Eduardo Gascon

Clinically Relevant Concentrations of Propofol but Not Midazolam Alter In Vitro Dendritic Development of Isolated γ-Aminobutyric Acid-positive Interneurons

Background: Recent laboratory studies showed that exposure to supraclinical concentrations of propofol can induce cell death of immature neurons. However, no data are available regarding the effects of clinically relevant concentrations of this agent on neuronal development. The authors addressed this issue by evaluating the effect of propofol on dendritic growth and arbor expansion of developing &ggr;-aminobutyric acid–positive (GABAergic) interneurons. Methods: Immature neuroblasts were isolated from the newborn rat subventricular zone and differentiated into GABAergic interneurons in culture. In addition to cell death, the effects of increasing concentrations and durations of propofol exposure on neuronal dendritic development were evaluated using the following morphologic parameters: total dendritic length, primary dendrites, branching point, and Scholl analysis. Results: The authors demonstrate that propofol induced cell death of GABAergic neurons at concentrations of 50 &mgr;g/ml or greater. As little as 1 &mgr;g/ml propofol significantly altered several aspects of dendritic development, and as little as 4 h of exposure to this agent resulted in a persistent decrease in dendritic growth. In contrast, application of midazolam did not affect neuronal development. Conclusion: Short-term exposure of immature developing GABAergic neurons to clinically relevant concentrations of propofol can induce long-term changes in dendritic arbor development. These results suggest that propofol anesthesia during central nervous system development could interfere with the molecular mechanisms driving the differentiation of GABAergic neurons and thus could potentially lead to impairment of neural networks.

PSA-NCAM in postnatally generated immature neurons of the olfactory bulb: a crucial role in regulating p75 expression and cell survival

In the mammalian brain, ongoing neurogenesis via the rostral migratory stream (RMS) maintains neuronal replacement in the olfactory bulb throughout life. Mechanisms that regulate the final number of new neurons in this system include proliferation, migration and apoptosis. Here we show that the polysialylated isoforms of the neural cell adhesion molecule (PSA-NCAM) act as a pro-survival molecule in immature newborn neurons. Confocal microscopic analysis revealed a threefold increase in TUNEL-positive cells in the subventricular zone (SVZ) and the RMS of transgenic animals lacking the gene encoding NCAM (NCAM-/-), as compared with wild types. The enhanced apoptotic cell death occurred specifically in the population of mCD24-positive newborn neurons, but not in GFAP-positive astrocytes. Using in vitro cultures of purified SVZ-derived neurons, we demonstrate that the loss or inactivation of PSA on NCAM, as well as the deletion of NCAM, lead to reduced survival in response to neurotrophins including BDNF and NGF. These changes in cell survival are accompanied by an upregulation of p75 neurotrophin receptor expression in vitro as well as in vivo. Furthermore, the negative effects of PSA-NCAM inactivation on cell survival could be prevented by the pharmacological blockade of the p75 receptor-signaling pathway. We propose that PSA-NCAM may promote survival by controlling the expression of the p75 receptor in developing neurons.

Adverse effects of methylene blue on the central nervous system

Background:An increasing number of clinical observations suggest adverse neurologic outcome after methylene blue (MB) infusion in the setting of parathyroid surgery. Hence, the aim of the current study was to investigate the potentially neurotoxic effects of MB using a combination of in vivo and in vitro experimental approaches. Methods:Isoflurane-anesthetized adult rats were used to evaluate the impact of a single bolus intravascular administration of MB on systemic hemodynamic responses and on the minimum alveolar concentration (MAC) of isoflurane using the tail clamp test. In vivo, MB-induced cell death was evaluated 24 h after MB administration using Fluoro-Jade B staining and activated caspase-3 immunohistochemistry. In vitro, neurotoxic effects of MB were examined in hippocampal slice cultures by measuring excitatory field potentials as well as propidium iodide incorporation after MB exposure. The impact of MB on dendritic arbor was evaluated in differentiated single cell neuronal cultures. Results:Bolus injections of MB significantly reduced isoflurane MAC and initiated widespread neuronal apoptosis. Electrophysiologic recordings in hippocampal slices revealed a rapid suppression of evoked excitatory field potentials by MB, and this was associated with a dose-dependent effect of this drug on cell death. Dose–response experiments in single cell neuronal cultures revealed that a 2-h-long exposure to MB at non–cell-death-inducing concentrations could still induce significant retraction of dendritic arbor. Conclusions:These results suggest that MB exerts neurotoxic effects on the central nervous system and raise questions regarding the safety of using this drug at high doses during parathyroid gland surgery.

