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The cochleo-vestibular ganglion contains neural crest-derived glial cells and sensory neurons that are derived from the neurogenic otic placode. Little is known about the molecular mechanisms that regulate the tightly orchestrated development of this structure. Here, we report that Sox10, a high-mobility group DNA-binding domain transcription factor that is required for the proper development of neural crest cell derivatives, is specifically expressed in post-migratory neural crest cells in the cochleo-vestibular ganglion. Using Sox10-deficient mice, we demonstrate that this transcription factor is essential for the survival, but not the generation, of the post-migratory neural crest cells within the inner ear. In the absence of these neural crest-derived cells, we have investigated the survival of the otocyst-derived auditory neurons. Surprisingly, auditory neuron differentiation, sensory target innervation and survival are conserved despite the absence of glial cells. Moreover, brain-derived neurotrophic factor expression is increased in the hair cells of Sox10-deficient mice, a compensatory mechanism that may prevent spiral ganglion neuronal cell death. Taken together, these data suggest that in the absence of neural crest-derived glial cells, an increase trophic support from hair cells promotes the survival of spiral ganglion neurons in Sox10 mutant mice.

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, Go6976 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.

In the embryonic day 19 organs of Corti, we showed that roscovitine, a chemical inhibitor of cyclin-dependent kinases (CDKs), significantly increased the number of hair cells (HCs) and corresponding supporting cells (SCs) by triggering differentiation of precursor cells without interacting with cell proliferation. The effect of roscovitine was mimicked by other CDK1, 2, 5, and 7 inhibitors but not by CDK4/6 and mitogen-activated protein kinase pathway antagonists. Immunohistochemical analysis indicated that roscovitine-specific intracellular targets, CDK1, 2, 5, and 7, were expressed in the organ of Corti and especially in Hensen's cells. Affinity chromatography studies showed a tight correlation between the protein levels of CDK1/2 and 5 and the rate of roscovitine-induced supernumerary cells in the organ of Corti. In addition, we demonstrated that basal CDK activity was higher and more roscovitine-sensitive at developmental stages that are selectively permissive for the emergence of supernumerary cells. These results suggest that CDKs are involved in the normal development of the organ of Corti and that, at least in E19 embryos, inhibition of CDKs is sufficient to trigger the differentiation of HCs and corresponding SCs, presumably from the Hensen's cell progenitors and/or from progenitors located in the greater epithelial ridge area.

1. 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. 2. 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. 3. 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. 4. These minor direct effects contrasted with a potent ability of LEV (EC(50)=1 - 10 microM) to reverse the inhibitory effects of the negative allosteric modulators zinc and beta-carbolines on both GABA(A) and glycine receptor-mediated responses. 5. Clonazepam, phenobarbital and valproate showed a similar ability to reverse the inhibition of beta-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. 6. 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 beta-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. 7. 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.

Systemic administration of nitroglycerin, a nitric oxide donor, triggers in migraine patients a delayed attack of unknown mechanism. After puberty migraine is more prevalent in women. Attacks can be triggered by abrupt falls in plasma estrogen levels, which accounts in part for sexual dimorphism, but lacks an established neurobiological explanation. We studied the effect of nitroglycerin on the innervated area of calcitonin gene-related peptide (CGRP) and serotonin-immunoreactive afferents to the superficial laminae of the spinal portion of trigeminal nucleus caudalis, and its modulation by estrogen. In male rats, nitroglycerin produced after 4 h a significant decrease of the area innervated by CGRP-immunoreactive afferents and an increase of that covered by serotonin-immunoreactive fibres. These effects were not observed in the superficial laminae of thoracic dorsal horns. The effect of nitroglycerin was similar in ovariectomized females. In estradiol-treated ovariectomized females the area in the spinal portion of trigeminal nucleus caudalis laminae I-II covered by CGRP-immunoreactive fibres was lower and that of serotonin-immunoreactive fibres was higher than in males and for both transmitters not significantly changed after nitroglycerin. The bouton size of CGRP profiles was smaller in estradiol-treated ovariectomized females, whereas after nitroglycerin it decreased significantly but only in males and ovariectomized females. Nitroglycerin, i.e. nitric oxide, is thus able to differentially influence afferent fibres in the superficial laminae of rat spinal trigeminal nucleus caudalis. Estradiol modulates the basal expression of these transmitters and blocks the nitroglycerin effect. These data may contribute to understanding the mechanisms by which estrogens influence migraine severity and the triggering of attacks by nitric oxide.

