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The postsynaptic density protein PSD-95 is a major element of synapses. PSD-95 is involved in aging, Alzheimer's disease (AD) and numerous psychiatric disorders. However, contradictory data about PSD-95 expression in aging and AD have been reported. Indeed in AD versus control brains PSD-95 varies according to regions, increasing in the frontal cortex, at least in a primary stage, and decreasing in the temporal cortex. In contrast, in transgenic mouse models of aging and AD PSD-95 expression is decreased, in behaviorally aged impaired versus unimpaired rodents it can decrease or increase and finally, it is increased in rodents grown in enriched environments. Different factors explain these contradictory results in both animals and humans, among others concomitant psychiatric endophenotypes, such as depression. The possible involvement of PSD-95 in reactive and/or compensatory mechanisms during AD progression is underscored, at least before the occurrence of important synaptic elimination. Thus, in AD but not in AD transgenic mice, enhanced expression might precede the diminution commonly observed in advanced aging. A two-compartments cell model, separating events taking place in cell bodies and synapses, is presented. Overall these data suggest that AD research will progress by untangling pathological from protective events, a prerequisite for effective therapeutic strategies.

Superficial layers I to III of the human cerebral cortex are more vulnerable toward Aβ peptides than deep layers V to VI in aging. Three models of layers were used to investigate this pattern of frailty. First, primary neurons from E14 and E17 embryonic murine cortices, corresponding respectively to future deep and superficial layers, were treated either with Aβ(1-42), okadaic acid, or kainic acid. Second, whole E14 and E17 embryonic cortices, and third, in vitro separated deep and superficial layers of young and old C57BL/6J mice, were treated identically. We observed that E14 and E17 neurons in culture were prone to death after the Aβ and particularly the kainic acid treatment. This was also the case for the superficial layers of the aged cortex, but not for the embryonic, the young cortex, and the deep layers of the aged cortex. Thus, the aged superficial layers appeared to be preferentially vulnerable against Aβ and kainic acid. This pattern of vulnerability corresponds to enhanced accumulation of senile plaques in the superficial cortical layers with aging and Alzheimer's disease.

Firstly discovered in rete testis fluid, clusterin is a glycoprotein present in most of the other biological fluids. Several isoforms of clusterin are encoded from a single gene located on chromosome 8 in human species. Among the different isoforms, the secreted form of clusterin is expressed by a variety of tissues, including the nervous system under normal conditions. This form is presumed to play an anti-apoptotic role and seems to be a major determinant in cell survival and neuroplasticity after stroke. In animal models of this pathology, both neuronal and astroglial subpopulations express high levels of clusterin early after the ischemic damage. Recent lines of evidence point also to its possible involvement in neurodegenerative disorders. It is thought that in Alzheimer's disease the association between amyloidogenic peptides and clusterin contributes to limit Aβ species misfolding and facilitates their clearance from the extracellular space. Thus, intercellular and intracellular factors that modulate local clusterin expression in the nervous system may represent potent targets for neurodegenerative disease therapies. In this review we provide a critical overview of the most recent data on the involvement of clusterin in neurodegenerative diseases with special reference to their putative clinical relevance.

The presenilin-1 gene is mutated in early-onset familial Alzheimer’s disease. The mutation Pro117Leu is implicated in a very severe form of the disease, with an onset of less than thirty years. The consequences of this mutation on neurogenesis in the hippocampus of adult transgenic mice have already been studied in situ. The survival of neural progenitor cells was impaired resulting in decreased neurogenesis in the dentate gyrus. Our intention was to verify if similar alterations could occur in vitro in progenitor cells from the murine ganglionic eminences isolated from embryos of this same transgenic mouse model. These cells were grown in culture as neurospheres and after differentiation the percentage of neurons generated as well as their morphology were analysed. The mutation results in a significant decrease in neurogenesis compared to the wild type mice and the neurons grow longer and more ramified neurites. A shift of differentiation towards gliogenesis was observed that could explain decreased neurogenesis despite increased proliferation of neural precursors in transgenic neurospheres. A diminished survival of the newly generated mutant neurons is also proposed. Our data raise the possibility that these alterations in embryonic development might contribute to increase the severity of the Alzheimer’s disease phenotype later in adulthood.

