Glutamate versus GABA in neuron-oligodendroglia communication.

In the central nervous system (CNS), myelin sheaths around axons are formed by glial cells named oligodendrocytes (OLs). In turn, OLs are generated by oligodendrocyte precursor cells (OPCs) during postnatal development and in adults, according to a process that depends on the proliferation and differentiation of these progenitors. The maturation of OL lineage cells as well as myelination by OLs are complex and highly regulated processes in the CNS. OPCs and OLs express an array of receptors for neurotransmitters, in particular for the two main CNS neurotransmitters glutamate and GABA, and are therefore endowed with the capacity to respond to neuronal activity. Initial studies in cell cultures demonstrated that both glutamate and GABA signaling mechanisms play important roles in OL lineage cell development and function. However, much remains to be learned about the communication of glutamatergic and GABAergic neurons with oligodendroglia in vivo. This review focuses on recent major advances in our understanding of the neuron-oligodendroglia communication mediated by glutamate and GABA in the CNS, and highlights the present controversies in the field. We discuss the expression, activation modes and potential roles of synaptic and extrasynaptic receptors along OL lineage progression. We review the properties of OPC synaptic connectivity with presynaptic glutamatergic and GABAergic neurons in the brain and consider the implication of glutamate and GABA signaling in activity-driven adaptive myelination.

A specific GABAergic synapse onto oligodendrocyte precursors does not regulate cortical oligodendrogenesis.

In the brain, neurons establish bona fide synapses onto oligodendrocyte precursor cells (OPCs), but the function of these neuron-glia synapses remains unresolved. A leading hypothesis suggests that these synapses regulate OPC proliferation and differentiation. However, a causal link between synaptic activity and OPC cellular dynamics is still missing. In the developing somatosensory cortex, OPCs receive a major type of synapse from GABAergic interneurons that is mediated by postsynaptic γ2-containing GABAA receptors. Here we genetically silenced these receptors in OPCs during the critical period of cortical oligodendrogenesis. We found that the inactivation of γ2-mediated synapses does not impact OPC proliferation and differentiation or the propensity of OPCs to myelinate their presynaptic interneurons. However, this inactivation causes a progressive and specific depletion of the OPC pool that lacks γ2-mediated synaptic activity without affecting the oligodendrocyte production. Our results show that, during cortical development, the γ2-mediated interneuron-to-OPC synapses do not play a role in oligodendrogenesis and suggest that these synapses finely tune OPC self-maintenance capacity. They also open the interesting possibility that a particular synaptic signaling onto OPCs plays a specific role in OPC function according to the neurotransmitter released, the identity of presynaptic neurons or the postsynaptic receptors involved.

TASK-2 channels contribute to pH sensitivity of retrotrapezoid nucleus chemoreceptor neurons.

Phox2b-expressing glutamatergic neurons of the retrotrapezoid nucleus (RTN) display properties expected of central respiratory chemoreceptors; they are directly activated by CO2/H(+) via an unidentified pH-sensitive background K(+) channel and, in turn, facilitate brainstem networks that control breathing. Here, we used a knock-out mouse model to examine whether TASK-2 (K2P5), an alkaline-activated background K(+) channel, contributes to RTN neuronal pH sensitivity. We made patch-clamp recordings in brainstem slices from RTN neurons that were identified by expression of GFP (directed by the Phox2b promoter) or ß-galactosidase (from the gene trap used for TASK-2 knock-out). Whereas nearly all RTN cells from control mice were pH sensitive (95%, n = 58 of 61), only 56% of GFP-expressing RTN neurons from TASK-2(-/-) mice (n = 49 of 88) could be classified as pH sensitive (>30% reduction in firing rate from pH 7.0 to pH 7.8); the remaining cells were pH insensitive (44%). Moreover, none of the recorded RTN neurons from TASK-2(-/-) mice selected based on ß-galactosidase activity (a subpopulation of GFP-expressing neurons) were pH sensitive. The alkaline-activated background K(+) currents were reduced in amplitude in RTN neurons from TASK-2(-/-) mice that retained some pH sensitivity but were absent from pH-insensitive cells. Finally, using a working heart-brainstem preparation, we found diminished inhibition of phrenic burst amplitude by alkalization in TASK-2(-/-) mice, with apneic threshold shifted to higher pH levels. In conclusion, alkaline-activated TASK-2 channels contribute to pH sensitivity in RTN neurons, with effects on respiration in situ that are particularly prominent near apneic threshold.

