Clusterin expression during fetal and postnatal CNS development in mouse.

Clusterin (or apolipoprotein J) is a widely distributed multifunctional glycoprotein involved in CNS plasticity and post-traumatic remodeling. Using biochemical and morphological approaches, we investigated the clusterin ontogeny in the CNS of wild-type (WT) mice and explored developmental consequences of clusterin gene knock-out in clusterin null (Clu-/-) mice. A punctiform expression of clusterin mRNA was detected through the hypothalamic region, neocortex and hippocampus at embryonic stages E14/E15. From embryonic stage E16 to the first week of the postnatal life, the vast majority of CNS neurons expressed low levels of clusterin mRNA. In contrast, a very strong hybridizing signal mainly localized in pontobulbar and spinal cord motor nuclei was observed from the end of the first postnatal week to adulthood. Astrocytes expressing clusterin mRNA were often detected through the hippocampus and neocortex in neonatal mice. Real-time polymerase chain amplification and clusterin-immunoreactivity dot-blot analyses indicated that clusterin levels paralleled mRNA expression. Comparative analyses between WT and Clu-/- mice during postnatal development showed no significant differences in brain weight, neuronal, synaptic and astrocyte markers as well myelin basic protein expression. However, quantitative estimation of large motor neuron populations in the facial nucleus revealed a significant deficit in motor cells (-16%) in Clu-/- compared with WT mice. Our data suggest that clusterin expression is already present in fetal life mainly in subcortical structures. Although the lack of this protein does not significantly alter basic aspects of the CNS development, it may have a negative impact on neuronal development in certain motor nuclei.

Permanent cerebral ischemia induces sustained procaspase 9L increase not controlled by Bcl-2.

We have investigated how transgenic overexpression of human Bcl-2 (Hu-Bcl-2) modifies cell death proteins activation in the long-term in a model of permanent cerebral ischemia induced by middle cerebral artery occlusion. Hu-Bcl-2, cytochrome c, caspases 9 and 3 expression were examined by immunoblotting and immunohistochemistry. In wild type mice, 1 day after middle cerebral artery occlusion, cytochrome c released from the mitochondria was detected. Middle cerebral artery occlusion induces a lasting activation of caspases in WT mice from day 3 post-injury. Increased level of caspase 3 is accompanied by a decrease in procaspase 3. In contrast, middle cerebral artery occlusion induced a sustained increase of procaspase 9L and a decrease in procaspase 9S concomitant to caspase 9 production. These events were observed in the operated but not in the unoperated hemisphere. Bcl-2 overexpression blocks cytochrome c release and delays caspases activation. Consequently procaspase 3 decrease was no more observed. However, Bcl-2 overexpression did not influence the middle cerebral artery occlusion-induced changes in procaspases 9 L and S. Fourteen days after middle cerebral artery occlusion the apoptotic cascade was no longer blocked in transgenic mice. Caspases 9 and 3 were increased, procaspase 3 was decreased but procaspase 9L and procaspase 9S remained increased and decreased respectively. Hu-Bcl-2 overexpression delays the activation of the cell death molecular machinery but does not control the ischemia-induced change in procaspase 9 L and S. Procaspase 9L increase is a potentially harmful event threatening cells of a rapid destruction when anti-apoptotic treatments by Bcl-2, or caspases inhibitors, are overrun.

Prevention of apoptotic neuronal death by controlling procaspases? A point of view.

