Tonic GABAA Receptors as Potential Target for the Treatment of Temporal Lobe Epilepsy.

Tonic GABAA receptors are a subpopulation of receptors that generate long-lasting inhibition and thereby control network excitability. In recent years, these receptors have been implicated in various neurological and psychiatric disorders, including Parkinson's disease, schizophrenia, and epilepsy. Their distinct subunit composition and function, compared to phasic GABAA receptors, opens the possibility to specifically modulate network properties. In this review, the role of tonic GABAA receptors in epilepsy and as potential antiepileptic target will be discussed.

Glycine receptors control the generation of projection neurons in the developing cerebral cortex.

The development of the cerebral cortex requires coordinated regulation of proliferation, specification, migration and differentiation of cortical progenitors into functionally integrated neurons. The completion of the neurogenic program requires a dynamic interplay between cell intrinsic regulators and extrinsic cues, such as growth factor and neurotransmitters. We previously demonstrated a role for extrasynaptic glycine receptors (GlyRs) containing the α2 subunit in cerebral cortical neurogenesis, revealing that endogenous GlyR activation promotes interneuron migration in the developing cortical wall. The proliferative compartment of the cortex comprises apical progenitors that give birth to neurons directly or indirectly through the generation of basal progenitors, which serve as amplification step to generate the bulk of cortical neurons. The present work shows that genetic inactivation of Glra2, the gene coding the α2 subunit of GlyRs, disrupts dorsal cortical progenitor homeostasis with an impaired capability of apical progenitors to generate basal progenitors. This defect results in an overall reduction of projection neurons that settle in upper or deep layers of the cerebral cortex. Overall, the depletion of cortical neurons observed in Glra2-knockout embryos leads to moderate microcephaly in newborn Glra2-knockout mice. Taken together, our findings support a contribution of GlyR α2 to early processes in cerebral cortical neurogenesis that are required later for the proper development of cortical circuits.

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.

Frequency-dependent modulation of glycine receptor activation recorded from the zebrafish larvae hindbrain.

In vertebrates, most glycinergic inhibitory neurons discharge phasically at a relatively low frequency. Such a pattern of glycine liberation from presynaptic terminals may affect the kinetics of post-synaptic glycine receptors. To examine this influence, we have analyzed the behavior of glycine receptors in response to repetitive stimulation at frequencies at which consecutive outside-out currents did not superimpose (0.5-4 Hz). Neurotransmitter release was mimicked on outside-out patches from zebrafish hindbrain Mauthner cells using fast flow application techniques. The amplitude of outside-out currents evoked by short (1 ms) repetitive applications of a saturating concentration (3 mM) of glycine remained unchanged for application frequencies1 Hz. Glycine-evoked current simulations using a simple Markov model describing zebrafish glycine receptor kinetic behavior, indicates that this down-regulation of glycine receptor efficacy is due to a progressive accumulation of the receptors in a long lasting desensitization state. Our simulations suggest that this down-regulation can occur even when spontaneous inhibitory currents were generated randomly at a frequency>1 Hz.

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 4 beta-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.

Kinetic properties of the alpha2 homo-oligomeric glycine receptor impairs a proper synaptic functioning.

Ionotropic glycine receptors (GlyRs) are present in the central nervous system well before the establishment of synaptic contacts. Immature nerve cells are known, at least in the spinal cord, to express alpha2 homomeric GlyRs, the properties of which are relatively unknown compared to those of the adult synaptic form of the GlyR (mainly alpha1/beta heteromeres). Here, the kinetics properties of GlyRs at the single-channel level have been recorded in real-time by means of the patch-clamp technique in the outside-out configuration coupled with an ultra-fast flow application system (< 100 micros). Recordings were performed on chinese hamster ovary (CHO) cells stably transfected with the alpha2 GlyR subunit. We show that the onset, the relaxation and the desensitisation of alpha2 homomeric GlyR-mediated currents are slower by one or two orders of magnitude compared to synaptic mature GlyRs and to other ligand-gated ionotropic channels involved in fast synaptic transmission. First latency analysis performed on single GlyR channels revealed that their slow activation time course was due to delayed openings. When synaptic release of glycine was mimicked (1 mM glycine; 1 ms pulse duration), the opening probability of alpha2 homomeric GlyRs was low (P(o) approximately = 0.1) when compared to mature synaptic GlyRs (Po = 0.9). This low Po is likely to be a direct consequence of the relatively slow activation kinetics of alpha2 homomeric GlyRs when compared to the activation kinetics of mature alpha1/beta GlyRs. Such slow kinetics suggest that embryonic alpha2 homomeric GlyRs cannot be activated by fast neurotransmitter release at mature synapses but rather could be suited for a non-synaptic paracrine-like release of agonist, which is known to occur in the embryo.

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

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.

Neurotransmitters as early signals for central nervous system development.

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.

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.

A cell type-specific and gap junction-independent mechanism for the herpes simplex virus-1 thymidine kinase gene/ganciclovir-mediated bystander effect.

