Lurcher and a response to peripheral administration of LPS in staggerer. The association of IL-1beta expression abnormalities with the only brain structure where a massive neurodegeneration occurs supports the role of proinflammatory cytokines in this process.

Sustained delivery of immunoglobulins from polymer microsources on a narrow surface of the developing rat brain.

Studies of postnatal neurogenesis have benefited from the use of a relatively non-invasive method for chronic delivery of bioactive substances to a restricted area of cortex. This method consists of the implantation of an Elvax polymer microsource of active substances close to the targeted brain surface. Receptor ligands, as well as macromolecules such as proteins, peptides and enzymes have been shown to be released by the implants in a sustained manner over weeks. Here we describe the kinetics and immunoreactivity of different immunoglobulins released in vitro and in vivo by Elvax polymer. In vitro, the immunoglobulins first diffuse during a burst phase from the pore network of the polymer matrix. Release continues during a slow phase depending on loading, porosity and volume of the matrix but also on intrinsic properties of immunoglobulins. Elvax microsources loaded either with anti-TAG-1 or with anti-HNK-1 antibodies according to the release data in vitro, are implanted on the posterior cerebellar cortex of postnatal rats during the period when the targeted antigens are expressed by the differentiating cells. After several days, the released immunoreactive antibodies are located at the antigenic sites within the cerebellar cortex close to the implants. The sustained local delivery of immunoglobulins using the Elvax implant method allows access to cell surface and matrix molecules and thereby to the mechanisms they control during postnatal neurogenesis.

Differential roles of cerebellar cortex and deep cerebellar nuclei in learning and retention of a spatial task: studies in intact and cerebellectomized lurcher mutant mice.

Lurcher mutant mice (+/Lc) exhibit a massive loss of neurons in the cerebellar cortex and the inferior olivary nucleus, while deep cerebellar nuclei are essentially intact. To discriminate the relative participation of the cerebellar cortex and deep structures in learning and memory, 3 to 6-month-old +/Lc mice were subjected to a spatial learning task derived from the Morris water escape. They were able to learn to escape as well as their strain-matched controls (+/+). Seven days later, their scores showed that they had memorized the spatial environment but not as accurately as +/+ mice. Cerebellectomy before training did not significantly alter the escape learning capabilities of either group, whereas cerebellectomy performed after learning completely abolished retention in +/+, as well as in +/Lc, mice. These results suggest that the cerebellum, although not necessary for learning a spatial task, plays a crucial role in its retention, and that the storing structure of spatial information differs in +/+ and +/Lc mice.

The cerebellum and postural sensorimotor learning in mice and rats.

Animals with cerebellar damage caused by gene mutations, surgical ablations and irradiation by X-rays during developmental stages are impaired in maintaining posture and equilibrium. For most tests, even in animals with total cerebellectomy, postural sensorimotor learning is not abolished. Simpler compensatory movements may be adopted. The acquisition of simple sensorimotor skills occurring after massive damage of the cerebellar cortex may be explained by the modulatory role of the cerebellar deep nuclei during learning processes or by the influence of extracerebellar regions taking over lost cerebellar function.

Neurotrophin-3 promotes cerebellar granule cell exit from the EGL.

In the cerebellum, the mRNAs for neurotrophin-3 (NT-3) and its high-affinity tyrosine kinase receptor trkC are expressed by both the differentiated granule cells of the internal granule cell layer (IGL) and their precursors in the external germinal layer (EGL). We have investigated the effects of chronic application of exogenous NT-3 in vivo on cerebellar granule cell genesis and differentiation. NT-3 was applied to the posterior surface of the rat cerebellum from P6 onwards using Elvax implants. At P10 the EGL of cerebellar lobules VII and VIII was significantly reduced in thickness in NT-3 implanted rats when compared with controls. Immunocytochemical analysis of the EGL using antibodies to proliferating cell nuclear antigen (PCNA) revealed that the number of postmitotic, premigratory (PCNA-immunonegative) granule cell precursors was preferentially reduced in the NT-3 implanted rats. In situ DNA fragmentation labelling confirmed that this was not accompanied by increased cell death in the EGL. These results suggest that NT-3 promotes the differentiation of postmitotic, premigratory granule cell precursors, accelerating cell exit from the EGL.

Overexpression of a Hu-bcl-2 transgene in Lurcher mutant mice delays Purkinje cell death.