The neuropathology of Alzheimer disease is characterized by senile plaques, neurofibrillary tangles and cell death. These hallmarks develop according to the differential vulnerability of brain networks, senile plaques accumulating preferentially in the associative cortical areas and neurofibrillary tangles in the entorhinal cortex and the hippocampus. We suggest that the main aetiological hypotheses such as the beta-amyloid cascade hypothesis or its variant, the synaptic beta-amyloid hypothesis, will have to consider neural networks not just as targets of degenerative processes but also as contributors of the disease's progression and of its phenotype. Three domains of research are highlighted in this review. First, the cerebral reserve and the redundancy of the network's elements are related to brain vulnerability. Indeed, an enriched environment appears to increase the cerebral reserve as well as the threshold of disease's onset. Second, disease's progression and memory performance cannot be explained by synaptic or neuronal loss only, but also by the presence of compensatory mechanisms, such as synaptic scaling, at the microcircuit level. Third, some phenotypes of Alzheimer disease, such as hallucinations, appear to be related to progressive dysfunction of neural networks as a result, for instance, of a decreased signal to noise ratio, involving a diminished activity of the cholinergic system. Overall, converging results from studies of biological as well as artificial neural networks lead to the conclusion that changes in neural networks contribute strongly to Alzheimer disease's progression.

Early studies showed that the administration of the anti-inflammatory cytokine interleukin-10 (IL10) protects against permanent middle cerebral artery occlusion (MCAO) in mice. In this study, transgenic mice expressing murine IL10 (IL10T) directed by the major histocompatibility complex Ea promoter were produced and used to explore the effect of chronically increased IL10 levels on MCAO-related molecular mechanisms. IL10 was over-expressed in astrocytes, microglia, and endothelial brain cells in IL10T compared with wild type mice. Four days following MCAO, IL10T mice showed a 40% reduction in infarct size which was associated to significantly reduced levels of active caspase 3 compared with wild type mice. Under basal conditions, anti-inflammatory factors such as nerve growth factor and GSH were up-regulated and the pro-inflammatory cytokine IL1beta was down-regulated in the brain of IL10T animals. In addition, these mice displayed increased basal GSH levels in microglial and endothelial cells as well as a marked increase in manganese superoxide dismutase in endothelial lining blood vessels. Following ischemia, IL10T mice showed a marked reduction in pro-inflammatory cytokines, including tumor necrosis factor-alpha, interferon-gamma, and IL1beta. Our data indicate that constitutive IL10 over-expression is associated with a striking resistance to cerebral ischemia that may be attributed to changes in the basal redox properties of glial/endothelial cells.

The discovery that a common polymorphism (5-HTTLPR, short variant) in the human serotonin transporter gene (SLC6A4) can influence personality traits and increase the risk for depression in adulthood has led to the hypothesis that a relative increase in the extracellular levels of serotonin (5-HT) during development could be critical for the establishment of brain circuits. Consistent with this idea, a large body of data demonstrate that 5-HT is a strong neurodevelopmental signal that can modulate a wide variety of cellular processes. In humans, serotonergic fibers appear in the developing cortex as early as the 10th gestational week, a period of intense neuronal migration. In this study we hypothesized that an excess of 5-HT could affect embryonic cortical interneuron migration. Using time-lapse videometry to monitor the migration of interneurons in embryonic mouse cortical slices, we discovered that the application of 5-HT decreased interneuron migration in a reversible and dose-dependent manner. We next found that 5-HT6 receptors were expressed in cortical interneurons and that 5-HT6 receptor activation decreased interneuron migration, whereas 5-HT6 receptor blockade prevented the migratory effects induced by 5-HT. Finally, we observed that interneurons were abnormally distributed in the cerebral cortex of serotonin transporter gene (Slc6a4) knockout mice that have high levels of extracellular 5-HT. These results shed new light on the neurodevelopmental alterations caused by an excess of 5-HT during the embryonic period and contribute to a better understanding of the cellular processes that could be modulated by genetically controlled differences in human 5-HT homeostasis.

A 23-year-old male with clinically diagnosed Lowe syndrome had bilateral cataracts, glaucoma, pendulous nystagmus, severe mental and growth retardation, hypotonia, areflexia, joints hyperextensibility, proteinuria, aminoaciduria, and metabolic acidosis. There was also severe epileptic activity (Lennox-Gastaut syndrome). The neuropathological examination revealed a marked cerebellar atrophy and central chromatolysis in the cerebral cortex. These observations do not confirm the hypothesis of dysmyelination as formulated in previous studies. The reported case rather suggests the existence of a dynamic process starting as a still-undefined metabolic abnormality that, in turn, causes various and inconsistent lesions at the microscopic level.