In various animal models of neurodegenerative diseases the long-lasting control of cell death by anti-apoptotic therapies is not successful. We present here our view on the control of procaspase expression in a model of cerebral stroke. We have investigated how Hu-Bcl-2 overexpression modifies cell death protein activation in a model of cerebral ischemia induced by permanent middle cerebral artery occlusion (MCAO). In wild type mice MCAO induced release of cytochrome c from the mitochondria, and activation of caspases 9 and 3. In parallel with caspases activation, procaspase 9 and procaspase 3 were, respectively, increased and decreased. In Hu-Bcl-2 transgenic mice cytochrome c release and caspases 9 and 3 activation were blocked. However procaspase 9 increased, like in wt mice, but procaspase 3 remained unchanged. By 2 weeks after MCAO caspases were no longer blocked in Hu-Bcl-2 transgenic mice. Procaspase 9 increase could represent a time bomb in Hu-Bcl-2 mice where caspase 9 activation is blocked. Indeed, cellular accumulation of procaspase 9 is a potentially harmful event able to overcome anti-apoptotic protection by Bcl-2 and threaten cells with rapid destruction. Through understanding of the upstream regulation of procaspase 9, early targets for the pharmacological control of apoptotic cell death may be revealed.

Intracerebroventricular infusion of leptin decreases serotonin transporter binding sites in the frontal cortex of the rat.

We demonstrate that chronic intracerebroventricular infusion of leptin dramatically decreases the number of [(3)H]paroxetine binding sites in the frontal cortex of the rat brain. In contrast, the density in paroxetine binding sites estimated in the region containing raphe projecting cell bodies (i.e., the dorsal and median raphe nuclei) remains unchanged. Since leptin treatment significantly decreases food intake, [(3)H]paroxetine binding parameters were also estimated in the frontal cortex of pair-fed control rats. No significant difference in [(3)H]paroxetine binding was observed between pair-fed and ad libitum fed control rats. These data indicate that leptin treatment could regionally down-regulate serotonin transporter binding sites in the brain. Although the cellular and molecular mechanisms underlying such an effect of leptin need further investigation, our observations support the notion of a possible interaction between leptin and the serotonergic system of potential interest in the pathophysiology of depression.

Mapping of cocaine and amphetamine regulated transcript (CART) mRNA expression in the hypothalamus of elderly human.

The expression of cocaine and amphetamine regulated transcript (CART) in the hypothalamus of aged humans was studied by in situ hybridization histochemistry. Formalin fixed coronal sections through the hypothalamus were hybridized with [35S]dATP 3' end-labeled deoxyoligonucleotide probes complementary to sequences of the CART encoding gene. Large populations of cells expressing CART messenger RNA were mainly detected in the paraventricular nucleus of the hypothalamus. Other hypothalamic subdivisions displaying numerous radiolabeled cells were the dorsomedial, ventromedial, infundibular and tuberomammillary nuclei, and the dorsal hypothalamic area. Additionally, a few labeled cells were observed in the perifornical and lateral hypothalamic areas. Hybridizing cells were occasionally seen in the supraoptic nucleus. Overall, the pattern of distribution of CART expressing cells observed throughout the human hypothalamus, with few exception, e.g. the supraoptic nucleus, is comparable to that previously reported in the rat brain. These anatomical results suggest that in human, hypothalamic CART expression could be involved a variety of neuroendocrine functions including food-intake.

Distinct patterns of neuronal loss and Alzheimer's disease lesion distribution in elderly individuals older than 90 years.

To explore the characteristics of brain aging in very old individuals, we performed a quantitative analysis of neurofibrillary tangle (NFT) and senile plaque (SP) distribution and neuron densities in 13 nondemented patients, 15 patients with very mild cognitive impairment, and 22 patients with Alzheimer's disease (AD), all older than 96 years of age. Non-demented cases displayed substantial NFT formation in the CAI field and entorhinal cortex only. Very mild cognitive impairment cases were characterized by the presence of high NFT densities in layers V and VI of area 20, and AD cases had very high NFT densities in the CAI field compared to nondemented cases. Moreover, high SP densities were found in areas 9 and 20 in AD, but not in cases with very mild cognitive impairment and nondemented cases. In contrast to previous reports concerning younger demented patients, neuron densities were preserved in the CAI field, dentate hilus, and subiculum in centenarians with AD. In these cases, there was a marked neuronal loss in layers II and V of the entorhinal cortex, and in areas 9 and 20. In the present series, no correlation was found between neurofibrillary tangle and neuron densities in the areas studied, whereas there was a negative correlation between senile plaque and neuron densities in area 20. The comparison between the present data and those reported previously concerning younger cohorts suggests that there is a differential cortical vulnerability to the degenerative process near the upper age-limit of life.

