Excess of serotonin affects embryonic interneuron migration through activation of the serotonin receptor 6.

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.

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.

Decreased infarct size after focal cerebral ischemia in mice chronically infected with Toxoplasma gondii.

To determine whether Toxoplasma gondii infection could modify biological phenomena associated with brain ischemia, we investigated the effect of permanent middle cerebral artery occlusion (MCAO) on neuronal survival, inflammation and redox state in chronically infected mice. Infected animals showed a 40% to 50% decrease of infarct size compared with non-infected littermates 1, 4 and 14 days after MCAO. The resistance of infected mice may be associated with increased basal levels of anti-inflammatory cytokines and/or a marked reduction of the MCAO-related brain induction of two pro-inflammatory cytokines, tumor necrosis factor-alpha and interferon-gamma (IFNgamma). In addition, potential anti-inflammatory/neuroprotective factors such as nerve growth factor, suppressor of cytokine signaling-3, superoxide dismutase activity, uncoupling protein-2 and glutathione (GSH) were upregulated in the brain of infected mice. Consistent with a role of GSH in central cytokine regulation, GSH depletion by diethyl maleate inhibited Toxoplasma gondii lesion resistance by increasing the proinflammatory cytokine IFNgamma brain levels. Overall, these findings indicate that chronic toxoplasmosis decisively influences both the inflammatory molecular events and outcome of cerebral ischemia.

Neuronal expression of the NADPH oxidase NOX4, and its regulation in mouse experimental brain ischemia.

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.

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.

Neural lobe of pituitary modulates corticotropin release in the rat.

The hypothesis was tested that the neural lobe of the pituitary may modulate the release of anterior pituitary hormones. The neural lobe of anesthetized lactating rats was electrically stimulated at 30 Hz (5 sec on and 5 sec off) for 3 min while taking blood samples for RIA of ACTH. Plasma ACTH increased within 3 min by 22 +/- 9% (average +/- SEM; P less than 0.025) in intact rats and by 38 +/- 17% (P less than 0.025) in rats where the nerve supply to the median eminence and neural lobe was interrupted. Electrical stimulation of the anterior pituitary was ineffective. No significant changes in plasma ACTH were observed in rats with coagulated hypophysial portal vessels or in Brattleboro rats with congenital diabetes insipidus. Apparently, neither peripheral plasma vasopressin (estimated at 150 microU/ml maximum) nor intermediate lobe ACTH could account for the observed rise in ACTH. Results suggest a vasopressin dependent modulation of ACTH release by the neural lobe, mediated either by axon collaterals to the median eminence or by a vascular interconnection between posterior and anterior pituitaries.

Vasopressin binding in the cerebral cortex of the Mongolian gerbil is reduced by transient cerebral ischemia.

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.

Postnatal distribution of cpp32/caspase 3 mRNA in the mouse central nervous system: an in situ hybridization study.

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.

[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.

Immunohistochemical colocalization of delta sleep-inducing peptide and luteinizing hormone-releasing hormone in rabbit brain neurons.

The anatomical distributions of luteinizing hormone-releasing hormone and delta sleep-inducing peptide immunoreactivity in the rabbit brain were studied by indirect immunofluorescence technique. The comparison of adjacent serial sections, one being immunolabeled with an antiserum to luteinizing hormone-releasing hormone, the other with an antiserum to delta sleep-inducing peptide, showed that the respective distribution patterns of immunoreactivity exhibited a remarkable overlap through the basal forebrain and hypothalamic regions. A sequential double-immunolabelling (elution-restaining method) clearly indicated that all the luteinizing hormone-releasing hormone-immunoreactive cell bodies displayed delta sleep-inducing peptide immunoreactivity. These cell bodies were sparse and mainly located throughout the septal-preoptico-suprachiasmatic region and the ventrolateral hypothalamus. The colocalization of luteinizing hormone-releasing hormone and delta sleep-inducing peptide immunoreactivity was also observed in many fibres supplying all these brain regions and terminal areas such as the organum vasculosum of the lamina terminalis, the subfornical organ, the median eminence and the pituitary stalk. These neuroanatomical findings are suggestive of interaction between delta sleep-inducing peptide and luteinizing hormone-releasing hormone in various brain areas including some circumventricular organs.

Distribution of mRNA for the alpha4 subunit of the nicotinic acetylcholine receptor in the human fetal brain.

Neuronal nicotinic acetylcholine receptors (nAChRs) present in the central nervous system (CNS), are multimeric proteins constituted of two different subunits, alpha and beta, with different subtype arrangements and different pharmacological and functional properties. By in situ hybridization, we studied the distribution of the mRNA for the alpha4 subunit of nAChRs in brains of human 25-week old normal and fragile X fetuses. A strong hybridization signal was detected throughout the thalamus, cortex, pyramidal layer of the Ammon's horn, and the granular layer of the dentate gyrus. Several other areas including the claustrum, caudate nucleus, putamen, globus pallidus, subthalamic nucleus, subiculum, entorhinal cortex, and Purkinje cell layer displayed a low to moderate radiosignal. With few exceptions, our data in the human brain agree those previously reported in the rat. Also, our data indicate that the alpha4 subunit mRNA is produced early in the development, in the more differentiated cells, and in a site-specific manner. Additionally, the alpha4 mRNA is produced in the brain of fragile X fetuses with the same pattern and same intensity than in the normal fetal brain suggesting that alpha4 subunit mRNA production is not altered in the fragile X syndrome. High levels of alpha4 subunit mRNA in human fetal brain support the hypothesis of a morphogenic role of nAChRs during the early CNS development.

The importance of nuclear magnetic relaxation dispersion (NMRD) profiles in MRI contrast media development.

Observation of the relaxivity of MRI contrast media over a wide range of magnetic fields is not only necessary for predicting their efficiency at any field but also compulsory for understanding and improving their mechanisms of action. The best experimental approach to this problem is the field cycling method, which allows the exploration of nuclear relaxation over a broad interval of magnetic field intensity but requires a specially dedicated instrument called a relaxometer. Particularly relevant are the relaxivity profiles of the two chelates Gd-DOTA and Gd-DTPA. Both show an important decrease from low to high fields within the current imaging range (0.02 T to 1.5 T). Although high field relaxivities of these chelates are similar, Gd-DTPA becomes less efficient in facilitating water protons relaxation at fields lower than 0.15 T. This behavior has to be related to different electronic relaxation times due to a different chelate symmetry.

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.

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.

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.

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.

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.

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.

Spinal afferents for peripheral osmoreceptors in the rat.

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.