Philippe Leroux

Central Role of Microglia in Neonatal Excitotoxic Lesions of the Murine Periventricular White Matter

Periventricular leukomalacia (PVL) is the main cause of neurologic handicap in pre‐term infants. The understanding of cellular and molecular mechanisms leading to white matter damage is critical for development of innovative therapeutic strategies for PVL. The pathogenesis of PVL remains unclear but possibly involves glutamate excitotoxicity as an important molecular pathway. We previously described a neonatal mouse model of excitotoxic white matter lesion mimicking human PVL. In the present study, we used this experimental tool to investigate the cellular populations and the glutamate receptor subtypes involved in excitotoxic white matter lesions. Combined immunohistochemical, electron microscopic, and cell death detection data revealed that microglial activation and astrocytic death were the primary responses of white matter to excitotoxic insult. In vitro experiments suggested that microglia activated by ibotenate released soluble factors that kill astrocytes. The use of selective agonists and antagonists of glutamate receptors revealed that N‐methyl‐D‐aspartate (NMDA) receptor activation was essential and sufficient to produce cystic white matter lesions. NMDA receptor immunohistochemistry labeled microglial cells in the neonatal periventricular white matter. The developing white matter displayed a window of sensitivity to excitotoxic damage that was paralleled by the transient presence of NMDA receptor‐expressing white matter cells. Assuming that similar pathophysiologic mechanisms are present in human pre‐ term infants, microglia and NMDA receptors could represent key targets for treatment of PVL.

Localization and characterization of PACAP receptors in the rat cerebellum during development: Evidence for a stimulatory effect of PACAP on immature cerebellar granule cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are abundant in the brain and particularly in the cerebellum of adult rats. In contrast, the occurrence of PACAP binding sites has not been investigated during ontogenesis. The aim of the present study was to determine the distribution and biochemical characteristics of PACAP binding sites in the rat cerebellum during postnatal development, and to examine the effect of PACAP on immature cerebellar granule cells. Autoradiographic studies revealed that PACAP binding sites are transiently expressed in a germinative matrix of the cerebellar cortex, the external granule cell layer, and in the medulla, from postnatal days 8 to 25. A population of PACAP binding sites persisted in the internal granule cell layer in the mature cerebellum. Emulsion-coated cytoautoradiography, performed on cultured immature granule cells from eight-day-old rat cerebellum, demonstrated that transient PACAP binding sites are expressed by cerebellar immature granule cells. Biochemical characterization of binding revealed the occurrence of two classes of PACAP recognition sites exhibiting, respectively, high (Kd = 0.39 +/- 0.08 nM) and low (Kd = 21.2 +/- 9.4 nM) affinity for PACAP27. The two naturally occurring forms PACAP38 and PACAP27 were equipotent in competing for [125I]PACAP27 binding. In contrast, the [Des-His1]PACAP38 analog was eight times less efficient and vasoactive intestinal polypeptide only induced weak displacement of the binding. Exposure of cultured immature granule cells to PACAP27 resulted in a dose-dependent stimulation of the production of cAMP, indicating that PACAP binding sites represent authentic receptors positively coupled to adenylate cyclase. These results show that PACAP receptors are actively expressed in the cerebellum of rats during postnatal development. The presence of functional PACAP receptors in the external granule cell layer suggests that PACAP may play a role in the control of proliferation and/or differentiation of granule cells.

Translent expression of somatostatin receptors in the rat cerebellum during development

Somatostatin and somatostatin receptors have not been identified in adult rat cerebellum. In contrast, during the development, somatostatin-containing neurons have been visualized in the deep layers of the cerebellum. The present study shows that during ontogenesis, somatostatin receptors are present in close association with the external granule cell layer of the cerebellum. No correlation was found between the location of immunoreactive somatostatin and the distribution of somatostatin receptors. The disappearance of somatostatin receptors, from postnatal day 13 to 23, was concomitant with the involution of the external germinal layer.

Ontogeny of somatostatin receptors in the rat brain: Biochemical and autoradiographic study

The ontogeny of somatostatin receptors in the rat brain has been studied by both membrane binding assays and in vitro receptor autoradiographic techniques. High levels of somatostatin binding sites were detected in brain of 15-day-old fetuses (E15). The pharmacological characterization of somatostatin binding sites and the regulatory effect of GTP on somatostatin binding at E15 suggest that somatostatin recognition sites correspond to authentic receptors. The values of maximal binding showed important variations throughout pre- and postnatal development. Globally, a marked increase in the total binding capacity was observed between E15 and postnatal day 8 (P8), with a transient fall at birth and P1. After P8, the concentration of somatostatin receptors progressively decreased and the weaning imposed at P21 accentuated the decline of receptor concentration. Although the density of somatostatin binding sites varied considerably, KD values did not change during brain development. Autoradiographic studies showed marked differences in the distribution of somatostatin receptors during ontogenesis. In the cortex, the cortical plate and the subplate zone appeared to contain high densities of binding sites from E15 to P1. However, the cortical layer which exhibited the higher labelling was the intermediate zone, located just beneath the subplate zone. On the contrary, the germinal epithelium bordering the lateral ventricle appeared virtually devoid of somatostatin binding sites. This laminar distribution of binding sites in the cortex disappeared from P4 to P8, in coincidence with the evolution of the underlying histological organization. At these stages, a homogeneous distribution was observed in almost all cortical layers, contrasting with the distribution of somatostatin receptors in the adult, which was restricted to layers IV-VI. In the cerebellar cortex, autoradiographic labelling was first seen at E15. After birth, the density of somatostatin receptors increased dramatically between P4 and P13, while, at P23, the labelling vanished in most lobes of the cerebellum. Taken together, these results show the early appearance of somatostatin receptors in the rat brain. The high density of somatostatin receptors observed in proliferative or pre-migratory areas suggests that somatostatin may be an important factor involved in the organization of the central nervous system.

