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.

Lipoprotein lipase and endothelial lipase expression in mouse brain: regional distribution and selective induction following kainic acid-induced lesion and focal cerebral ischemia.

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.

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.

Neuroserpin, a neuroprotective factor in focal ischemic stroke.

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 middle 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.

Cell death is prevented in thalamic fields but not in injured neocortical areas after permanent focal ischaemia in mice overexpressing the anti-apoptotic protein Bcl-2.

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.

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.

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.

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.

[Effects of CO2 and adrenaline on 1% lidocaine in axillary block]. / Effets du CO2 et de l'adrénaline sur la lidocaïne 1% dans le bloc axillaire.

OBJECTIVES: To compare lidocaine hydrocarbonate and lidocaine hydrochloride, with and without adrenaline, in the axillary block obtained with a neurostimulator. STUDY DESIGN: Prospective, randomized, double blind study. PATIENTS: Sixty-six patients undergoing surgery of the upper limb under axillary block, allocated into four groups. METHODS: The criteria for evaluation were: onset time, duration and quality of sensory and motor blockades, and blood concentrations of lidocaine in 39 patients. In all patients musculocutaneous, radial, median and ulnar nerves were stimulated and the volume of local anaesthetic administered was 25 mL per square meter of body surface. Group 1 received lidocaine hydrocarbonate 1% (n = 17), group 2, lidocaine hydrocarbonate 1% with adrenaline 1/200,000 (n = 17), group 3, lidocaine hydrochloride 1% (n = 16) and group 4, lidocaine hydrochloride 1% with adrenaline 1/200,000 (n = 16). RESULTS: No significant inter-group differences were found concerning sensory and motor blockades and onset time. The duration of analgesia was longer in groups CO2 + A and HCL + A. The lidocaine blood concentrations were globally lower in group HCL + A. CONCLUSIONS: Considering the cost/benefit ratio and the absence of clinical benefits of lidocaine hydrocarbonate, lidocaine hydrochloride should be preferred.

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.

Nuclear magnetic relaxation dispersion studies of water-soluble gadolinium(III)-texaphyrin complexes.

Water proton 1/T1 nuclear magnetic relaxation dispersion (NMRD) profiles were measured for a water-soluble gadolinium(III) texaphyrin (Gd-tex) complex as a function of temperature and in the presence and absence of 5% human serum albumin (HSA). Upon dissolving the complex in water (0.259 mM), the water relaxivity values decreased with time but remained higher than those of free GD3+(aq) at all fields. Concurrent measurements of free Gd3+ using metallochromic dyes indicated that demetallation of the texaphyrin did not occur over a period of several days at 37 degrees C. The high relaxivity values and shape of the NMRD profile of this complex may be ascribed to a combination of large water coordination number (q estimated at 3.5) and long tau R. Upon mixing an aqueous solution of the complex with 5% HSA, the low-field water relaxivity slightly decreased whereas the high-field relaxivity increased relative to the free complex in water, and the relaxivities became nearly independent of temperature. These observations indicate that water exchange between the inner coordination sphere of Gd-tex and bulk water becomes limiting in the presence of HSA.