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.

Antagonism of serotonergic 5-HT2A/2C receptors: mutual improvement of sleep, cognition and mood?

Serotonin [5-hydroxytryptamine (5-HT)] and 5-HT receptors are involved in sleep and in waking functions such as cognition and mood. Animal and human studies support a particular role for the 5-HT(2A) receptor in sleep, which has led to renewed interest in this receptor subtype as a target for the development of novel pharmacological agents to treat insomnia. Focusing primarily on findings in healthy human volunteers, a review of the available data suggests that antagonistic interaction with 5-HT(2A) receptors (and possibly also 5-HT(2C) receptors) prolongs the duration of slow wave sleep and enhances low-frequency (< 7 Hz) activity in the sleep electroencephalogram (EEG), a widely accepted marker of sleep intensity. Despite certain differences, the changes in sleep and the sleep EEG appear to be remarkably similar to those of physiologically more intense sleep after sleep deprivation. It is currently unclear whether these changes in sleep are associated with improved vigilance, cognition and mood during wakefulness. While drug-induced interaction with sleep must be interpreted cautiously, too few studies are available to provide a clear answer to this question. Moreover, functional relationships between sleep and waking functions may differ between healthy controls and patients with sleep disorders. A multimodal approach investigating subjective and objective aspects of sleep and wakefulness provides a promising research avenue for shedding light on the complex relationships among 5-HT(2A/2C) receptor-mediated effects on sleep, the sleep EEG, cognition and mood in health and various diseases associated with disturbed sleep and waking functions.

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.

Brain activation and hypothalamic functional connectivity during human non-rapid eye movement sleep: an EEG/fMRI study.

Regional differences in sleep EEG dynamics indicate that sleep-related brain activity involves local brain processes with sleep stage specific activity patterns of neuronal populations. Macroscopically, it is not fully understood which cerebral brain regions are involved in the successive discontinuation of wakefulness. We simultaneously used EEG and functional MRI on 9 subjects (6 female: mean = 24.1 years, 3 male: mean = 26.0 years) and analyzed local blood oxygenation level dependent signal changes linked to the transition from wakefulness to different non-rapid eye movement (NREM) sleep stages (according to Rechtschaffen and Kales) of the first sleep cycles after 36 h of total sleep deprivation. Several brain regions throughout the cortex, the limbic lobe, the thalamus, the caudate nucleus, as well as midbrain structures, such as the mammillary body/hypothalamus, showed reduced activity during NREM sleep across all sleep stages. Additionally, we found deactivation patterns specific to NREM sleep stages compared with wakefulness suggesting that a synchronized sleeping state can be established only if these regions interact in a well-balanced way. Sleep stage 2, which is usually linked to the loss of self-conscious awareness, is associated with signal decreases comprising thalamic and hypothalamic regions, the cingulate cortex, the right insula and adjacent regions of the temporal lobe, the inferior parietal lobule and the inferior/middle frontal gyri. The hypothalamic region known to be of particular importance in the regulation of the sleep-wake cycle shows specific temporally correlated network activity with the cortex while the system is in the sleeping state, but not during wakefulness. We describe a specific pattern of decreased brain activity during sleep and suggest that this pattern must be synchronized for establishing and maintaining sleep.

Neuronal activity and brain-derived neurotrophic factor regulate the density of inhibitory synapses in organotypic slice cultures of postnatal hippocampus.

Hippocampal interneurons inhibit pyramidal neurons through the release of the neurotransmitter GABA. Given the importance of this inhibition for the proper functioning of the hippocampus, the development of inhibitory synapses must be tightly regulated. In this study, the possibility that neuronal activity and neurotrophins regulate the density of GABAergic inhibitory synapses was investigated in organotypic slice cultures taken from postnatal day 7 rats. In hippocampal slices cultured for 13 d in the presence of the GABA(A) receptor antagonist bicuculline, the density of glutamic acid decarboxylase (GAD) 65-immunoreactive terminals was increased in the CA1 area when compared with control slices. Treatment with the glutamate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione decreased the density of GAD65-immunoreactive terminals in the stratum oriens of CA1. These treatments had parallel effects on the density of GABA-immunoreactive processes. Electron microscopic analysis after postembedding immunogold labeling with antibodies against GABA indicated that bicuculline treatment increased the density of inhibitory but not excitatory synapses. Application of exogenous BDNF partly mimicked the stimulatory effect of bicuculline on GAD65-immunoreactive terminals. Finally, antibodies against BDNF, but not antibodies against nerve growth factor, decrease the density of GAD65-immunoreactive terminals in bicuculline-treated slices. Thus, neuronal activity regulates the density of inhibitory synapses made by postnatal hippocampal interneurons, and BDNF could mediate part of this regulation. This regulation of the density of inhibitory synapses could represent a feedback mechanism aimed at maintaining an appropriate level of activity in the developing hippocampal networks.