Fibroblast KATP currents modulate myocyte electrophysiology in infarcted hearts.

Cardiac metabolism remains altered for an extended period of time after myocardial infarction. Studies have shown fibroblasts from normal hearts express KATP channels in culture. It is unknown whether fibroblasts from infarcted hearts express KATP channels and whether these channels contribute to scar and border zone electrophysiology. KATP channel subunit expression levels were determined in fibroblasts isolated from normal hearts (Fb), and scar (sMI-Fb) and remote (rMI-Fb) regions of left anterior descending coronary artery (LAD) ligated rat hearts. Whole cell KATP current density was determined with patch clamp. Action potential duration (APD) was measured with optical mapping in myocyte-only cultures and heterocellular cultures with fibroblasts with and without 100 µmol/l pinacidil. Whole heart optical mapping was used to assess KATP channel activity following LAD ligation. Pinacidil activated a potassium current (35.4 ± 7.5 pA/pF at 50 mV) in sMI-Fb that was inhibited with 10 µmol/l glibenclamide. Kir6.2 and SUR2 transcript levels were elevated in sMI-Fb. Treatment with Kir6.2 short interfering RNA decreased KATP currents (87%) in sMI-Fb. Treatment with pinacidil decreased APD (26%) in co-cultures with sMI-Fb. APD values were prolonged in LAD ligated hearts after perfusion with glibenclamide. KATP channels are present in fibroblasts from the scar and border zones of infarcted hearts. Activation of fibroblast KATP channels could modulate the electrophysiological substrate beyond the acute ischemic event. Targeting fibroblast KATP channels could represent a novel therapeutic approach to modify border zone electrophysiology after cardiac injury.

A distinct de novo expression of Nav1.5 sodium channels in human atrial fibroblasts differentiated into myofibroblasts.

Fibroblasts play a major role in heart physiology. They are at the origin of the extracellular matrix renewal and production of various paracrine and autocrine factors. In pathological conditions, fibroblasts proliferate, migrate and differentiate into myofibroblasts leading to cardiac fibrosis. This differentiated status is associated with changes in expression profile leading to neo-expression of proteins such as ionic channels. The present study investigates further electrophysiological changes associated with fibroblast differentiation focusing on the activity of voltage-gated sodium channels in human atrial fibroblasts and myofibroblasts. Using the patch clamp technique we show that human atrial myofibroblasts display a fast inward voltage gated sodium current with a density of 13.28 ± 2.88 pA pF(-1) whereas no current was detectable in non-differentiated fibroblasts. Quantitative RT-PCR reveals a large amount of transcripts encoding the Na(v)1.5 α-subunit with a fourfold increased expression level in myofibroblasts when compared to fibroblasts. Accordingly, half of the current was blocked by 1 µm of tetrodotoxin and immunocytochemistry experiments reveal the presence of Na(v)1.5 proteins. Overall, this current exhibits similar biophysical characteristics to sodium currents found in cardiac myocytes except for the window current that is enlarged for potentials between -100 and -20 mV. Since fibrosis is one of the fundamental mechanisms implicated in atrial fibrillation, it is of great interest to investigate how this current could influence myofibroblast properties. Moreover, since several Na(v)1.5 mutations are related to cardiac pathologies, this study offers a new avenue on the fibroblasts involvement of these mutations.

