Jean-Michel Rigo

Plasticity of Cultured Mesenchymal Stem Cells: Switch from Nestin‐Positive to Excitable Neuron‐Like Phenotype

Bone marrow mesenchymal stem cells (MSCs) can differentiate into several types of mesenchymal cells, including osteocytes, chondrocytes, and adipocytes, but, under appropriate experimental conditions, can also differentiate into nonmesenchymal cells—for instance, neural cells. These observations have raised interest in the possible use of MSCs in cell therapy strategies for various neurological disorders. In the study reported here, we addressed the question of in vitro differentiation of MSCs into functional neurons. First, we demonstrate that when they are co‐cultured with cerebellar granule neurons, adult MSCs can express neuronal markers. Two factors are needed for the emergence of neuronal differentiation of the MSCs: the first one is nestin expression by MSCs (nestin is a marker for the responsive character of MSCs to extrinsic signals), and the second one is a direct cell–cell interaction between neural cells and MSCs that allows the integration of these extrinsic signals. Three different approaches suggest that neural phenotypes arise from MSCs by a differentiation rather than a cell fusion process, although this last phenomenon can also coexist. The expression of several genes—including sox, pax, notch, delta, frizzled, and erbB—was analyzed by quantitative reverse transcription polymerase chain reaction (RT‐PCR) in order to further characterize the nestin‐positive phenotype compared to the nestin‐negative one. An overexpression of sox2, sox10, pax6, fzd, erbB2, and erbB4 is found in nestin‐positive MSCs. Finally, electrophysiological analyses demonstrate that MSC‐derived neuron‐like cells can fire single‐action potentials and respond to several neurotransmitters such as GABA, glycine, and glutamate. We conclude that nestin‐positive MSCs can differentiate in vitro into excitable neuron‐like cells.

Neurotransmitters as Early Signals for Central Nervous System Development

Abstract. During brain ontogenesis, the temporal and spatial generation of the different types of neuronal and glial cells from precursors occurs as a sequence of successive progenitor stages whose proliferation, survival and cell-fate choice are controlled by environmental and cellular regulatory molecules. Neurotransmitters belong to the chemical microenvironment of neural cells, even at the earliest stages of brain development. It is now established that specific neurotransmitter receptors are present on progenitor cells of the developing central nervous system and could play, during neural development, a role that has remained unsuspected until recently. The present review focuses on the occurrence of neurotransmitters and their corresponding ligand-gated ion channel receptors in immature cells, including neural stem cells of specific embryonic and neonatal brain regions. We summarize in vitro and in vivo data arguing that neurotransmitters could regulate morphogenetic events such as proliferation, growth, migration, differentiation and survival of neural precursor cells. The understanding of neurotransmitter function during early neural maturation could lead to the development of pharmacological tools aimed at improving adult brain repair strategies.

The anti-epileptic drug levetiracetam reverses the inhibition by negative allosteric modulators of neuronal GABA- and glycine-gated currents

In this study in vitro and in vivo approaches were combined in order to investigate if the anti‐epileptic mechanism(s) of action of levetiracetam (LEV; Keppra®) may involve modulation of inhibitory neurotransmission. GABA‐ and glycine‐gated currents were studied in vitro using whole‐cell patch‐clamp techniques applied on cultured cerebellar granule, hippocampal and spinal neurons. Protection against clonic convulsions was assessed in vivo in sound‐susceptible mice. The effect of LEV was compared with reference anti‐epileptic drugs (AEDs): carbamazepine, phenytoin, valproate, clonazepam, phenobarbital and ethosuximide. LEV contrasted the reference AEDs by an absence of any direct effect on glycine‐gated currents. At high concentrations, beyond therapeutic relevance, it induced a small reduction in the peak amplitude and a prolongation of the decay phase of GABA‐gated currents. A similar action on GABA‐elicited currents was observed with the reference AEDs, except ethosuximide. These minor direct effects contrasted with a potent ability of LEV (EC50=1 – 10 μM) to reverse the inhibitory effects of the negative allosteric modulators zinc and β‐carbolines on both GABAA and glycine receptor‐mediated responses. Clonazepam, phenobarbital and valproate showed a similar ability to reverse the inhibition of β‐carbolines on GABA‐gated currents. Blockade of zinc inhibition of GABA responses was observed with clonazepam and ethosuximide. Phenytoin was the only AED together with LEV that inhibited the antagonism of zinc on glycine‐gated currents and only clonazepam and phenobarbital inhibited the action of DMCM. LEV (17 mg kg−1) produced a potent suppression of sound‐induced clonic convulsions in mice. This protective effect was significantly abolished by co‐administration of the β‐carboline FG 7142, from a dose of 5 mg kg−1. In contrast, the benzodiazepine receptor antagonist flumazenil (up to 10 mg kg−1) was without any effect on the protection afforded by LEV. The results of the present study suggest that a novel ability to oppose the action of negative modulators on the two main inhibitory ionotropic receptors may be of relevance for the anti‐epileptic mechanism(s) of action of LEV.