Tumor cells expressing the herpes simplex virus type 1 thymidine kinase (HSV-tk) gene are killed by nucleoside analogues such as ganciclovir (GCV). GCV affects not only the cells expressing HSV-tk but also neighboring cells that do not express the gene; this phenomenon commonly is called "bystander effect." GCV metabolites transfer via gap junctional intercellular communication (GJIC) accounts for the bystander effect in different cell lines, but other mechanisms have also been described. In this study, we analyzed the mechanisms of the bystander effect in two cell lines exhibiting different capacities of communication (DHD/K12 and 9L). The 9L cells exhibited a very good bystander effect, which was completely blocked by a long-term inhibitor of GJIC, 18 alpha-glycyrrhetinic acid. DHD/K12 cells exhibited a moderate bystander effect that was not abolished by 18 alpha-glycyrrhetinic acid or 1-octanol, another strong inhibitor of GJIC. Interestingly, we also observed a bystander effect in cultures where HSV-tk-expressing DHD/K12 cells were physically separated from their untransfected counterparts but grown in the same medium. Moreover, the transfer of filtered conditioned medium from GCV-treated HSV-tk-expressing DHD/K12 cells to DHD/K12 parental cells induced a decrease of survival in a concentration-dependent manner, suggesting that the bystander effect in this cell line was mediated by a soluble factor.

Growth factor therapy to the damaged inner ear: clinical prospects.

Most hearing loss results from lesions of the sensory cells and/or of the neurons of the auditory part of the inner ear. There is currently no treatment able to stop the progression of a hearing loss or to restore a lost auditory function. In this paper, we review the progress which has been made with respect to the regeneration and the protection of the hair cells and of the auditory neurons in the cochlea. In particular, we emphasize the control by growth factors of the protection/repair mechanisms of the neurosensory structures within the inner ear, in the prospect of the possible clinical use of these molecules. Finally, we discuss the different approaches which can be used to deliver these therapeutic agents to the inner ear.

Neurotransmitter-mediated regulation of CNS myelination: a review.

In addition to treatments aimed at preventing or limiting damage to myelin and oligodendrocytes, there is a crucial need for repair strategies in human demyelinating disorders. There is increasing evidence that besides growth factors, neurotransmitters can regulate different steps of the oligodendrogliogenesis. The present review on neurotransmitter receptor expression and function in the oligodendrocyte lineage emphasizes the concept that in this lineage cell proliferation and differentiation can be controlled through the modulation of the functional state of channel proteins and receptors, such as the delayed K+ rectifier, the AMPA/kainate, dopamine or muscarinic receptors, and, most likely, others yet to be found. We anticipate that a better understanding of the neurotransmitter-mediated neuronal oligodendroglial communication network opens prospects in the field of central nervous system (CNS) myelin repair, allowing the recruitment of the myelinating machinery that is known to remain present but quiescent in the CNS of multiple sclerosis patients.

Cultured oligodendrocyte progenitors derived from cerebral cortex express a glycine receptor which is pharmacologically distinct from the neuronal isoform.

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.

Expression of growth factors and their receptors in the postnatal rat cochlea.

RT-PCR was used to assay for growth factors and receptors from seven different protein families in cochlea tissues of the juvenile rat. There was a broad representation of the growth factor families in all the cochlea tissues examined, though the organ of Corti and stria vascularis expressed a greater variety than the spiral ganglion. This broad expression suggests that a variety of known growth factors play significant roles in the development, maintenance, and repair of the inner ear. The results of this survey serve as a basis for the design of future in vitro experiments that will address the ability of growth factors to protect hair cells from damage and to evoke a repair-regeneration response by injured hair cells.

Developmental regulation of neuroligand-induced responses in cultured oligodendroglia.

Using whole-cell patch-clamp techniques, we show that oligosphere-derived oligodendrocyte progenitor cells (OP) display GABA-, glutamate-, 5-HT-, glycine- and acetylcholine-gated inward currents. When OP differentiate into oligodendrocytes (ODC), the amplitude of peak currents elicited by saturating concentrations of these transmitters decreases except for 5-HT. Intracellular Ca2+ concentration changes induced by microperfusion of glutamate, 5-HT, TRH, met-enkephalin and substance P were monitored using a fluo-3-based calcium imaging system. When OP cells differentiate into ODC, a global decrease of the proportion of responding cells is observed. During type-2 astrocytes commitment, this proportion decreases for 5-HT, TRH- and metenkephalin stimulations whereas it remains constant for substance P and glutamate. These data demonstrate a development regulation of neurotransmitter- and neuropeptide-induced responses within the oligodendroglial lineage.

In Vitro Ototoxicity of aminoglycosides and platin derivatives. A semi-automatic assay for sensory hair cell damage in explanted rat organ of corti.

The ototoxic damage that drugs such as neomycin, kanamycin, colistin, cisplatin, transplatin and carboplatin cause on outer and inner hair cells in postnatal day 3 rat cochlear explants was investigated. Phalloidin-fluorescein conjugate-stained stereocilia bundles of sensory hair cells were quantified by video image analysis as a measurement of ototoxic effect. The video image quantification system established dose-response curves for ototoxic drugs (e.g. calculation of an IC(50)) and allowed comparisons between several ototoxins from the same family. This methodology provided the means to assess the efficacy of otoprotectant agents in preventing ototoxicity. Poly-l-aspartate (10(-5)m) and poly-l-glutamate (10(-5)m) protected auditory hair cells from neomycin (10(-3)m) toxicity while reduced glutathione (10(-3)m) provided protection against cisplatin (10(-4)m)-induced hair cell damage.