Enhanced fibroblast-myocyte interactions in response to cardiac injury.

RATIONALE: A critical event in the development of cardiac fibrosis is the transformation of fibroblasts into myofibroblasts. The electrophysiological consequences of this phenotypic switch remain largely unknown. OBJECTIVE: Determine whether fibroblast activation following cardiac injury results in a distinct electrophysiological phenotype that enhances fibroblast-myocyte interactions. METHODS AND RESULTS: Neonatal rat myocyte monolayers were treated with media (CM) conditioned by fibroblasts isolated from normal (Fb) and infarcted (MI-Fb) hearts. Fb and MI-Fb were also plated on top of myocyte monolayers at 3 densities. Cultures were optically mapped after CM treatment or fibroblast plating to obtain conduction velocity and action potential duration (APD(70)). Intercellular communication and connexin43 expression levels were assessed. Membrane properties of Fb and MI-Fb were evaluated using patch clamp techniques. MI-Fb CM treatment decreased conduction velocity (11.1%) compared to untreated myocyte cultures. APD(70) was reduced by MI-Fb CM treatment compared to homocellular myocyte culture (9.4%) and Fb CM treatment (6.4%). In heterocellular cultures, MI-Fb conduction velocities were different from Fb at all densities (+29.8%, -23.0%, and -16.7% at 200, 400, and 600 cells/mm(2), respectively). APD(70) was reduced (9.6%) in MI-Fb compared to Fb cultures at 200 cells/mm(2). MI-Fb had more hyperpolarized resting membrane potentials and increased outward current densities. Connexin43 was elevated (134%) in MI-Fb compared to Fb. Intercellular coupling evaluated with gap fluorescence recovery after photobleaching was higher between myocytes and MI-Fb compared to Fb. CONCLUSIONS: These data demonstrate cardiac injury results in significant electrophysiological changes that enhance fibroblast-myocyte interactions and could contribute to the greater incidence of arrhythmias observed in fibrotic hearts.

The SNARE protein SNAP-25 is required for normal exocytosis at auditory hair cell ribbon synapses.

Hearing depends on fast and sustained calcium-dependent synaptic vesicle fusion at the ribbon synapses of cochlear inner hair cells (IHCs). The implication of the canonical neuronal SNARE complex in this exocytotic process has so far remained controversial. We investigated the role of SNAP-25, a key component of this complex, in hearing, by generating and analyzing a conditional knockout mouse model allowing a targeted postnatal deletion of Snap-25 in IHCs. Mice subjected to IHC Snap-25 inactivation after hearing onset developed severe to profound deafness because of defective IHC exocytosis followed by ribbon degeneration and IHC loss. Viral transfer of Snap-25 in these mutant mice rescued their hearing function by restoring IHC exocytosis and preventing synapses and hair cells from degeneration. These results demonstrate that SNAP-25 is essential for normal hearing function, most likely by ensuring IHC exocytosis and ribbon synapse maintenance.

Electrophysiological and functional effects of sphingosine-1-phosphate in mouse ventricular fibroblasts.

The aim of this study was to characterize the effects of sphingosine-1-phosphate (S1P) on cardiac ventricular fibroblasts. Impacts of S1P on fibroblast excitability, cell migration, proliferation and secretion were characterized. The patch-clamp technique in the whole-cell configuration was used to study the S1P-induced current from mouse ventricular fibroblasts. The expression level of the S1P receptor during cell culture duration was evaluated by western-blot. Fibroblast proliferation and migration were quantified using the methylene blue assay and the Boyden chamber technique, respectively. Finally, fibroblast secretion properties were estimated by quantification of the IL-6 and collagen levels using ELISA and SIRCOL collagen assays, respectively. We found that S1P activated SUR2/Kir6.1 channel and that this effect was sensitive to specific inhibition of the S1P receptor of type 3 (S1P3R). In contrast, S1P1R receptor inhibition had no effect. Moreover, the S1P-induced current increased with cell culture duration whereas S1P3R expression level remained constant. The activation of SUR2/Kir6.1 channel by S1P via S1P3R stimulated cell proliferation and decreased IL-6 and collagen secretions. S1P also stimulated fibroblast migration via S1P3R but independently from SUR2/Kir6.1 channel activation. This study demonstrates that S1P, via S1P3R, affects cardiac ventricular fibroblasts function independently or through activation of SUR2/Kir6.1 channel. The latter effect occurs after fibroblasts differentiate into myofibroblasts, opening a new potential therapeutic strategy to modulate fibrosis after cardiac physiopathological injury.