Neuronotrophic Effect of Developing Otic Vesicle on Cochleo-Vestibular Neurons: Evidence for Nerve Growth Factor Involvement

In the developing inner ear, the existence of a neuronal death and of a peripheral target-derived trophic effect on cochleovestibular neurons has been documented. Using cultures of rat cochleovestibular neurons, we show that the E12 otic vesicle releases a factor promoting the survival and the neuritogenesis of these neurons, and that this effect is mimicked by NGF. The effect of the optic vesicle conditioned medium (OVCM) on cochleovestibular neurons is suppressed by anti-NGF antibodies. OVCM is neuronotrophic for NGF-sensitive sympathetic neurons, an effect that is also suppressed by anti-NGF antibodies, further demonstrating the presence of biologically active nerve growth factor.

Complex invasion pattern of the cerebral cortex bymicroglial cells during development of the mouse embryo.

Microglia are the immune cells of the central nervous system. They are suspected to play important roles in adult synaptogenesis and in the development of the neuronal network. Microglial cells originate from progenitors in the yolk sac. Although it was suggested that they invade the cortex at early developmental stages in the embryo, their invasion pattern remains largely unknown. To address this issue we analyzed the pattern of cortical invasion by microglial cells in mouse embryos at the onset of neuronal cell migration using in vivo immunohistochemistry and ex vivo time‐lapse analysis of microglial cells. Microglial cells begin to invade the cortex at 11.5 days of embryonic age (E11.5). They first accumulate at the pial surface and within the lateral ventricles, after which they spread throughout the cortical wall, avoiding the cortical plate region in later embryonic ages. The invasion of the cortical parenchyma occurs in different phases. First, there is a gradual increase of microglial cells between E10.5 and E14.5. From E14.5 to E15.5 there is a rapid phase with a massive increase in microglia, followed by a slow phase again from E15.5 until E17.5. At early stages, many peripheral microglia are actively proliferating before entering the parenchyma. Remarkably, activated microglia accumulate in the choroid plexus primordium, where they are in the proximity of dying cells. Time‐lapse analysis shows that embryonic microglia are highly dynamic cells.

Substance P protects spiral ganglion neurons from apoptosis via PKC-Ca2+-MAPK/ERK pathways

In the current study, we have investigated the ability of substance P (SP) to protect 3‐day‐old (P3) rat spiral ganglion neurons (SGNs) from trophic factor deprivation (TFD)‐induced cell death. The presence of SP high affinity neurokinin‐1 receptor (NK1) transcripts was detected in the spiral ganglion and the NK1 protein localized to SGNs both ex vivo and in vitro. Treatment with SP increased cytoplasmic Ca2+ in SGNs, further arguing for the presence of functional NK1 on these neurons. Both SP and the agonist [Sar9,Met(O2)11]‐SP significantly decreased SGN cell death induced by TFD, with no effect on neurite outgrowth. The survival promoting effect of SP was blocked by the NK1 antagonist, WIN51708. Both pan‐caspase inhibitor BOC‐D‐FMK and SP treatments markedly reduced activation of caspases and DNA fragmentation in trophic factor deprived‐neurons. The neuroprotective action of SP was antagonised by specific inhibitors of second messengers, including 1.2‐bis‐(O‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA‐AM) to chelate cytosolic Ca2+, the protein kinase C (PKC) inhibitors bisindolylmaleimide I, Gö6976 and LY333531 and the MAPK/ERK inhibitor U0126. In contrast, nifedipine, a specific inhibitor of l‐type Ca2+ channel, and LY294002, a phosphatidylinositol‐3‐OH kinase (PI3K) inhibitor, had no effect on SP trophic support of SGNs. Moreover, activation of endogenous PKC by 4β‐phorbol 12‐myristate 13‐acetate (PMA) also reduced the loss of trophic factor‐deprived SGNs. Thus, NK1 expressed by SGNs transmit a survival‐promoting regulatory signal during TFD‐induced SGN cell death via pathways involving PKC activation, Ca2+ signalling and MAPK/ERK activation, which can be accounted for by an inhibition of caspase activation.