Clusterin is a glycoprotein highly expressed in response to tissue injury. Using clusterin-deficient (Clu-/-) mice, we investigated the role of clusterin after permanent middle cerebral artery occlusion (MCAO). In wild-type (WT) mice, clusterin mRNA displayed a sustained increase in the peri-infarct area from 14 to 30 days post-MCAO. Clusterin transcript was still present up to 90 days post-ischemia in astrocytes surrounding the core infarct. Western blot analysis also revealed an increase of clusterin in the ischemic hemisphere of WT mice, which culminates up to 30 days post-MCAO. Concomitantly, a worse structural restoration and higher number of GFAP-reactive astrocytes in the vicinity of the infarct scar were observed in Clu-/- as compared to WT mice. These findings go beyond previous data supporting a neuroprotective role of clusterin in early ischemic events in that they demonstrate that this glycoprotein plays a central role in the remodeling of ischemic damage.

Peroxisome proliferator-activated receptors (PPARs) are involved in energy expenditure, regulation of inflammatory processes, and cellular protection in peripheral tissues. Among the different types of PPARs, PPARbeta is the only one to be widely expressed in cortical neurons. Using PPARbeta knockout (KO) mice, we report here a detailed investigation of the role of PPARbeta in cerebral ischemic damage, associated inflammatory and antioxidant processes as well as food intake regulation after middle cerebral artery occlusion (MCAO). The PPARbeta KO mice had a two-fold increase in infarct size compared with wild-type (WT) mice. Brain oxidative stress was dramatically enhanced in these KO mice, as documented by an increased content of malondialdehyde, decreased levels of glutathione and manganese superoxide dismutase, and no induction of uncoupling protein 2 (UCP2) mRNA. Unlike WT mice, PPARbeta KO mice showed a marked increase of prooxidant interferon-gamma but no induction of nerve growth factor and tumor necrosis factor alpha after MCAO. In WT mice, MCAO resulted in inflammation-specific transient hyperphagia from day 3 to day 5 after ischemia, which was associated with an increase in neuropeptide Y (NPY) mRNA. This hyperphagic phase and NPY mRNA induction were not observed in PPARbeta KO mice. Furthermore, our study also suggests for the first time that UCP2 is involved in MCAO food intake response. These data indicate that PPARbeta plays an important role in integrating and regulating central inflammation, antioxidant mechanisms, and food intake after MCAO, and suggest that the use of PPARbeta agonists may be of interest for the prevention of central ischemic damage.

A new β-microprobe (βP) has been used to locally measure the time–concentration curve of a radiolabeled substance. The βP, analogous to positron emission tomography methodology, is useful for in vivo animal studies because it can acquire time–concentration curves with high temporal and spatial resolution. Using [18F]fluoro-2-deoxy-d-glucose and βP, we evaluated the reliability of the biologic parameters and we compared this method with the [14C]2-deoxy-d-glucose autoradiography. βP time–concentration curves in three regions of the brain were obtained from 24 rats. Four kinetic parameters (K1-k4) were estimated from 60-minute experimental periods using a three-compartment model. Best fits were obtained when the vascular fraction (Fv) was estimated simultaneously with the four kinetic parameters (K1-k4). The mean estimated Fv values were about 5.5% for the frontal cortex regions and 8.0% for the cerebellum. Correlation coefficients higher than 0.830 were observed between regional cerebral metabolic rates for glucose (rCMRglc) values obtained by βP and autoradiography. In addition, the βP-derived input function was similar to that obtained by manual sampling. Our findings show that reliable rCMRglc values can be obtained using βP.

Uncoupling protein 2 (UCP2) is suggested to be a regulator of reactive oxygen species production in mitochondria. We performed a detailed study of brain injury, including regional and cellular distribution of UCP2 mRNA, as well as measures of oxidative stress markers following permanent middle cerebral artery occlusion in UCP2 knockout (KO) and wild-type (WT) mice. Three days post ischemia, there was a massive induction of UCP2 mRNA confined to microglia in the peri-infarct area of WT mice. KO mice were less sensitive to ischemia as assessed by reduced brain infarct size, decreased densities of deoxyuridine triphosphate nick end-labelling (TUNEL)-labelled cells in the peri-infact area and lower levels of lipid peroxidation compared with WT mice. This resistance may be related to the substantial increase of basal manganese superoxide dismutase levels in neurons of KO mice. Importantly, we found a specific decrease of mitochondrial glutathione (GSH) levels in UCP2 expressing microglia of WT, but not in KO mice after ischemia. This specific association between UCP2 and mitochondrial GSH levels regulation was further confirmed using lipopolysaccharide models of peripheral inflammation, and in purified peritoneal macrophages. Moreover, our data imply that UCP2 is not directly involved in the regulation of ROS production but acts by regulating mitochondrial GSH levels in microglia.