The cardiac fibroblast: functional and electrophysiological considerations in healthy and diseased hearts.

Cardiac fibrosis occurs in a number of cardiovascular diseases associated with a high incidence of arrhythmias. A critical event in the development of fibrosis is the transformation of fibroblasts into an active phenotype or myofibroblast. This transformation results in functional changes including increased proliferation and changes in the release of signaling molecules and extracellular matrix deposition. Traditionally, fibroblasts have been considered to affect cardiac electrophysiology indirectly by physically isolating myocytes and creating conduction barriers. There is now increasing evidence that cardiac fibroblasts may play a direct role in modulating the electrophysiological substrate in diseased hearts. The purpose of this review is to summarize the functional changes associated with fibroblast activation, the membrane currents that have been identified in adult cardiac fibroblasts, and describe recent studies of fibroblast-myocyte electrical interactions with emphasis on the changes that occur with cardiac injury. Further analysis of fibroblast membrane electrophysiology and their interactions with myocytes will lead to a more complete understanding of the arrhythmic substrate. These studies have the potential to generate new therapeutic approaches for the prevention of arrhythmias associated with cardiac fibrosis.

The cerebral cortex is a substrate of multiple interactions between GABAergic interneurons and oligodendrocyte lineage cells.

In the cerebral cortex, GABAergic interneurons and oligodendrocyte lineage cells share different characteristics and interact despite being neurons and glial cells, respectively. These two distinct cell types share common embryonic origins and are born from precursors expressing similar transcription factors. Moreover, they highly interact with each other through different communication mechanisms during development. Notably, cortical oligodendrocyte precursor cells (OPCs) receive a major and transient GABAergic synaptic input, preferentially from parvalbumin-expressing interneurons, a specific interneuron subtype recently recognized as highly myelinated. In this review, we highlight the similarities and interactions between GABAergic interneurons and oligodendrocyte lineage cells in the cerebral cortex and suggest potential roles of this intimate interneuron-oligodendroglia relationship in cortical construction. We also propose new lines of research to understand the role of the close link between interneurons and oligodendroglia during cortical development and in pathological conditions such as schizophrenia.

Myelination of parvalbumin interneurons shapes the function of cortical sensory inhibitory circuits.

Myelination of projection neurons by oligodendrocytes is key to optimize action potential conduction over long distances. However, a large fraction of myelin enwraps the axons of parvalbumin-positive fast-spiking interneurons (FSI), exclusively involved in local cortical circuits. Whether FSI myelination contributes to the fine-tuning of intracortical networks is unknown. Here we demonstrate that FSI myelination is required for the establishment and maintenance of the powerful FSI-mediated feedforward inhibition of cortical sensory circuits. The disruption of GABAergic synaptic signaling of oligodendrocyte precursor cells prior to myelination onset resulted in severe FSI myelination defects characterized by longer internodes and nodes, aberrant myelination of branch points and proximal axon malformation. Consequently, high-frequency FSI discharges as well as FSI-dependent postsynaptic latencies and strengths of excitatory neurons were reduced. These dysfunctions generated a strong excitation-inhibition imbalance that correlated with whisker-dependent texture discrimination impairments. FSI myelination is therefore critical for the function of mature cortical inhibitory circuits.

Molecular and functional characterization of a new potassium conductance in mouse ventricular fibroblasts.