Organization of spinocerebellar projection map in three types of agranular cerebellum: Purkinje cells vs. granule cells as organizer element.

The organization of the spinocerebellar projection was analysed by the anterograde axonal WGA-HRP (horseradish peroxidase-wheat germ agglutinin conjugate) tracing method in three different types of agranular cerebellar cortex either induced experimentally by X-irradiation or occurring spontaneously in weaver (wv/wv) and staggerer (sg/sg) mutant mice. The results of this study show that in the X-irradiated rat and weaver mouse, in both of which the granule cells are directly affected and die early in development, the spinal axons reproduce, with few differences, the normal spinocerebellar pattern. Conversely, in staggerer mouse, in which the Purkinje cells are intrinsically affected and granule neurons do not seem to be primarily perturbed by the staggerer gene action, the spinocerebellar organization is severely modified. These findings appear somewhat paradoxical because if granule cells, the synaptic targets of mossy spinocerebellar fibers, were necessary for the organization of spinocerebellar projection, the staggerer cerebellum would exhibit a much more normal projectional map than the weaver and the X-irradiated cerebella. It is, therefore, obvious that granule cells, and even specific synaptogenesis, are not essential for the establishment of the normal spinocerebellar topography. On the other hand, the fact that the Purkinje cells are primarily affected in the unique agranular cortex in which the spinocerebellar organization is severely modified suggests that these neurons could be the main element in the organization of the spinocerebellar projection map. This hypothesis is discussed in correlation with already-reported findings on the zonation of the cerebellar cortex by biochemically different clusters of Purkinje cells.

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.

Transient developmental expression of monoamine transporters in the rodent forebrain.

Neurons in first-order sensory thalamic nuclei have been shown to express functional plasma membrane serotonin (SERT) and vesicular monoamine (VMAT2) transporters during early postnatal development. In the present study, we provide an extensive description of the spatial and the temporal patterns of VMAT2 and SERT expression, during early embryonic development and postnatal life, by using in situ hybridization and immunocytochemistry. VMAT2 and SERT genes are transiently expressed in a wide population of non-monoaminergic neurons in the central and peripheral nervous system with a large overlap in the temporal and spatial pattern of expression of both genes. A selective pattern of expression of both genes was observed in the thalamus with expression limited to the dorsal thalamus and more particularly to primary sensory relay nuclei that convey point to point projection maps. Transient expression of the transporters was also observed in sensory cranial nerves, in the hippocampus, cerebral cortex, septum, and amygdala. VMAT2 and SERT gene expression was not necessarily linked, as some neural populations expressed only VMAT2, while others only contained SERT. Since VMAT2 serves to transport catecholamines besides serotonin, we examined the developmental expression of the plasma membrane dopamine and norepinephrine transporters but found no transient expression of these genes. Despite minor temporal disparities, VMAT2 and SERT extinguished almost simultaneously during the second and third weeks of post-natal life. These expressions did not seem to be dependent on peripheral neural inputs, since monocular enucleations and infraorbital nerve cuts effected on the day of birth, did not modify the period of transporter expression or of extinction.

Distribution pattern and ultrastructural localization of Rxt1, an orphan Na+/Cl(-)-dependent transporter, in the central nervous system of rats and mice.