Lipoprotein and endothelial lipases are members of the triglyceride lipase gene family. These genes are expressed in the brain, where the encoded proteins are fulfilling functions that have yet to be elucidated. In this study, we examined the distribution of their respective mRNAs in the C57BL/6 mouse brain by in situ hybridization. In control mice, we observed widespread expression of lipoprotein lipase (LPL) mRNA mainly in pyramidal cells of the hippocampus (CA1, CA2 and CA3 areas), in the striatum and in several cortical areas. Endothelial lipase (EL) mRNA expression was restricted to CA3 pyramidal cells of the hippocampus, to ependymal cells in the ventral part of the third ventricle and to some cortical cell layers. To gain insight into the role played by lipases in the brain, neurodegeneration was induced by intraperitoneal injection of kainic acid (KA) or by occlusion of the middle cerebral artery (MCA). Upon injection of KA, a rapid increase in EL mRNA expression was observed in the piriform cortex, hippocampus, thalamus and neocortex. However, the levels of LPL mRNA were unaffected by KA injection. Remarkably, after focal cerebral ischemia, the expression of EL was unaffected whereas a dramatic increase in LPL expression was observed in neocortical areas of the lesioned side of the brain. These results show that LPL and EL transcripts are selectively upregulated in function of the type of brain injury. LPL and EL could thus fulfill a function in the pathophysiological response of the brain to injury.

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.

Previous studies have suggested that various apoptotic-related proteins could be involved in the death process induced by cerebral ischaemia. In order to further clarify their role and examine how the anti-apoptotic protein Bcl-2 could influence this process, the time-course of mRNA expression of various cell death genes was studied from 1 to 14 days following permanent occlusion of the middle cerebral artery in wild-type (WT) and Bcl-2 transgenic mice, within and outside the area of infarction. No differences of the infarct sizes were observed between the two groups of mice, showing that the extent of neuronal injury could not have been lowered by the Bcl-2 transgene. Seven days after the ischaemic insult, the mRNA expression of the cell death gene effector cpp32 was dramatically upregulated in the penumbra of WT and Bcl-2 transgenic mice. Interestingly, the cpp32 transcript was markedly induced from 3 days in the ipsilateral thalamus of the two groups of mice. However, apoptotic bodies were observed in the thalamic field of WT but not transgenic mice. This suggests that cpp32 mRNA may be induced in an attempt to kill the injured cells and, in contrast to the penumbra, cell death in the thalamus may be prevented in Bcl-2 transgenic mice. Based on these results, the pathophysiological mechanisms that underly neuronal damage following ischaemia need consideration in order to evaluate the extent of neuroprotection that may be afforded by the Bcl-2 anti-apoptotic protein. Although the present study does not confirm previous data showing a protective role of Bcl-2 in neocortical infarcted areas, it suggests that anti-apoptotic therapies may constitute a possible treatment for areas of the brain remote from those directly affected by ischaemia.

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.

Apoptotic cell death is a major feature of the developing nervous system and of certain neurodegenerative diseases. Various gene effectors and repressors of this type of cell death have been identified. Among them, bcl-xl and bax, which encode for antiapoptotic and proapoptotic proteins, respectively, play major roles during development. The gene cpp32 encodes for the caspase 3 cysteine protease and is a critical mediator of cell death during embryonic development in the mammalian brain. To gain insight into the possible implications of these cell death genes during the postnatal development, we investigated the expression of bax, bcl-xl, and cpp32 mRNAs by in situ hybridization in the mouse brain from birth to adulthood. Whereas bax and bcl-xl mRNAs were expressed widely in neonates and adult mice, our results showed that cpp32 mRNA levels were decreased strongly from 12 postnatal days. From 1 postnatal day to 12 postnatal days, cpp32 mRNA was expressed ubiquitously in all brain nuclei, including areas where neurogenesis occurred. A positive correlation between areas displaying high levels of mRNA and apoptotic nuclei also was shown. In the adult, cpp32 mRNA was restricted to the piriform and entorhinal cortices, the neocortex, and to areas where neurogenesis is observed (e.g., olfactory bulb and dentate gyrus). The same pattern of expression was observed in adult mice over-expressing the antiapoptotic protein Bcl-2. These results demonstrate that the expression of cpp32 mRNA is highly regulated during the mouse postnatal period, leading to a specific distribution in the adult central nervous system. Moreover, the prevention of cell death by Bcl-2 likely is not linked to the regulation of caspase mRNA levels.