Localization and identification of α-melanocyte-stimulating hormone (α-MSH) in the frog brain

Abstract The distribution of α-melanocyte-stimulating hormone (α-MSH) in the central nervous system of the frog Rana ridibunda was determined by immunofluorescence using a highly specific antiserum. α-MSH-like containing perikarya were localized in the infundibular region, mainly in the ventral hypothalamic nucleus. A rich plexus of immunoreactive fibers directed towards the ventral telencephalic region was detected. Reverse-phase high-performance liquid chromatography and radioimmunoassay were used to characterize α-MSH-like peptides in the frog brain. Chromatographic separation revealed that immunoreactive α-MSH coeluted with synthetic des-Nα-acetyl α-MSH, authentic α-MSH and their sulfoxide derivatives. The heterogeneity of α-MSH-like material in the frog brain was in marked contrast with the figure observed in the intermediate lobe of the pituitary gland where only des-Nα-acetyl α-MSH is present. These findings support the existence of discrete α-MSH immunoreactive neurons in the frog brain containing both desacetyl and authentic α-MSH.

Transient expression of somatostatin receptors in the rat visual system during development

The ontogeny of somatostatin receptors in the rat visual system was studied by auto-radiography, using [125I-Tyr0,DTrp8]S14 as a radioligand. The binding sites showed high affinity for somatostatin and somatostatin analogues, and were regulated by GTP as early as day 16 of fetal life (E16), indicating that they represent functional somatostatin receptors. The density of somatostatin receptors was quantified by computerized image-analysis of film autoradiograms, and by grain counting on emulsion-coated slides. During fetal life, somatostatin receptors were observed in the retina, optic nerve, optic chiasma, optic tract, and lateral geniculate nucleus. The highest densities of somatostatin receptors were measured from E16 to E18 in the retina and primary optic pathways. During the first postnatal days, the density of somatostatin receptors decreased dramatically in the retina. In both the optic pathways and dorsal lateral geniculate nucleus, somatostatin receptors gradually disappeared, and the levels of somatostatin receptors were almost undetectable at postnatal day 21 (P21). Conversely, the density of somatostatin receptors remained stable in the ventral lateral geniculate nucleus during the early postnatal life (P0-P7). The timing of expression and the localization of somatostatin receptors in the developing visual system suggest that the immature ganglion cells are responsible for the expression of these evanescent somatostatin receptors. After eye opening, the distribution patterns of somatostatin receptors in the retina and the lateral geniculate nucleus were similar to those observed in adults. In particular, from P14 onwards, somatostatin receptors were concentrated in the inner plexiform layer and, to a lesser extent, in the ganglion cell and photoreceptor layers. In the ventral lateral geniculate nucleus, a heterogeneous distribution of somatostatin receptors was noted, the highest densities being found in the intergeniculate leaflet and the medial zone limiting the parvo-magnocellular interface. The distribution of somatostatin receptors in the retina and the ventral lateral geniculate nucleus after the second postnatal week, together with the presence of somatostatin-like immunoreactive elements in these structures, provide support for the involvement of somatostatin as a neurotransmitter or neuromodulator in the visual system of the adult rat. Conversely, the transient expression of somatostatin receptors observed before maturation and complete organization of the optic pathways suggests that somatostatin plays a trophic role during development of the visual system.

Chronic Mild Stress during Gestation Worsens Neonatal Brain Lesions in Mice

Cerebral palsy remains a public health priority. Recognition of factors of susceptibility to perinatal brain lesions is key for the prevention of cerebral palsy. In most cases, the pathophysiology of these lesions is thought to involve prior exposure to predisposing factors that make the developing brain more vulnerable to perinatal events. The present study tested the hypothesis that exposure to chronic minimal stress throughout gestation would sensitize the offspring to neonatal excitotoxic brain lesions, which mimic lesions observed in cerebral palsy. Pregnant mice were exposed to chronic, ultramild stress, applied throughout gestation. Neonatal brain lesions were induced by intracerebral injection of glutamate analogs. Excitotoxic lesions were significantly worsened in pups exposed to gestational stress. Stress induced a significant rise of circulating corticosterone levels both in pregnant mothers and in newborn pups. The deleterious effects of stress on excitotoxicity were totally suppressed in mice with reduced levels of glucocorticoid receptors. Stress induced a significant increase of neopallial NMDA binding sites in the offspring. At adulthood, animals exposed to stress and neonatal excitotoxic challenge showed a significant impairment in the Morris water maze test when compared with animals exposed to the excitotoxic challenge but not the gestational stress. These findings suggest that stress during gestation, which may mimic low-level stress in human pregnancy, could be a novel risk factor for cerebral palsy.