Mapping of serotonin transporter messenger RNA-containing nerve cell populations in the cat brainstem.

The anatomical distribution of nerve cells populations expressing serotonin transporter messenger RNA was investigated in the cat brain by means of in situ hybridization histochemistry. Formalin fixed coronal sections were hybridized with [35S]dATP 3' end-labeled oligoprobes complementary to three nucleotide sequences taken from the human and serotonin transporter. A strong hybridization signal was found in nerve cells populations exclusively localized within the brainstem. These positive cells mainly resided in the nuclei of the raphe, especially in the nuclei of the raphe dorsalis and raphe centralis superior. A small number of labeled cells was also observed in various areas including the dorsal part of the interpeduncular nucleus, in the midbrain, and the region ventrolateral to the inferior olive, the ventral midline and around the central canal, in the medulla oblongata. Overall, these data agree with the notion that in the cat, as previously suggested in the human and in the rat brain, the serotonin membrane transporter messenger RNA is predominantly expressed in areas known to contain serotonergic cell bodies.

[3H]Nisoxetine binding sites in the cat brain: an autoradiographic study.

The binding of [3H]nisoxetine, a selective inhibitor of the high-affinity noradrenaline uptake sites, was studied on frontal frozen sections of the cat brain. The highest densities in autoradiographic signal were observed in the nucleus locus coeruleus and its ascending pathways, in the area postrema and in the dorsal part of the inferior olive, the pontine nuclei, the raphe nuclei, the colliculi, the periventricular and lateral areas of the hypothalamus, the suprachiasmatic nucleus, the nucleus accumbens and the olfactory bulb. A moderately high concentration of binding sites was observed in the hippocampal formation, especially in the molecular layer of Ammon's horn, in the superficial layers of the entorhinal cortex and in the indusium griseum. Binding sites were visualized in all the subdivisions of the neocortex. The highest density of binding was generally detected in the outer edge of the superficial layer I. In some cortical areas, especially in the visual cortex, labeling with a prevalent laminar distribution in the superficial layers I-III and in the deep layers V-VI was clearly observed. Moderate to low densities of binding sites were seen in most other areas of the brain except in the white matter, the caudate nucleus and putamen, which were devoid of labeling. Overall these findings indicate a good correlation between the distribution of [3H]nisoxetine binding sites and the noradrenergic systems. Furthermore, data suggest that in several areas, high-affinity noradrenaline reuptake mechanisms could play an important role in local interactions between the noradrenergic system and the other monoaminergic systems.

Quantitative analysis of neuropathologic changes in the cerebral cortex of centenarians.

1. The quantitative distribution of neurofibrillary tangles and senile plaques was studied in the brains of 65 elderly patients aged from 96 to 104 years by immunohistochemistry. 2. According to the clinical and neuropathological diagnoses, three groups of cases were considered: 19 patients with Alzheimer's disease, 22 patients with mixed dementia (vascular and degenerative) and 24 patients with no or very mild cognitive impairment. 3. Moderate to high neurofibrillary tangle densities were always present in the hippocampus and entorhinal cortex. The inferior temporal cortex was very frequently affected in demented and non-demented cases whereas the superior frontal cortex was spared in the majority of cases independently of the clinical diagnosis. Quantitatively, Alzheimer's disease cases showed significantly higher NFT densities than cases with no clinical findings of dementia only in the CA1 field of the hippocampus. 4. The hippocampus and entorhinal cortex were often devoid of senile plaques in non-demented cases while the vast majority of Alzheimer's disease cases had few SP in these regions. The frontal and temporal cortex were more frequently involved than the limbic structures in both non-demented and Alzheimer's disease cases. The SP densities in layers II and III of the inferior temporal and superior frontal cortex were significantly higher in Alzheimer's disease than in non-demented cases. 5. These observations suggest that the dementing process in nonagenarians and centenarians may differ to that described in younger demented individuals in that neurofibrillary tangles involve principally the hippocampal formation with relative sparing of the neocortex. Furthermore, they indicate that both the neurofibrillary tangle densities in the CA1 field and senile plaque densities in the superficial layers of the neocortex must be considered for the neuropathological diagnosis of Alzheimer's disease in this age group.