The Lou/C rat, an inbred strain of Wistar origin, was described as a model of resistance to age- and diet-induced obesity. Although such a resistance involves many metabolic parameters described in our previous studies, Lou/C rats also exhibit a spontaneous food restriction due to decreased food consumption during the nocturnal period. We then attempted to delineate the leptin sensitivity and mechanisms implicated in this strain, using different protocols of acute central and peripheral leptin administration. A first analysis of the meal patterns revealed that Lou/C rats eat smaller meals, without any change in meal number compared to age-matched Wistar animals. Although the expression of the recognized leptin transporters (leptin receptors and megalin) measured in the choroid plexus was normal in Lou/C rats, the decreased triglyceridemia observed in these animals is compatible with an increased leptin transport across the blood brain barrier. Improved hypothalamic leptin signaling in Lou/C rats was also suggested by the higher pSTAT3/STAT3 (signal transducer and activator of transcription 3) ratio observed following acute peripheral leptin administration, as well as by the lower hypothalamic mRNA expression of the suppressor of cytokine signaling 3 (SOCS3), known to downregulate leptin signaling. To conclude, spontaneous hypophagia of Lou/C rats appears to be related to improved leptin sensitivity. The main mechanism underlying such a phenomenon consists in improved leptin signaling through the Ob-Rb leptin receptor isoform, which seems to consequently lead to overexpression of brain-derived neurotrophic factor (BDNF) and thyrotropin-releasing hormone (TRH).

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.

Central oxytocin (OT) receptors were labelled in 3-month-old and 20-month-old rats with an iodinated OT antagonist. Comparison of the autoradiograms by quantitative image analysis revealed in the old animals a significant reduction of binding in three regions; the number of labelled OT receptors was decreased by 90% in the head of the caudate putamen, by 68% in the olfactory tubercle, and by 41% in the ventromedial hypothalamic nucleus. Previous studies had shown that the expression of OT receptors in the olfactory tubercle and in the ventromedial hypothalamic nucleus was dependant upon gonadal steroids. Therefore we hypothesize that the reduced number of OT receptors in the latter two structures of aged rats was the consequence of the 4-fold decrease of plasma testosterone that we found in this age. Another mechanism may be responsible for the marked reduction of OT receptors in the caudate putamen.

Patients with the early-onset Alzheimer's disease P117L mutation in the presenilin-1 gene (PS-1) present pathological hallmarks in the hippocampus, the frontal cortex and the basal ganglia. In the present work we determined by immunohistochemistry which brain regions were injured in the transgenic PS-1 P117L mice, in comparison to their littermates, the B6D2 mice. Furthermore, as these regions are involved in novelty detection, we investigated the behavior of these mice in tests for object and place novelty recognition. Limited numbers of senile plaques and neurofibrillary tangles were detected in aged PS-1 P117L mice in the CA1 only, indicating that the disease is restrained to an initial neuropathological stage. Western blots showed a change in PSD-95 expression (p=0.03), not in NR2A subunit, NR2B subunit and synaptophysin expressions in the frontal cortex, suggesting specific synaptic alterations. The behavioral tests repeatedly revealed, despite a non-significant preference for object or place novelty, maladaptive exploratory behavior of the PS-1 P117L mice in novel environmental conditions, not due to locomotor problems. These mice, unlike the B6D2 mice, were less inhibited to visit the center of the cages (p=0.01) and they continued to move excessively in the presence of a displaced object (p=0.021). Overall, the PS-1 P117L mice appear to be in an initial Alzheimer's disease-like neuropathological stage, and they showed a lack of reaction toward novel environmental conditions.

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.

Substance P was visualized in the rat and the guine 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.

The immunocytochemical distribution of CLIP (corticotropin-like intermediate lobe peptide) or ACTH(18–39), a small biologically active peptide, was examined in the human brain, using a monoclonal antibody against this peptide. Groups of CLIP-immunoreactive cell bodies, small to medium size and bipolar or triangular in shape, were found in the basal hypothalamus extending from the retrochiasmatic region to the premammillary nuclei area. Immunoreactive fibers with varicosities, terminals and “pipe shape” structures, were distributed within the hypothalamus, limbic structures, the brainstem and spinal cord nuclei, forming a particularly rich network in the hypothalamus, the preoptic area, the septal region, the amygdala and the upper brainstem periaqueductal gray matter. The above neuroanatomical observations confirm and extend previous findings in animals, strengthening even more the possibility that this peptide may be involved in numerous behavioral, autonomic and physiological functions such as regulation of sleep-waking cycle, pain control and respiratory and cardiovascular regulation.