The cellular and subcellular localization of Rxt1 protein, an orphan Na+/Cl(-)-dependent transporter, was investigated in the central nervous system of rats and mice, with rabbit polyclonal antibodies specifically directed against its C-terminal region. At the light microscope level, the distribution of Rxt1, visualized by the immunoperoxidase method, was found to be similar in rats and mice. Labelled elements were present in numerous gray matter regions of the central nervous system, from the olfactory bulb to the spinal cord. In all labelled regions, immunoreactivity was confined to the neuropil where both a diffuse labelling of low intensity and an intense punctate staining were noted. To further identify the nature of the cellular elements bearing the punctate staining, possible changes in this labelling pattern were investigated: (i) in deep cerebellar nuclei and lateral vestibular nucleus of the Lurcher mutant mouse, in which all cerebellar Purkinje cells are missing and (ii) in the rat cervical spinal cord, 10 days after multiple resections of dorsal roots. The vast majority of the punctate structures, delineating the neuronal perikaryal and stem dendritic contours, had disappeared in the mutant mouse, providing evidence that they belong to Purkinje cell axon terminals. In rhizotomized rats, the intense labelling in laminae I and III had disappeared, demonstrating that it occurred in subclasses of axonal projections of primary afferent fibres. These results strongly suggest that Rxt1 is present in presynaptic axon terminals. The electron microscopic study was carried out in the hippocampus, cerebellum and lateral vestibular nucleus of control mice, where Rxt1-labelled punctate structures were found to be abundant. Immunostaining was confined to axon terminals, particularly in hippocampal and cerebellar mossy fibres and in Purkinje cell axonal terminations of the cerebellar deep nuclei and lateral vestibular nucleus. In the cerebellar cortex, axon terminals belonging to inhibitory local circuit neurons (basket and Golgi cells), were free of labelling. The observations reported in this study have shown that: (1) The Rxt1 transporter is neuron-specific, and is expressed by only some classes or even subclasses of neuronal systems. (2) This transporter can be encountered in excitatory axons using glutamate as neurotransmitter (hippocampal and cerebellar mossy fibres: primary afferent fibres), as well as in inhibitory axons known by their GABAergic nature (Purkinje cell axon terminals) where it might be involved in the re-uptake process of one or several molecules released from corresponding terminals.

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.

A morphometric study of mouse trigeminal roots after unilateral destruction of vibrissae follicles at birth.

Trigeminal sensory roots were studied in neonatal mice. On the deafferented side, the surface area of the cross-section through the sensory root is diminished by 31% and the number of myelinated fibers is reduced by 21%, but the proportion between myelinated and unmyelinated fibers remains unchanged. The distribution of axonal diameters, analysed in 7 dorso-ventral scanning bands through the sensory roots, indicates a loss or eventually an atrophy of large myelinated axons in the medial two thirds of the sensory root. In both control and deafferented sides the diameter of the myelinated fiber (outside the myelin sheath) is proportional to the axon diameter (inside the myelin sheath). Our results confirm the loss of most of the neurons innervating vibrissae and the lack of regeneration or sprouting in the deafferented root in the newborn mouse.

Delay in the maturation of muscle fibers in infants with congenital hypotonia.

Muscle biopsies of hypotonic children have shown delayed maturation of a fetal type of muscle fibers: subsarcolemmal halo devoid of activity for mitochondrial dehydrogenases, type II predominance and in some cases abnormal dispersion of fiber diameter. Fiber subtypes within group II were also abnormal. One case has definite embryonic characteristics with presence of myoblasts. Not a single clinical pattern was present in these patients and a variety of associated disorders were recognized. Some patients had a clinical picture corresponding to congenital benign hypotonia as described by Walton.

Immaturity of muscle fibers in the congenital form of myotonic dystrophy: its consequences and its origin.

Skeletal muscle maturation is impaired in children with congenital myotonic dystrophy. This immaturity is characterized at the light microscopy level by an abnormal presence of myotubes, small fascicles of muscle fibers, thin myofibers, and delayed muscle fiber type differentiation with a peripheral halo lacking mitochondrial oxidative enzyme activity. At an ultrastructural level, the characteristics are a paucity of myofibrils with a peripheral rim devoid of mitochondria and myofibrils in the fibers. In time the muscle is able to gain a certain degree of maturity as shown in one of our cases who had two successive muscle biopsies. The muscle, however, never becomes normal but retains discrepancies in fiber size and fiber type distribution and shows some fiber necrosis. Maturation of the motoneurons is normal, which may explain necrosis of immature muscle fibers. In an experimental study carried out to look for evidence of a circulatory factor in mothers of children with congenital myotonic dystrophy, it was found that sera from these mothers administered intra-peritoneally to newborn rats does in fact impair muscle maturation, whereas rats injected similarly with sera from control women showed normal muscle maturation.