Glycine Release from Radial Cells Modulates the Spontaneous Activity and Its Propagation during Early Spinal Cord Development

Rhythmic electrical activity is a hallmark of the developing embryonic CNS and is required for proper development in addition to genetic programs. Neurotransmitter release contributes to the genesis of this activity. In the mouse spinal cord, this rhythmic activity occurs after embryonic day 11.5 (E11.5) as waves spreading along the entire cord. At E12.5, blocking glycine receptors alters the propagation of the rhythmic activity, but the cellular source of the glycine receptor agonist, the release mechanisms, and its function remain obscure. At this early stage, the presence of synaptic activity even remains unexplored. Using isolated embryonic spinal cord preparations and whole-cell patch-clamp recordings of identified motoneurons, we find that the first synaptic activity develops at E12.5 and is mainly GABAergic. Using a multiple approach including direct measurement of neurotransmitter release (i.e., outside-out sniffer technique), we also show that, between E12.5 and E14.5, the main source of glycine in the embryonic spinal cord is radial cell progenitors, also known to be involved in neuronal migration. We then demonstrate that radial cells can release glycine during synaptogenesis. This spontaneous non-neuronal glycine release can also be evoked by mechanical stimuli and occurs through volume-sensitive chloride channels. Finally, we find that basal glycine release upregulates the propagating spontaneous rhythmic activity by depolarizing immature neurons and by increasing membrane potential fluctuations. Our data raise the question of a new role of radial cells as secretory cells involved in the modulation of the spontaneous electrical activity of embryonic neuronal networks.

Pattern of invasion of the embryonic mouse spinal cord by microglial cells at the time of the onset of functional neuronal networks

Microglial cells invade the central nervous system during embryonic development, but their developmental functional roles in vivo remain largely unknown. Accordingly, their invasion pattern during early embryonic development is still poorly understood. To address this issue, we analyzed the initial developmental pattern of microglial cell invasion in the spinal cord of CX3CR1‐eGFP mouse embryos using immunohistochemistry. Microglial cells began to invade the mouse embryonic spinal cord at a developmental period corresponding to the onset of spontaneous electrical activity and of synaptogenesis. Microglial cells reached the spinal cord through the peripheral vasculature and began to invade the parenchyma at 11.5 days of embryonic age (E11.5). Remarkably, at E12.5, activated microglial cells aggregated in the dorsolateral region close to terminals of dying dorsal root ganglia neurons. At E13.5, microglial cells in the ventral marginal zone interacted with radial glial cells, whereas ramified microglial cells within the parenchyma interacted with growing capillaries. At this age, activated microglial cells (Mac‐2 staining) also accumulated within the lateral motor columns at the onset of the developmental cell death of motoneurons. This cell aggregation was still observed at E14.5, but microglial cells no longer expressed Mac‐2. At E15.5, microglial cells were randomly distributed within the parenchyma. Our results provide the essential basis for further studies on the role of microglial cells in the early development of spinal cord neuronal networks in vivo.

Functional glycine receptors are expressed by postnatal nestin-positive neural stem/progenitor cells.

Multipotent neural stem and progenitor cells (NS/PCs) are well‐established cell subpopulations occurring in the developing, and also in the mature mammalian nervous systems. Trophic and transcription factors are currently the main signals known to influence the development and the commitment of NS/PCs and their progeny. However, recent studies suggest that neurotransmitters could also contribute to neural development. In that respect, rodent‐cultured embryonic NS/PCs have been reported to express functional neurotransmitter receptors. No similar investigation has, however, been made in postnatal and/or in adult rodent brain stem cells. In this study, using RT‐PCR and immunocytochemical methods, we show that α1‐, α2‐ and β‐subunit mRNAs and α‐subunit proteins of the glycine ionotropic receptor are expressed by 80.5 ± 0.9% of postnatal rat striatum‐derived, nestin‐positive cells within cultured neurospheres. Whole‐cell patch‐clamp experiments further demonstrated that glycine triggers in 33.5% of these cells currents that can be reversibly blocked by strychnine and picrotoxin. This demonstrates that NS/PCs express functional glycine receptors, the consequence(s) of their activation remaining unknown.

Glycine triggers an intracellular calcium influx in oligodendrocyte progenitor cells which is mediated by the activation of both the ionotropic glycine receptor and Na+‐dependent transporters

Using fluo‐3 calcium imaging, we demonstrate that glycine induces an increase in intracellular calcium concentration ([Ca2+]i) in cortical oligodendrocyte progenitor (OP) cells. This effect results from a calcium entry through voltage‐gated calcium channels (VGCC), as it is observed only in OP cells expressing such channels, and it is abolished either by removal of calcium from the extracellular medium or by application of an l‐type VGCC blocker. Glycine‐triggered Ca2+ influx in OP cells actually results from an initial depolarization that is the consequence of the activation of both the ionotropic glycine receptor (GlyR) and Na+‐dependent transporters, most probably the glycine transporters 1 (GLYT1) and/or 2 (GLYT2) which are colocalized in these cells. Through this GlyR‐ and transporter‐mediated effect on OP intrcellular calcium concentration [Ca2+]i, glycine released by neurons may, as well as other neurotransmitters, serve as a signal between neurons and OP during development.