1. Caerulein, an analogue of cholecystokinin (CCK-8), like CCK-8, has been shown to produce hypnotic effects similar to those of benzodiazepine (flunitrazeparo). 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.

Correspondance: Panteleimon.Giannakopoulos@medecine.unige.ch; International audience; 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 El 6 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

Correspondance auteur: Panteleimon G. Email: Panteleimon.Giannakopoulos@medecine.unige.ch; International audience; Clusterin or apolipoprotein J is a heterodimeric glycoprotein which is known to be increased during tissue involution in response to hormonal changes or injury and under circumstances leading to apoptosis. Previous studies in wild-type (WT) and clusterin-null (Clu-/-) mice indicated a protective role of clusterin over-expression in astrocytes lasting up to 90 days post-ischemia. However, in in vitro and in vivo models of neonatal hypoxia-ischemia, clusterin exacerbates necrotic cell death. We developed recombinant forms of clusterin and examined their effect on propidium iodide uptake, neuronal and synaptic markers as well as electrophysiological recordings in hippocampal slice cultures from Clu-/- and WT mice subjected to oxygen-glucose deprivation (OGD). WT mice displayed a marked up-regulation of clusterin associated with electrophysiological deficits and dramatic increase of propidium iodide uptake 5 days post-OGD. Immunocytochemical and western blot analyses revealed a substantial decrease of neuronal nuclei and synaptophysin immunoreactivity that predominated in WT mice. These findings contrasted with the relative post-OGD resistance of Clu-/- mice. The addition of biologically active recombinant forms of human clusterin for 24 h post-OGD led to the abolishment of the ischemic tolerance in Clu-/- slices. This deleterious effect of clusterin was reverted by the concomitant administration of the NMDA receptor antagonist, D-2-amino-5-phosphonopentanoate. The present data indicate that in an in vitro model of ischemia characterized by the predominance of NMDA-mediated cell death, clusterin exerts a negative effect on the structural integrity and functionality of hippocampal neurons

Summary Substance P-like immunoreactivity was visualized by immunohistochemical methods in 20 postmortem brains: 6 senile, 4 presenile Alzheimer dementia (AD), 3 AD with interhemispheric asymmetric cortical atrophy, and 7 control cases. For all pathological cases, the SP-like immunoreactivity was significantly reduced in the neocortical areas and in the hippocampus. This contrasted with an enhanced SP-like immunoreactivity in the pallidum and the substantia nigra in AD brains and a more pronounced SP-like immunoreactivity in the more atrophic side in the asymmetrically atrophied brains.

Ischemia-induced neuronal damage has been linked to elevated production of reactive oxygen species (ROS) both in animal models and in humans. NADPH oxidase enzymes (NOX-es) are a major enzymatic source of ROS, but their role in brain ischemia has not yet been investigated. The present study was carried out to examine the expression of NOX4, one of the new NADPH oxidase isoforms in a mouse model of focal permanent brain ischemia. We demonstrate that NOX4 is expressed in neurons using in situ hybridization and immunohistochemistry. Ischemia, induced by middle cerebral artery occlusion resulted in a dramatic increase in cortical NOX4 expression. Elevated NOX4 mRNA levels were detectable as early as 24 h after the onset of ischemia and persisted throughout the 30 days of follow-up period, reaching a maximum between days 7 and 15. The early onset suggests neuronal reaction, while the peak period corresponds to the time of neoangiogenesis occurring mainly in the peri-infarct region. The occurrence of NOX4 in the new capillaries was confirmed by immunohistochemical staining. In summary, our paper reports the presence of the ROS producing NADPH oxidase NOX4 in neurons and demonstrates an upregulation of its expression under ischemic conditions. Moreover, a role for NOX4 in ischemia/hypoxia-induced angiogenesis is suggested by its prominent expression in newly formed capillaries.