[Molecular biology of beta A4 protein and Alzheimer disease]. / Biologie moléculaire de la protéine bêta A4 et maladie d'Alzheimer.

Senile plaques, neurofibrillary tangles and neuronal loss are the major histopathologic hallmarks of Alzheimer's disease. A prominent component of the senile plaques is a polypeptide (beta A4) of about 40 amino acids derived via proteolytic cleavage from a set of larger protein isoforms collectively referred to as the amyloid precursor protein (APP). The protein APP is a widely distributed transmembrane glycoprotein structurally related to a cell surface receptor. APP is encoded by a single gene on chromosome 21 in which missense mutations have been demonstrated in several cases of familial Alzheimer's disease. It is thought that various factors fostering the APP-processing pathway by which the polypeptide beta A4 is generated might be pathogenic. Thus the mechanisms that govern the rate of transcription of the APP gene, the differential splicing of the precursor messenger and the proteolytic processing of APP are current subjects of intensive investigation. Theoretically, each of these events represents a potential target for a therapeutic intervention. However, the relationships between amyloidogenesis and the formation of the neurofibrillary tangles associated to the neuronal loss remain to be elucidated.

[Cerebral hippocampic ischemia, metabolic disorders and neuronal death]. / Ischémie cérébrale hippocampique, troubles métaboliques et mort cellulaire.

Ischemia, hypoxia and anoxia are the most affecting causes in the neuropathology of the central nervous system because energy is produced almost entirely from the oxidative metabolism of glucose (60 mg/min) and neurons are more vulnerable to oxygen deficiency than astrocytes, oligodendroglia and microglia. Even though the reduction of blood supply and the decrease of oxygen content are the predominant factors to induce pathological processes, intrinsic factors of neuronal organization have become the center of interest, such as the anatomical connectivity, neurotransmitters and receptors operating in specific vulnerable areas. Among brain areas, the hippocampus is a classical predilection site for ischemic injury as for selective vulnerability. Short ischemic period is sufficient to damage CA1 sector whereas the adjacent CA3 sector and the gyrus dentatus are resistant. This differential pattern of resistance to damage between CA1 and other brain areas allows to compare specific properties of these hippocampic cells.

Production and immunohistochemical application of monoclonal antibodies against delta sleep-inducing peptide.

Monoclonal antibodies were produced following immunization of rats with delta sleep-including peptide (DSIP). The spleen cells of the rats were fused with the myeloma cell line SP2/0. The supernatants of hybridomas were screened on a solid-phase immunoassay using dot-immunobinding of DSIP and some DSIP fragments. The supernatants of six stable producer clones were found to react with DSIP. From this procedure it was also deduced that all these monoclonal antibodies recognized epitope(s) of the penta carboxy-terminal region of DSIP (DSIP5-9). Application of these monoclonal antibodies to rat median eminence sections gave a strong immunolabelling of a large population of fibres and terminal-like structures, mainly localized through the lateral areas. Elution-restaining experiments using a monoclonal antibody to DSIP and a polyclonal antiserum to luteinizing hormone-releasing hormone (LHRH) showed that the patterns of immunoreactivity respectively visualized overlap almost completely. Although numerous LHRH-immunoreactive neuronal elements were also easily demonstrated in the median eminence of the mouse, the hamster and the gerbil species, incubation of sections with monoclonal antibodies to DSIP failed to give any immunoreaction. Taken together these data argue for the independence of the DSIP/LHRH immunolabelling systems. Furthermore, it was demonstrated that DSIP5-9-related epitopes detected in the rat median eminence have no counterpart in the three other rodent species investigated. These species differences may reflect the fact that the carboxy-terminal sequence of the nonapeptide DSIP originally discovered in the rabbit is not conserved in all rodent species.