GABA Regulates Dendritic Growth by Stabilizing Lamellipodia in Newly Generated Interneurons of the Olfactory Bulb

The initial formation and growth of dendrites is a critical step leading to the integration of newly generated neurons into postnatal functional networks. However, the cellular mechanisms and extracellular signals regulating this process remain mostly unknown. By directly observing newborn neurons derived from the subventricular zone in culture as well as in olfactory bulb slices, we show that ambient GABA acting through GABAA receptors is essential for the temporal stability of lamellipodial protrusions in dendritic growth cones but did not interfere with filopodia dynamics. Furthermore, we provide direct evidence that ambient GABA is required for the proper initiation and elongation of dendrites by promoting the rapid stabilization of new dendritic segments after their extension. The effects of GABA on the initial formation of dendrites depend on depolarization and Ca2+ influx and are associated with a higher stability of microtubules. Together, our results indicate that ambient GABA is a key regulator of dendritic initiation in postnatally generated olfactory interneurons and offer a mechanism by which this neurotransmitter drives early dendritic growth.

Sequential activation of p75 and TrkB is involved in dendritic development of subventricular zone-derived neuronal progenitors in vitro

Dendritic arbor development of subventricular zone‐derived interneurons is a critical step in their integration into functional circuits of the postnatal olfactory bulb. However, the mechanism and molecular control of this process remain unknown. In this study, we have developed a culture model where dendritic development of purified subventricular zone cells proceeds under serum‐free conditions in the absence of added growth factors and non‐neural cells. We demonstrate that the large majority of these cells in culture express GABA and elaborate dendritic arbors with spine‐like protrusions but they do not possess axons. These neurons expressed receptors for neurotrophins including p75, TrkB and TrkC but not TrkA. Application of exogenous neurotrophins, including brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT3) and nerve growth factor (NGF), to cultures stimulated dendritic growth and led to more complex dendritic arbors during the initial 3 days in culture. Our results suggest that these effects are independent of Trk receptors and mediated by the p75/ceramide signaling pathway. We also show that brain‐derived neurotrophic factor is the only neurotrophin that is able to influence late‐phase dendritic development via TrkB receptor activation. These results suggest that dendritic arbor development of subventricular zone‐derived cells may be regulated by neurotrophins through the activation of p75 and the TrkB receptor signaling pathways in a sequentially defined temporal pattern.

Role of matrix metalloproteinases in apoptosis after transient focal cerebral ischemia in rats and mice

The involvement of matrix metalloproteinases (MMPs) in cerebral ischemia‐induced apoptosis was investigated in a model of transient focal cerebral ischemia in rats treated intracerebroventricularly (i.c.v.) with 4‐((3‐(4‐phenoxylphenoxy)propylsulfonyl)methyl)‐tetrahydropyran‐4‐carboxylic acid N‐hydroxy amide, a broad spectrum non‐peptidic hydroxamic acid MMP inhibitor, and in MMP‐9‐deficient mice. Our results showed that MMP inhibition reduced DNA fragmentation by 51% (P < 0.001) and cerebral infarct by 60% (P < 0.05) after ischemia. This protection was concomitant with a 29% reduction of cytochrome c release into the cytosol (P < 0.005) and a 54% reduction of calpain‐related α‐spectrin degradation (P < 0.05), as well as with an 84% increase in the immunoreactive signal of the native form of poly(ADP) ribose polymerase (P < 0.01). By contrast, specific targeting of the mmp9 gene in mice did reduce cerebral damage by 34% (P < 0.05) but did not modify the apoptotic response after cerebral ischemia. However, i.c.v. injection of MMP‐9‐deficient mice with the same broad‐spectrum inhibitor used in rats significantly reduced DNA degradation by 32% (P < 0.05) and contributed even further to the protection of the ischemic brain. Together, our pharmacological and genetic results indicate that MMPs other than MMP‐9 are actively involved in cerebral ischemia‐induced apoptosis.