We have generated a mouse strain carrying a transgene driven by a strong and ubiquitous promoter (human cytomegalovirus hCMV/beta-actin) and containing an enhanced green fluorescent protein (eGFP) coding sequence upstream of the 3' untranslated region (3'UTR) of tissue-type plasminogen activator (t-PA) mRNA. The 3'UTR of t-PA mRNA is known to be involved in the reversible deadenylation and translational repression of transcripts in mouse oocytes. hCMV/beta-actin-eGFP-3'UTR t-PA transgenic mice express eGFP mRNA in all brain structures analyzed but lack eGFP fluorescence, with the exception of blood vessels, choroid plexus, and Purkinje cells. Taking advantage of these features, we tested whether certain pathological conditions, in particular injuries of the nervous system, might trigger eGFP fluorescence in traumatized cells or neurons. From this perspective, we analyzed eGFP mRNA expression and eGFP fluorescence in experimental models of nervous system lesions, such as motoneuron axotomy and cerebral stroke induced by middle cerebral artery occlusion. We found an increase in eGFP fluorescence in specific brain areas in cells suffering or reacting to these injuries. This increased fluorescence is correlated with an increased transcription of eGFP in lesioned cells, presumably enhanced by a release of the translational silencing mediated by the 3'UTR region of the t-PA mRNA. This transgenic mouse model may prove useful to study the development of neurodegenerative lesions.

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.

Because recent studies have indicated that tissue plasminogen activator (tPA) aggravates neurodegenerative processes in many neural pathologies, we studied whether the endogenous tPA antagonist neuroserpin has a neuroprotective effect in an animal model of focal ischemic stroke. After induction of a focal ischemic stroke in the mouse by occlusion of the midddle cerebral artery, we found that microglial cells accumulated in the marginal zone of the infarct are the most important source for both plasminogen activators, tPA and uPA. To investigate the effect of neuroserpin on the size and the histology of the infarct we produced transgenic mice overexpressing neuroserpin approximately sixfold in the nervous system. In the brain of these mice the total tPA activity in the uninjured tissue was strongly reduced. After induction of a focal ischemic stroke in the transgenic mice by a permanent occlusion of the middle cerebral artery (MCA), the infarcts were 30% smaller than in the wild-type mice. Immunohistochemical analyses and in situ hybridization revealed an attenuation of the microglial activation in the reactive zone. Concomitantly, the microglial production of tPA and uPA, as well as the PA-activity in the infarct region was markedly reduced. Thus, our results indicate that neuroserpin reduces microglial activation and, therefore, the PA activity and has a neuroprotective role after focal ischemic stroke.

The exponential increase in the prevalence of Alzheimer's disease with age, as well as the consistent presence of Alzheimer's disease pathologic changes in elderly people, has induced much debate regarding the relationships between brain aging and Alzheimer's disease. In this context, the study of nonagenarians and centenarians is of particular interest because it allows one to define the spectrum and extent of changes in brain morphology that occur near the upper age limit of life and to assess whether Alzheimer's disease is on a continuum with normal brain aging. The epidemiological and neuropathological evidence summarized in this chapter supports that Alzheimer's disease is an age-related pathologic condition, which does not represent an inevitable consequence of ageing. Moreover, it suggests the presence of a particular subgroup of oldest-old individuals with high resistance to the neurodegenerative process. These observations are discussed within the theoretical framework of human longevity.

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.

Alzheimer’s disease (AD) is characterized neuropathologically by the presence of two classical histologic hallmarks, neurofibrillary tangles (NFT) and senile plaques (SP). Recent analyses of the distribution of the pathologic changes in AD suggest that this disease affects select populations of neurons within the cerebral cortex that are characterized by specific regional and laminar distribution and connectivity patterns, whereas other neuronal subgroups are remarkably resistant to the degenerative process. Based on the distribution of NFT and SP, it has been proposed that a global corticocortical disconnection leads to the loss of integrated functions exhibited by AD patients. Furthermore, it appears that lesion distribution follows certain organization schemes and that these regional and laminar patterns may relate cortical circuits to clinical symptoms. Several neuropathologic studies have documented the presence of NFT and SP in the cerebral cortex of non-demented elderly individuals (Mountjoy et al., 1983; Tomlinson et al., 1968). More recently, it has been shown that amyloid deposition and NFT formation are not only common features of brain aging, but that they are present within certain regions of the cerebral cortex with densities comparable to those observed in demented patients (Bouras et al., 1993; Hof et al., 1992). Thus, high NFT densities have been demonstrated in the hippocampal formation and temporal neocortex, but sparse NFT in the other neocortical regions in cases presenting with very mild signs of cognitive decline (Bouras et al., 1993; Hof et al., 1992).

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.

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.