A comparative study of histological and immunohistochemical methods for neurofibrillary tangles and senile plaques in Alzheimer's disease.

Several studies have demonstrated that the accurate visualization and quantification of pathological lesions in neurodegenerative disorders depend on the reliability of staining methods. In an attempt to gain a better assessment of the density and distribution of the neuropathological markers of Alzheimer's disease, we compared the staining efficiency of a modified thioflavine S protocol for neurofibrillary tangles (NFT) and senile plaques (SP) to different argentic impregnation techniques (Bielchowsky, Gallyas, Globus, Campbell-Switzer-Martin) and to immunohistochemical stainings obtained with two different antibodies against the amyloid beta protein A4 and the microtubule-associated tau protein. The modified thioflavine S technique (MTST) detects up to 60% more SP and up to 50% more NFT than the Bielschowsky and Globus methods, respectively. The results obtained with the specific antibodies are comparable to those obtained with the MTST, but these immunotechniques are more expensive and time consuming for routine neuropathological evaluation, and the appropriate antibodies are not always commercially available. Furthermore, the morphological appearance of NFT and SP with MTST is greatly improved when compared to the classical thioflavine S and the increased signal-to-noise ratio between specifically stained structures and background permits an accurate semi-automatic quantification.

An improved thioflavine S method for staining neurofibrillary tangles and senile plaques in Alzheimer's disease.

Large differences are usually observed when standard staining methods for a number of pathological lesions in neurodegenerative disorders are compared. With the modified thioflavine S method presented here (easy and cheap to perform), the morphological appearance of the stained neurofibrillary tangles (NFT) and senile plaques (SP) is greatly improved. Furthermore, the intense contrast between stained lesions and background obtained with this technique permits an accurate automatic quantification of NFT and SP using a computer-assisted image analysis system.

Asymmetric increase in substance P immunoreactivity in the rat and guinea pig substantia nigra after unilateral neocortical ablation.

Substance P was visualized in the rat and the guinea pig basal ganglia, mesencephalon and spinal cord after surgical unilateral cortical ablation. An increased density in substance P-like immunoreactive patterns in the substantia nigra was observed ipsilaterally to the lesioned hemisphere. These results, together with previous observations in cases of Alzheimer's disease presenting with asymmetric cortical atrophy, suggest an influence of the corticostriatal pathway on substance P-like immunoreactivity in these subcortical structures. Finally, these experiments proved to be a reliable animal model for the study of the effects of neocortical lesions on the neuropeptidergic innervation of certain subcortical regions.

Immunocytochemical demonstration of DSIP-like immunoreactivity in the hypothalamus of the rat.

The distribution of delta sleep-inducing peptide immunoreactivity (DSIP-IR) was studied in the rat diencephalon. Varicose nerve fibers exhibiting DSIP-IR were found throughout the mediobasal hypothalamus, most frequently in the hypothalamic arcuate nucleus and in the adjoining median eminence and pituitary stalk. This innervation provides a basis for the involvement of DSIP in neuroendocrine regulation at the hypothalamic level. In the hypothalamus, DSIP-IR innervation was also observed close to the third ventricle and within the mamillary complex. Despite pretreatment with colchicine, no evidence of immunoreactive cell bodies containing DSIP-IR could be found.

Colocalization of delta sleep inducing peptide and luteinizing hormone releasing hormone in neurosecretory vesicles in rat median eminence.