Cerebellar Purkinje cells in the heterozygous Lurcher mutant undergo cell autonomous degeneration beginning in the second week of postnatal development and becoming almost total around 30-45 days. The Lurcher mutation was recently identified as gain-of-function defect in the delta 2 glutamate receptor causing a constitutive current leak, suggesting that +/Lc Purkinje cells die by an excitotoxic mechanism. In previous studies we have shown that overexpression of bcl-2, a key regulator of cell death, in the heterozygous Lurcher mutant does not prevent +/Lc Purkinje cell death. To investigate further the mechanisms of +/Lc Purkinje cell death, we have crossed +/Lc mutants with a second line of Hu-bcl-2 transgenics (NSE73a) that shows an earlier onset of transgene expression and higher expression levels. Analysis of eight +/Lc-NSE73a mutants (4 at 2 months and 4 at 5-6 months) showed that Hu-bcl-2 overexpression delayed, but ultimately could not prevent +/Lc Purkinje cell death.

Role of the cerebellum in exploration behavior.

Compared to +/+ mice, Lurcher (+/Lc) mutant mice whose cerebellar cortex is lacking almost all Purkinje cells and granule cells, exhibit a low level of exploration; this deficit is not due to a low level of activity but to both a decreased motivation to explore a novel environment and to spatial deficits. The characteristics of exploration in cerebellectomized +/+ and +/Lc mice suggest that the cerebellum is involved not only in cognitive but also in motivational processes.

An animal model of autism: behavioural studies in the GS guinea-pig.

Autism is a human behavioural pathology marked by major difficulties in abnormal socialization, language comprehension and stereotypic motor patterns. These behavioural abnormalities have been associated with corticocerebral and cerebellar abnormalities in autistic patients, particularly in vermal folia VI and VII. Progress in understanding this disease has been hindered by the absence of a non-primate animal model. GS guinea-pigs are a partially inbred, non-ataxic guinea-pig strain with cerebellar and corticocerebral abnormalities similar to those reported to exist in human patients with autism. In order to determine if GS guinea-pigs represent an animal model of autism, their behaviour was compared with that of Hartley strain guinea-pigs. GS animals learned a motor task significantly more rapidly than Hartley guinea-pigs, but performed it in a more stereotypic manner and were less influenced by environmental stimuli than Hartleys. GS animals exhibited significantly less exploratory behaviour in a novel environment and were significantly less responsive to 50-95 dBA pure tones than Hartley guinea-pigs. In a social interaction assay, GS guinea-pigs interacted significantly less frequently with each other or with Hartley guinea-pigs than Hartleys did under the same conditions. GS behaviour thus exhibits autistic-like behaviour patterns: motor stereotypy, lack of exploration and response to environment and poor social interaction. Coupled with the neuropathological findings, this abnormal behaviour suggests that GS guinea-pigs could be a useful animal model of autism.

Quantitative analysis of cerebellar lobulation in normal and agranular rats.

Cerebellar pattern formation was investigated in rats treated with DNA modifying agents. Animals were subjected to combinations of daily injections of methylazoxymethanol acetate (MAM) for the last 6 days gestation and/or localised X-irradiation of the hindbrain on postnatal days 1 and 5 (P1 and P5). Animals were analysed on embryonic day 18 (E18), P0, P3, P7, and P14. Five parameters of the cerebellum were recorded from midsagittal sections: the number of primary lobules; the thickness of the external germinal layer (EGL); the density of cells in the internal granule cell layer (IGL) region; and the midsagittal area and perimeter. In addition, the laterolateral cerebellar distance was calculated. The data demonstrate that pre- and postnatal reduction of the EGL results in reduced cerebellar growth and folding. Cessation of the treatment at birth results in a recovery and eventual overproduction of EGL, but cerebellar growth and the development of fissures lags behind that of normal rats. Pre- and postnatal destruction of the EGL severely limited cerebellar growth and fissuration, and the cerebella contained only five primary lobules at P14. Rats subjected to postnatal X-irradiation alone had a similar low density of granule cells relative to those treated with a combination of prenatal MAM injections and postnatal X-irradiation, and yet the cerebella contained deeper fissures and more lobules (nine at P14). The data indicate that there are two phases of cerebellar folding: the establishment of five lobules that arise independent of granule cell production, and the granule cell-dependent expansion and partitioning of these five principal lobules during postnatal development. We propose that the lack of correlation between the severity of the granule cell loss and degree of lobulation in agranular rats indicates that granule cells exert an inductive influence over lobulation that is in part independent of the forces generated by their production and differentiation.