In Mongolian gerbils, the content of vasopressin in the cerebral cortex, the striatum, and the hypothalamus is increased after induction of acute cerebral ischemia. We used an iodinated vasopressin analogue and light microscopic autoradiography to study the distribution of vasopressin V1 receptors in the brain of adult male gerbils and to evaluate the effects of a transient bilateral cerebral ischemia (6 minutes) on the density of this receptor population. The animals were killed immediately or 10, 30, or 100 hours after transient bilateral occlusion of the common carotid arteries. In control animals, specific [125I]-VPA binding sites were present in various structures of the brain (olfactory bulb, anterior olfactory nucleus, lateral septum, bed nucleus of the stria terminalis, median preoptic area, ventral pallidum, substantia innominata, amygdala, thalamus, hypothalamic mammillary nuclei, superior colliculus, subiculum, central gray, nucleus of the solitary tract, hypoglossal nucleus). The strongest labeling was detected in the cerebral cortex, layers 5-6. After 30-100 hours of survival time following ischemia there was a marked decrease in [125I]-VPA binding site density in these cerebral cortex layers. To a lesser degree, a decrease was also detected in the lateral septal nucleus. In contrast, labeling in other noncortical structures remained unchanged. All animals with 100 hours recovery showed a loss of cells in hippocampus (CA1 layer) and striatum. In addition, ischemia induced concomitant and proliferative changes in cortical and hippocampal astrocytes assessed by glial fibrillary acid protein immunoreactivity. These observations indicate a role for vasopressin in the cerebral cortex either on neurons or on glial cells and the modulation of vasopressin receptor expression by transient cerebral ischemia.

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.

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.

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.

The superfusion of the hepatic portal vein of anaesthetized rats with hypertonic solutions of NaCl elicits a significant increase of neural activity measured in the hypothalamo-neurohypophysial tract. Superfusions of the liver or hepatic veins with hypertonic saline solutions had no effect, whereas superfusions of the portal vein also increased the firing rates of supraoptic endocrine neurones and the intramammary pressure. Results suggest that sodium chloride sensitive receptors (perhaps osmoreceptors) are located within the wall of the hepatic portal vein and mediate neurophypophysial hormone release.

Superfusion of the hepatic portal vein with hypertonic saline solutions increases the electrical activity of the hypothalamo-neurohypophysial tract. Bilateral cervical vagotomy had no effect on this response in all rats studied. Section of the hepatic branch of the vagus abolished the hypothalamic response in only two animals, but injection of xylocaine into the spinal cord at thoracic levels abolished the response in all remaining animals. The results suggest that peripheral osmoreceptors of the portal vein activate the hypothalamo-neurohypophysial system through a spinal afferent pathway.

Floating sections from human brains immersed for more than forty years in formalin, or from brains freshly fixed for a short time are treated by KMnO4-Pal's modified solutions to suppress the endogenous peroxidase activity before using the peroxidase-antiperoxidase method (PAP), or to remove the autofluorescence of lipofuscin, which is very intense in brains from old patients, before using the immunofluorescence method. Following this, immersion of sections in NaOH and H2O2 allows for the demasking of antigenic sites. These treatments enhance the immunolabelling considerably, with results comparable to those obtained with freshly fixed tissues, and facilitate the discrimination between specifically and unspecifically stained structures.

The distribution of delta sleep inducing peptide (DSIP) in the rabbit brain has been studied with immunohistochemical techniques. DSIP-like immunoreactivity was predominantly detected in the basal forebrain, hypothalamus and hypophysis. Even in colchicine-pretreated animals, immunolabeled cell bodies were relatively few. They were mostly scattered through the ventrolateral septum, the diagonal band of Broca and preoptic areas. Clusters of positive cell bodies were also found in the arcuate nucleus and adjacent lateral hypothalamic areas. Large populations of varicose fibers and terminal-like structures were observed in the juxtaventricular zone of the ventrolateral septum, in the preoptic areas and lamina terminalis especially around the preoptic recess of the third ventricle and more caudally, in the ventromedial nucleus of the hypothalamus. Dense networks of immunolabeled fibers were visualized in the median eminence and pituitary stalk where many fibers could be seen in close apposition to the capillaries. Many DSIP-immunoreactive fibers were observed in the subfornical organ. Other extra-hypothalamic regions displaying a low-to-moderate density of immunoreactive fibers were the indusium griseum, the hippocampus, the fimbria of the fornix, the subcommissural organ, the medial habenula and, occasionally, the medial periaqueductal gray. Most cells of the pars intermedia and a few cells of the pars distalis of the anterior pituitary were DSIP-immunoreactive. Taken together these results in the rabbit brain emphasize the predominant localization of DSIP-like immunoreactivity in areas related to the hypothalamic neurosecretory systems.