The colocalization of immunoreactivities similar to delta sleep inducing peptide (DSIP) and luteinizing hormone releasing hormone (LH-RH) was investigated by light and electron microscopic immunocytochemistry in the rat median eminence. At the light microscopic level, DSIP and LH-RH immunostained fibers, and varicosities exhibited a similar distribution pattern throughout the median eminence. Immunoreactive axons were mainly found in the lateral part of the external layer. Using an elution-restaining technique, the coexistence of LH-RH and DSIP immunoreactivities was observed in most labelled axons. To determine the intracellular localization of DSIP and LH-RH, we used double immunocytochemical labelling with species-specific antibodies and secondary antibodies conjugated to different sizes of gold particles. The two peptides were found colocalized in single axons. Immunoreactive terminals frequently showed direct membrane apposition with tanycyte processes but rare contacts with portal capillaries. No staining was observed in tanycytes and ependymal or glial elements. Moreover, we could demonstrate that LH-RH and DSIP (or a closely related molecular form) are contained not only in the same axons, but also in the same approximately 100-nm dense-core vesicles, suggesting cosecretion of these peptides.

Hypnotic action of flunitrazepam is reversed by proglumide in rats.

1. Caerulein, an analogue of cholecystokinin (CCK-8), like CCK-8, has been shown to produce hypnotic effects similar to those of benzodiazepine (flunitrazepam). 2. Proglumide antagonizes the action of CCK-8 and of its analogue. 3. The aim of the present study was to demonstrate whether proglumide would affect the potent hypnotic action of flunitrazepam in rats. 4. The association of proglumide with flunitrazepam suppress the increase of total sleep time and slow wave sleep seen after flunitrazepam alone. Proglumide alone has no effect on sleep stages. The authors report here for the first time that the hypnotic action of flunitrazepam is antagonized by proglumide in rat.

Immunohistochemical mapping of delta sleep-inducing peptide in the cat brain and hypophysis. Relationships with the LHRH system and corticotropes.

Using the indirect immunofluorescence method, the distribution of the delta sleep-inducing peptide was studied in the cat brain and hypophysis. Delta sleep-inducing peptide-like-immunoreactive cell bodies mostly visualized in colchicine-pretreated animals were mainly found scattered throughout the diagonal band of Broca, the ventral septum and the anterior hypothalamic areas. A few immunoreactive cell somata were also seen in the ventrolateral hypothalamic area and more occasionally in the triangular septal nucleus. The heaviest concentrations of delta sleep-inducing peptide-like-immunoreactive varicose fibres and terminal-like structures were observed in the septo-preoptic region, in the median eminence and pituitary stalk. Some other brain regions supplied with few delta sleep-inducing peptide-immunoreactive fibres included the fimbria-fornix, the dorsal part of the subfornical organ, the medial habenular nucleus and more caudally, the periaqueductal gray. Elution-restaining experiments revealed that delta sleep-inducing peptide-like immunoreactivity frequently occurred in luteinizing hormone-releasing hormone-immunoreactive neurons and vice versa. At the pituitary level, delta sleep-inducing peptide-like immunoreactivity was detected in most, if not all, melanocorticotropes of the pars intermedia and further in a large subpopulation of corticotropes mainly located in the zona tuberalis of the pars distalis. Taken together these anatomical findings support the view that delta sleep-inducing peptide (or a closely related molecular form) could play a modulatory role at various levels of the hypothalamo-pituitary system.

Distribution and colocalization of delta sleep-inducing peptide and luteinizing hormone-releasing hormone in the aged human brain: an immunohistochemical study.

The distributions of delta sleep-inducing peptide (DSIP)- and luteinizing hormone-releasing hormone (LHRH)-immunoreactive neurons were investigated in the human brain with special emphasis on the basal forebrain (from the septum to the hypothalamus), using indirect immunofluorescence. With a modified elution technique, sequential stainings on the same section showed that DSIP- and LHRH-immunoreactivities were often colocalized. Small numbers of LHRH/DSIP-immunoreactive cells were essentially detected in the diagonal band of Broca, the medial septum and the ventral hypothalamus. The richest areas displaying fibres and terminal-like structures were the preoptic area, the ventromedial and ventrolateral hypothalamic areas, the periventricular region and certain circumventricular organs (i.e. median eminence, vascular organ of the lamina terminalis). Few isolated fibres were observed in the subfornical organ. The topographical relationships between DSIP- and LHRH-immunoreactivities in the neurosecretory systems suggest that DSIP may play a role as important as that of LHRH.