[Histochemical, quantitative and ultrastructural maturation of human fetal muscle]. / Etude de la maturation histochimique, quantitative et ultrastructurale du muscle foetal humain

Histochemical studies of muscle specimens from human fetuses showed: (a) a uniform fiber type population having the properties of Type IIC fibres up to 19 weeks of development; (b) a progressive appearance of Type I fibres after that age; (c) a decrease in number of Type IIC fibres during the last 3 months of pregnancy, accompanied by the appearance of Type IIB and Type IIA fibres; (d) the presence after the myotube stage of fibers with a light peripheral halo in sections stained for mitochondrial dehydrogenases. Electron-microscopic examination of the muscle fibres confirmed the existence of a peripheral halo devoid of myofibrils and mitochondria and showed: (a) scarcity of myofibrils in comparison with mature muscle fibres and (b) irregularity in shape of the myofibrils. In addition, quantitative studies demonstrated an important variation of the fibre diameters up to 21 weeks and the increase of the mean diameter after this age. It is suggested that the persistence after birth of some features of immaturity identical to those described in this work may be considered as a pathological finding.

Hyponatremia: pathophysiology, differential diagnosis and new aspects of treatment.

Hyponatremia is the most frequent electrolyte disorder in clinical medicine. It is usually attributable to primary vasopressin excess, causing the syndrome of inappropriate antidiuresis (SIAD), or to secondary vasopressin stimulation, involving a baroreceptor mechanism. The latter is regularly found in the hyponatremia of liver cirrhosis, cardiac failure and volume contraction. In the first kind of setting the concentrations of creatinine, urea and urate in plasma will be low because of the associated volume expanded state. In the second type of setting they will be elevated because of the circulatory compromise of these patients. The hyponatremia of SIAD may be treated by water restriction, furosemide and substitution of the inadvertent sodium losses by giving 3% NaCl. Baroreceptor hyponatremia is best treated by fluid restriction together with judiciously administered saline. In correcting severe chronic hyponatremia, the rate of correction should not exceed 1 mM/l/h and the corrected serum sodium concentration should not be higher than 130 mM/l. In the foreseeable future oral non-peptide oral vasopressin antagonists will become available. They are expected to become new tools for the treatment of hyponatremia.

Experiments on anthelmintic control of Fasciola hepatica in Brazil.

Two separate field trials involving naturally infected cattle were carried out on two farms known to have a history of Fasciola hepatica infection. On the first farm, 15 animals per group were allocated as follows: G1, triclabendazole (TCBZ) four times a year; G2, TCBZ twice a year (May and September); G3, untreated control. All groups grazed together and after 3.5 years the animals were slaughtered and their livers examined by federal meat inspectors who condemned 100% of livers in G3 and 8.3% in G2 owing to the presence of lesions of fasciolosis. In G1 no livers were condemned. Significant differences in weight gains were not detected and fluke counts remained at low levels in the treated groups. Also, in the control group, egg counts started to decrease when animals were 2 years old. On the second farm, groups of 20 animals were treated as follows: G1, TCBZ three times a year (May, September and December); G2, TCBZ twice a year (May and September); G3, nitroxynil twice a year (May and September); G4, rafoxanide twice a year (May and September); G5, untreated controls. All animals were weighed and faecal samples examined at approximately 28-day intervals. During the period of the study, larger weight gains were detected in the TCBZ treated groups than in the others. TCBZ treatment kept F. hepatica egg counts at a lower level for longer periods than the other drugs and significant differences in weight gains were only obtained between the group receiving TCBZ three times a year and the control group.

Cell death and axon regeneration of Purkinje cells after axotomy: challenges of classical hypotheses of axon regeneration.

Although adult mammalian neurons are able to regenerate their axons in the peripheral nervous system under certain conditions, they are not able to do it in the central nervous system. The environment surrounding the severed axons appears to be a key factor for axon regeneration. Many studies aiming to enhance axon regeneration in the CNS of adult mammals have successfully manipulated this environment by adding growth permissive molecules and/or neutralizing growth inhibitory molecules. In both cases, the number of axons able to regenerate was low and the different neuronal populations were not equal in their regenerative response, suggesting that manipulation of the environment is not always sufficient. This is particularly well illustrated in the cerebellar system, in which axotomized inferior olivary neurons regenerate when confronted with a permissive environment, whereas mature Purkinje cells do not. The intrinsic ability of a neuron to regenerate its axon is generally correlated with the intensity of its reaction to axotomy (expression of molecules, probability to die). Furthermore, molecules such as GAP-43 (growth-associated molecule) and c-Jun are involved in both axon regeneration and cell death suggesting that these two processes are linked. Surprisingly, Purkinje cells lose their capacity to regenerate their axon (even in the absence of myelin) during development before losing their capacity to react to an axotomy by cell death. These results emphasize the different reactions to axotomy between neuron types and underline that in Purkinje cells, the two cell decisions (axon regeneration and cell death) are differently regulated and therefore not part of the same signaling pathway.

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.