Autoradiographic study of somatostatin receptors in the rat hypothalamus: validation of a GTP-induced desaturation procedure.

The radiolabelled somatostatin analogs [125I-Tyr0,DTrp8]S14 and [Leu8,DTrp22,125I-Tyr25]S28 were used as radioligands to study the distribution of somatostatin receptors in the rat hypothalamus. Previous studies have detected very few somatostatin-binding sites in the hypothalamus using in vitro autoradiography. Since the lack of autoradiographic labelling has been ascribed to the occupancy of the receptors by endogenous ligands, we have developed a method using guanosine triphosphate (GTP) pretreatment to unmask somatostatin receptors. Preincubation of brain slices with 10(-6) M GTP, by desaturating the occupied receptors, made it possible to reveal the wide distribution of somatostatin-binding sites in the rat hypothalamus. Somatostatin-14 binding site populations were observed in numerous hypothalamic areas including the preoptic area where the receptors likely account for self-inhibition of somatostatin release, the supraoptic nucleus, the bed nucleus of the stria terminalis, the anterior hypothalamic nucleus, the perifornical area, the zona incerta and a mediolateral area located laterally to the ventromedian and dorsomedian nuclei and limited laterally by the mammillo-thalamic tract, the fornix and the optic tract. All structures showing S14-binding sites were labelled by the S28 radioligand. In addition, the paraventricular parvocellular nucleus contained exclusively S28-binding sites, which could be involved in the inhibitory effect of S28 on CRF-mediated endocrine and sympathetic responses. A moderate density of S28-preferring sites was also detected in the periventricular nucleus. In summary, GTP preincubation of brain slices appeared to be a useful technique to reveal multiple somatostatin receptors populations in the brain. The widespread distribution of somatostatin receptors in the hypothalamus is in total agreement with the variety of physiological effects of the somatostatin peptide family.

Neuronal migration disorder in Zellweger mice is secondary to glutamate receptor dysfunction

Disorders of neuronal migration in cerebral cortex are associated with neurological impairments, including mental retardation and epilepsy. Their causes and pathophysiology remain largely unknown, however. In patients with Zellweger disease, a lethal panperoxisomal disorder, and in mice lacking the Pxr1 import receptor for peroxisomal matrix proteins, the absence of peroxisomes leads to abnormal neuronal migration. Analysis of Pxr1−/− mice revealed that the migration defect was caused by altered N‐methyl‐D‐aspartate (NMDA) glutamate receptor–mediated calcium mobilization. This NMDA receptor dysfunction was linked to a deficit in platelet‐activating factor, a phenomenon related to peroxisome impairment. These findings confirm NMDA receptor involvement in neuronal migration and suggest a link between peroxisome metabolism and NMDA receptor efficacy. Ann Neurol 2000;48:336–343

Pseudomonas fluorescens as a potential pathogen: adherence to nerve cells

In order to determine the infectious potential of the psychrotrophic bacterium Pseudomonas fluorescens, a species closely related to the opportunistic pathogen P. aeruginosa, we investigated the binding activity of this bacterium on primary cultures of rat neonate cortical neurons and glial cells, adrenal paraneurons and NG108-15 neuroblastoma cells. Incubated at concentrations of 10(6) and 10(8) CFU/mL, P. fluorescens MF37 exhibited a high binding activity on neurons in the same range as that of P. aeruginosa PAO1. A significant, but lower, adherence of P. fluorescens was also detected on glial cells and adrenal paraneurons. In contrast, when P. fluorescens MF37 or P. aeruginosa PAO1 were incubated with neuroblastoma cells, no binding was observed. In neurons, the association of P. fluorescens with the plasma membrane occurred both on neurites and cell body. Leakage of the cytoplasmic content was frequently noted. Studies performed using the fluorescent probe Hoechst 33258 revealed that in 10% of neurons, P. fluorescens induced the appearance of densely stained clusters of DNA that was typical of an early step of apoptosis. In glial cells exposed to P. fluorescens, marked changes in the morphology of the nucleus, including fragmentation into lobular structures and aggregation of DNA, were also reminiscent of the existence of a possible apoptotic mechanism. Taken together, these results reveal that P. fluorescens can bind to nerve cells and affect their physiology and, in agreement with recent clinical observations, suggest that P. fluorescens could behave as a pathogen.