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 alpha(1)-, alpha(2)- and beta-subunit mRNAs and alpha-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.

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.

Using the whole-cell patch-clamp technique, we demonstrate glycine-induced currents in oligosphere-derived oligodendrocyte progenitors cultured from newborn rats. Similar inward currents are also triggered by beta-alanine and taurine, two established glycine receptor agonists. In our recording conditions, glycine-gated currents in oligodendrocyte progenitors reverse about 0 mV and are reversibly inhibited by the glycine competitive antagonist strychnine, the Cl- channel blocker picrotoxinin and the non-competitive antagonist cyanotriphenylborate. The oligodendrocyte progenitors glycine receptor (GlyR) differs from the corresponding neuronal receptor: [3H]strychnine binding data and the strychnine inhibition curve of glycine-induced currents in oligodendrocyte progenitor cultures suggest the existence of two strychnine binding sites on the oligodendroglial GlyR. Using total RNA isolated from oligodendrocyte progenitors cultures, reverse transcription-polymerase chain reaction analysis of glycine receptor subunit expression shows the presence of alpha2 and beta subunits and immunocytochemical stainings confirm that this GlyR contains an alpha subunit which is not alpha1. The molecular structure of the oligodendroglial GlyR could be either homopentameric alpha2 or heteromeric alpha2beta but in both cases, the sequence of the alpha2 or beta subunits have to be different from the known neuronal sequences in order to explain, respectively, the cyanotriphenylborate (alpha2) and picrotoxinin (beta) sensitivities. This work thus demonstrates that GlyR are expressed by oligodendrocytes obtained not only from spinal cord but also from supraspinal structures. The pharmacological properties and presumably the molecular structure of oligodendroglial GlyR are original. The physiological meaning of the presence of such receptors on developing and mature oligodendrocytes remains unknown.

Macrophages (monocytes/microglia) could play a critical role in central nervous system repair. We have previously found a synchronism between the re- gression of spontaneous axonal regeneration and the deactivation of macrophages 3- 4 wk after a compres- sion-injury of rat spinal cord. To explore whether reactivation of endogenous macrophages might be ben- eficial for spinal cord repair, we have studied the effects of granulocyte-macrophage colony stimulating factor (GM-CSF) in the same paraplegia model and in cell cultures. There was a significant, though transient, improvement of locomotor recovery after a single delayed intraperitoneal injection of 2 g GM-CSF, which also increased significantly the expression of Cr3 and brain-derived neurotrophic factor (BDNF) by mac- rophages at the lesion site. At longer survival delays, axonal regeneration was significantly enhanced in GM- CSF-treated rats. In vitro, BV2 microglial cells ex- pressed higher levels of BDNF in the presence of GM-CSF and neurons cocultured with microglial cells activated by GM-CSF generated more neurites, an effect blocked by a BDNF antibody. These experiments suggest that GM-CSF could be an interesting treatment option for spinal cord injury and that its beneficial effects might be mediated by BDNF.—Bouhy, D., Mal- grange, B., Multon, S., Poirrier, A. L., Scholtes, F., Schoenen, J., Franzen, R. Delayed GM-CSF treatment stimulates axonal regeneration and functional recovery in paraplegic rats via an increased BDNF expression by endogenous macrophages. FASEB J. 20, 000 - 000 (2006)