Duality of glutamatergic and GABAergic control of pulsatile GnRH secretion by rat hypothalamic explants: I. Effects of antisense oligodeoxynucleotides using explants including or excluding the preoptic area.

Using antisense oligodeoxynucleotides we aimed to study the role of N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid (GABA) receptors in the mechanism of Gonadotrophin-releasing hormone (GnRH) secretion in vitro. Since GnRH cell bodies are located in the rat preoptic hypothalamus while most GnRH terminals are in the retrochiasmatic hypothalamus, we compared the effects of oligodeoxynucleotides on explants of the whole (preoptic area included) or retrochiasmatic hypothalamus. When GnRH secretion is evoked by muscimol and NMDA, a time-related reduction of GnRH secretion is caused by antisense oligodeoxynucleotides for the beta subunit of the GABAA receptor and the NR2A subunit of the NMDA receptor, respectively. After 6-7 h, binding studies of tritiated ligands show a decrease in GABA- and NMDA-receptor expression. While these antisense effects are observed using whole explants, no such effects are seen using retrochiasmatic explants, indicating that the facilitatory GABAA and NMDA receptors are encoded in the preoptic area. Using several missense oligodeoxynucleotides or antisense for the NR2B and NR2C subunits of the NMDA receptor, the muscimol- and NMDA-evoked release of GnRH is not affected. When spontaneous pulsatile GnRH secretion is studied, the NR2A antisense oligodeoxynucleotides cause an increase of the interpulse interval. This increase is seen using whole but not retrochiasmatic explants. In contrast, the GABAA and NR2C antisense oligodeoxynucleotides result in a reduction of GnRH interpulse interval. Such a reduction is seen using whole as well as retrochiasmatic explants, indicating that the GABAA and NMDA receptors which mediate inhibition of GnRH pulsatility are encoded in the retrochiasmatic hypothalamus. We conclude that NMDA receptors (NR2A subunit) encoded in the preoptic hypothalamus mediate a facilitatory effect on GnRH pulsatility while GABAA and NMDA (NR2C subunit) receptors encoded in the retrochiasmatic hypothalamus mediate an inhibition of GnRH pulsatility. Pulsatile GnRH secretion is affected differently than the agonist-evoked release of GnRH suggesting that the GnRH secretory neurons and the GnRH pulse generator consist of different cellular entities.

Duality of glutamatergic and GABAergic control of pulsatile GnRH secretion by rat hypothalamic explants: II. Reduced NR2C- and GABAA-receptor-mediated inhibition at initiation of sexual maturation.

N-methyl-D-aspartate (NMDA) receptors and gamma-aminobutyric acid (GABA) receptors are involved in the mechanism of pulsatile gonadotrophin-releasing hormone (GnRH) secretion. The aim of this study was to elucidate the role of those receptors in the acceleration of pulsatile GnRH secretion seen at onset of puberty. Using hypothalamic explants from prepubertal (15 days), early pubertal (25 days) and adult (50 days) male rats, we studied the effects of pharmacological antagonists and antisense oligodeoxynucleotides on GnRH release evoked by NMDA and GABA receptor agonists as well as the interval between spontaneous GnRH secretory pulses. At the three studied ages, the muscimol-evoked release of GnRh is similarly inhibited by the GABAA receptor antagonist bicuculline. In contrast, the frequency of pulsatility is stimulated by bicuculline as indicated by a reduction of the mean GnRh interpulse interval from 60 to 40 min and such an effect is seen at 15 days only. The GnRH interpulse interval is also reduced by GABAA receptor antisense oligodeoxynucleotides at 15 days while no effects are seen at 25 days. At the three studied ages, the NMDA-evoked release of GnRH and the GnRh interpulse interval are similarly inhibited by 100 or 500 microM of the NMDA receptor antagonist 7-chlorokynurenic acid (7CK). These effects are consistent with the increase of GnRH interpulse interval caused by NR2A antisense oligodeoxynucleotides at 15 days (86 vs 64 min in controls) as well as 25 days (44 vs 36 min). A low (5 microM) concentration of 7CK does not result in any effect except a reduction of GnRH interpulse interval which is seen at 15 days only. A similar reduction of GnRh interpulse interval is obtained using NR2C antisense oligodeoxynucleotides at 15 days (50 vs 64 min in controls) while no effects are seen at 25 days (35 vs 36 min). At 25 days, muscimol can prevent the developmental increase in frequency of pulsatile GnRH secretion. In summary, pulsatile GnRH secretion by the prepubertal hypothalamus characteristically involves an inhibition mediated through GABAA receptors and the NR2C subunit of NMDA receptors. Based on these data, we propose a model for the mechanism of the onset of puberty which involves the disappearance or inactivation of GABAergic neurons located in the retrochiasmatic hypothalamus and expressing the NR2C subtype of NMDA receptors.