The Role of PSA-NCAM in Adult Neurogenesis

Recruitment of new neurons to existing circuits is a fascinating form of adult plasticity. In the mammalian brain, this process occurs in two discrete regions, the subventricular zone of the lateral ventricule (SVZ) and the subgranular zone of dentate gyrus (SGZ) in the hippocampus. Several recent studies suggest that isoforms of the neuronal cell adhesion molecule (NCAM) carrying the linear homopolymer of alpha 2,8-linked sialic acid (polysialic acid, PSA), play a role in regulating several steps of adult neurogenesis including migration, survival and process outgrowth of newly generated neurons. Here, we will review recent evidence on how PSA-NCAM might regulate the biological properties of new neurons in sites of adult neurogenesis.

The Polysialylated Neural Cell Adhesion Molecule Promotes Neurogenesis in vitro

A characteristic feature of neurogenic sites in the postnatal brain is the expression of the polysialylated forms of the neural cell adhesion molecule (PSA-NCAM). To investigate the role of PSA-NCAM in generation of neuronal populations, we developed an in vitro model where neurogenesis occurs in primary cortical cultures following serum withdrawal. We show that removal or inactivation of the PSA tail of NCAM in these cultures leads to a significant decrease in the number of newly generated neurons. Similarly, cultures prepared from NCAM knock-out mice exhibit a significantly reduced neurogenesis. Pulse-chase experiments using the proliferation marker BrdU reveal that the lack of PSA does not affect the mitotic rate of neural progenitors but rather, it reduces the early survival of newly generated neurons. These results suggest that, in addition to its role in the migration of neuronal progenitors, PSA-NCAM is required for the adequate survival of these cells.

Removal of PSA from NCAM affects the survival of magnocellular vasopressin‐ and oxytocin‐producing neurons in organotypic cultures of the paraventricular nucleus

The expression of the polysialic acid neural cell adhesion molecule (PSA‐NCAM) in the hypothalamo‐neurohypophyseal system has been correlated with morphofunctional plasticity. In this study, we investigated the role of PSA‐NCAM in the survival of oxytocin (OT)‐ and vasopressin (VP)‐producing magnocellular cells of this system. We used a recently developed organotypic slice culture model of the rat hypothalamic paraventricular nucleus (PVN) in which ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) are potent survival factors for magnocellular neurons. We demonstrate by means of confocal microscopy that cultured magnocellular VP and OT neurons express strong immunoreactivity for PSA‐NCAM. Removal of PSA from NCAM by the enzyme Endo N leads to a significant loss of both VP and OT neurons in the presence of low concentrations of CNTF. Endo N treatment did not change cell survival in the presence of LIF. These results suggest that, in addition to its role in neuro‐glial plasticity, PSA‐NCAM might also influence the trophic factor responsiveness of hypothalamic VP and OT neurosecretory cells.

Hepatocyte Growth Factor-Met Signaling Is Required for Runx1 Extinction and Peptidergic Differentiation in Primary Nociceptive Neurons

Nociceptors in peripheral ganglia display a remarkable functional heterogeneity. They can be divided into the following two major classes: peptidergic and nonpeptidergic neurons. Although RUNX1 has been shown to play a pivotal role in the specification of nonpeptidergic neurons, the mechanisms driving peptidergic differentiation remain elusive. Here, we show that hepatocyte growth factor (HGF)-Met signaling acts synergistically with nerve growth factor-tyrosine kinase receptor A to promote peptidergic identity in a subset of prospective nociceptors. We provide in vivo evidence that a population of peptidergic neurons, derived from the RUNX1 lineage, require Met activity for the proper extinction of Runx1 and optimal activation of CGRP (calcitonin gene-related peptide). Moreover, we show that RUNX1 in turn represses Met expression in nonpeptidergic neurons, revealing a bidirectional cross talk between Met and RUNX1. Together, our novel findings support a model in which peptidergic versus nonpeptidergic specification depends on a balance between HGF-Met signaling and Runx1 extinction/maintenance.