To determine if transforming growth factor alpha (TGF alpha) pretreatment protects hair cells from aminoglycoside induced injury by modifying their intracellular calcium concentration, we assayed hair cell calcium levels in organ of Corti explants both before and after aminoglycoside (i.e. neomycin, 10(-3) M) exposure either with or without growth factor pretreatment. After TGF alpha (500 ng/ml) treatment, the intracellular calcium level of hair cells showed a five-fold increase as compared to the levels observed in the hair cells of control cultures. After ototoxin exposure, calcium levels in hair cells of control explants showed an increase relative to their baseline levels, while in the presence of growth factors pretreatment, hair cells showed a relative reduction in calcium levels. Pretreatment of organ of Corti explants afforded significant protection of hair cell stereocilia bundle morphology from ototoxic damage when compared to explants exposed to ototoxin alone. This study correlates a rise in hair cell calcium levels with the otoprotection of hair cells by TGF alpha in organ of Corti explants.

SUMMARY The neuronal survival promoting ability of brain derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF), individually and in combination, was evaluated in dissociated cell cultures of postnatal day 5 (P5) rat acoustic ganglia. The neuritogenic promoting effect of these same neurotrophic factors was examined in organotypic explants of P5 rat acoustic ganglia. The results showed that BDNF was maximally effective at a concentration of 10 ng /mL in promoting both survival and neuritogenesis of these postnatal auditory neurons in vifro. CNTF was maximally effective at a concentration of 0.01 ng/mL at promoting both survival and neuritogenesis in the acoustic ganglion cultures. BDNF had its strongest effect on neuronal survival while CNTF was most effective in stimulating neurite outgrowth. These two neurotrophic factors, when added together at their respective maximally effective concentrations, behave in an additive manner for promoting both survival and neu

This article reports the production of a surgical implant meeting several specific requirements such as biocompatibility, biodegradability, macroporosity, and flexibility. Porosity was controlled by an original method consisting of the aggregation of calibrated poly-D,L-lactide microparticles. The size of the interstices between the aggregated microspheres was in a direct relationship to the microsphere diameter. A first approach was based on coating the microspheres with poly(vinyl alcohol) followed by chemically crosslinking the coating layers that were in mutual contact. This method was disregarded because of the acute cytotoxicity of glutaraldehyde used as the crosslinking agent, the absence of macroporosity, and the complete lack of flexibility. A physical technique of aggregation was then tested, which relied on the plasticization of poly-D,L-lactide microspheres with triethylcitrate to the point where microspheres strongly adhered to each other when they were in contact. This method has proved to be straightforward and definitely superior to the chemical approach, particularly with respect to cytotoxicity, control of macroporosity, and flexibility. A polymer support was thus successfully produced which was biodegradable, macroporous (interconnected pores of 10–100 μm in diameter), and flexible. This potential medical device is presently being used for neuronal transplantation in the central nervous system. © 1995 John Wiley & Sons, Inc.

Media conditioned by cultured neonatal cerebral cortex microexplants (CCM) or astrocytes (ACM) contain low molecular weight (< 1,000 Da) substance(s) which inhibits the gamma aminobutyric acid (GABA)-induced inward current recorded in cerebellar granule cells and hippocampal neurons in culture using the whole-cell patch-clamp technique. This effect is specific for CCM and ACM, as medium conditioned by PC12 cells (PC12CM) does not affect the GABA response of these cells. It is also specific for GABA-induced currents because glutamate-induced currents do not change either in amplitude or in shape in the presence of CCM or ACM. The inhibitory effect on the GABA response in cerebellar granule cells of both ACM and CCM could be suppressed by flumazenil, a specific benzodiazepine (BZD) antagonist and could be mimicked by two BZD inverse agonists. These data thus demonstrate the presence of a BZD inverse agonist-like activity in CCM and ACM. This effect of ACM on different neuronal cell types was heterogenous since no detectable effect could be observed on the GABA-induced current in GABA-responsive dorsal root ganglion (DRG) neurons, presumably reflecting a functional heterogeneity of the GABAA receptors present in these different neuronal subsets. By the release of such an endogenous BZD inverse agonist-like activity, glia cells could possibly modulate GABAA receptor-mediated responses.