The present work is aimed at identifying and characterizing, at a molecular and functional level, new ionic conductances potentially involved in the excitation-secretion coupling and proliferation of cardiac ventricular fibroblasts. Among potassium channel transcripts which were screened by high-throughput real-time PCR, SUR2 and Kir6.1 mRNAs were found to be the most abundant in ventricular fibroblasts. The corresponding proteins were not detected by western blot following 5 days of cell culture, but had appeared at 7 days, increasing with extended cell culture duration as the fibroblasts differentiated into myofibroblasts. Using the inside-out configuration of the patch-clamp technique, single potassium channels could be recorded. These had properties similar to those reported for SUR2/Kir6.1 channels, i.e. activation by pinacidil, inhibition by glibenclamide and activation by intracellular UDP. As already reported for this molecular signature, they were insensitive to intracellular ATP. In the whole-cell configuration, these channels have been shown to be responsible for a glibenclamide-sensitive macroscopic potassium current which can be activated not only by pinacidil, but also by nanomolar concentrations of the sphingolipid sphingosine-1-phosphate (S1P). The activation of this current resulted in an increase in cell proliferation and a decrease in IL-6 secretion, suggesting it has a functional role in situations where S1P increases. Overall, this work demonstrates for the first time that SUR2/Kir6.1 channels represent a significant potassium conductance in ventricular fibroblasts which may be activated in physio-pathological conditions and which may impact on fibroblast proliferation and function.

Implication of connexins 40 and 43 in functional coupling between mouse cardiac fibroblasts in primary culture.

Cardiac fibroblasts contribute to the structure and function of the myocardium. However their involvement in electrophysiological processes remains unclear; particularly in pathological situations when they proliferate and develop fibrosis. We have identified the connexins involved in gap junction channels between fibroblasts from adult mouse heart and characterized their functional coupling. RT-PCR and Western blotting results show that mRNA and proteins of connexin40 and connexin43 are expressed in cultured cardiac fibroblasts, while Cx45 is not detected. Analysis of gap junctional communications established by these connexins with the gap-FRAP technique demonstrates that fibroblasts are functionally coupled. The time constant of permeability, k, calculated from the fluorescence recovery curves between cell pairs is 0.066+/-0.005 min(-1) (n = 65). Diffusion analysis of Lucifer Yellow through gap junction channels with the scrape-loading method demonstrates that when they are completely confluent, a majority of fibroblasts are coupled forming an interconnecting network over a distance of several hundred micrometers. These data show that cardiac fibroblasts express connexin40 and connexin43 which are able to establish functional communications through homo and/or heterotypic junctions to form an extensive coupled cell network. It should then be interesting to study the conditions to improve efficiency of this coupling in pathological conditions.

Developmental cell death regulates lineage-related interneuron-oligodendroglia functional clusters and oligodendrocyte homeostasis.

The first wave of oligodendrocyte precursor cells (firstOPCs) and most GABAergic interneurons share common embryonic origins. Cortical firstOPCs are thought to be replaced by other OPC populations shortly after birth, maintaining a consistent OPC density and making postnatal interactions between firstOPCs and ontogenetically-related interneurons unlikely. Challenging these ideas, we show that a cortical firstOPC subpopulation survives and forms functional cell clusters with lineage-related interneurons. Favored by a common embryonic origin, these clusters display unexpected preferential synaptic connectivity and are anatomically maintained after firstOPCs differentiate into myelinating oligodendrocytes. While the concomitant rescue of interneurons and firstOPCs committed to die causes an exacerbated neuronal inhibition, it abolishes interneuron-firstOPC high synaptic connectivity. Further, the number of other oligodendroglia populations increases through a non-cell-autonomous mechanism, impacting myelination. These findings demonstrate unprecedented roles of interneuron and firstOPC apoptosis in regulating lineage-related cell interactions and the homeostatic oligodendroglia density.