María Cecilia Angulo

Molecular and Physiological Diversity of Cortical Nonpyramidal Cells

The physiological and molecular features of nonpyramidal cells were investigated in acute slices of sensory-motor cortex using whole-cell recordings combined with single-cell RT-PCR to detect simultaneously the mRNAs of three calcium binding proteins (calbindin D28k, parvalbumin, and calretinin) and four neuropeptides (neuropeptide Y, vasoactive intestinal polypeptide, somatostatin, and cholecystokinin). In the 97 neurons analyzed, all expressed mRNAs of at least one calcium binding protein, and the majority (n = 73) contained mRNAs of at least one neuropeptide. Three groups of nonpyramidal cells were defined according to their firing pattern. (1) Fast spiking cells (n = 34) displayed tonic discharges of fast action potentials with no accommodation. They expressed parvalbumin (n = 30) and/or calbindin (n = 19) mRNAs, and half of them also contained transcripts of at least one of the four neuropeptides. (2) Regular spiking nonpyramidal cells (n = 48) displayed a firing behavior characterized by a marked accommodation and presented a large diversity of expression patterns of the seven biochemical markers. (3) Finally, a small population of vertically oriented bipolar cells, termed irregular spiking cells (n = 15), fired bursts of action potentials at an irregular frequency. They consistently co-expressed calretinin and vasoactive intestinal polypeptide. Additional investigations of these cells showed that they also co-expressed glutamic acid decarboxylase and choline acetyl transferase. Our results indicate that neocortical nonpyramidal neurons display a large diversity in their firing properties and biochemical patterns of co-expression and that both characteristics could be correlated to define discrete subpopulations.

Glutamate Released from Glial Cells Synchronizes Neuronal Activity in the Hippocampus

Glial cells of the nervous system directly influence neuronal and synaptic activities by releasing transmitters. However, the physiological consequences of this glial transmitter release on brain information processing remain poorly understood. We demonstrate here in hippocampal slices of 2- to 5-week-old rats that glutamate released from glial cells generates slow transient currents (STCs) mediated by the activation of NMDA receptors in pyramidal cells. STCs persist in the absence of neuronal and synaptic activity, indicating a nonsynaptic origin of the source of glutamate. Indeed, STCs occur spontaneously but can also be induced by pharmacological tools known to activate astrocytes and by the selective mechanical stimulation of single nearby glial cells. Bath application of the inhibitor of the glutamate uptake dl-threo-β-benzyloxyaspartate increases both the frequency of STCs and the amplitude of a tonic conductance mediated by NMDA receptors and probably also originated from glial glutamate release. By using dual recordings, we observed synchronized STCs in pyramidal cells having their soma distant by <100 μm. The degree of precision (<100 msec) of this synchronization rules out the involvement of calcium waves spreading through the glial network. It also indicates that single glial cells release glutamate onto adjacent neuronal processes, thereby controlling simultaneously the excitability of several neighboring pyramidal cells. In conclusion, our results show that the glial glutamate release occurs spontaneously and synchronizes the neuronal activity in the hippocampus.

Nonsynaptic junctions on myelinating glia promote preferential myelination of electrically active axons

The myelin sheath on vertebrate axons is critical for neural impulse transmission, but whether electrically active axons are preferentially myelinated by glial cells, and if so, whether axo-glial synapses are involved, are long-standing questions of significance to nervous system development, plasticity and disease. Here we show using an in vitro system that oligodendrocytes preferentially myelinate electrically active axons, but synapses from axons onto myelin-forming oligodendroglial cells are not required. Instead, vesicular release at nonsynaptic axo-glial junctions induces myelination. Axons releasing neurotransmitter from vesicles that accumulate in axon varicosities induces a local rise in cytoplasmic calcium in glial cell processes at these nonsynaptic functional junctions, and this signalling stimulates local translation of myelin basic protein to initiate myelination.

Oligodendrocyte Precursor Cells Are Accurate Sensors of Local K+ in Mature Gray Matter

Oligodendrocyte precursor cells (OPCs) are the major source of myelinating oligodendrocytes during development. These progenitors are highly abundant at birth and persist in the adult where they are distributed throughout the brain. The large abundance of OPCs after completion of myelination challenges their unique role as progenitors in the healthy adult brain. Here we show that adult OPCs of the barrel cortex sense fine extracellular K+ increases generated by neuronal activity, a property commonly assigned to differentiated astrocytes rather than to progenitors. Biophysical, pharmacological, and single-cell RT-PCR analyses demonstrate that this ability of OPCs establishes itself progressively through the postnatal upregulation of Kir4.1 K+ channels. In animals with advanced cortical myelination, extracellular stimulation of layer V axons induces slow K+ currents in OPCs, which amplitude correlates with presynaptic action potential rate. Moreover, using paired recordings, we demonstrate that the discharge of a single neuron can be detected by nearby adult OPCs, indicating that these cells are strategically located to detect local changes in extracellular K+ concentration during physiological neuronal activity. These results identify a novel unitary neuron–OPC connection, which transmission does not rely on neurotransmitter release and appears late in development. Beyond their abundance in the mature brain, the postnatal emergence of a physiological response of OPCs to neuronal network activity supports the view that in the adult these cells are not progenitors only.

Postnatal Switch from Synaptic to Extrasynaptic Transmission between Interneurons and NG2 Cells

NG2 cells, oligodendrocyte precursors, play a critical role in myelination during postnatal brain maturation, but a pool of these precursors is maintained in the adult and recruited to lesions in demyelinating diseases. NG2 cells in immature animals have recently been shown to receive synaptic inputs from neurons, and these have been assumed to persist in the adult. Here, we investigated the GABAergic synaptic activity of NG2 cells in acute slices of the barrel cortex of NG2-DsRed transgenic mice during the first postnatal month, which corresponds to the period of active myelination in the neocortex. Our data demonstrated that the frequency of spontaneous and miniature GABAergic synaptic activity of cortical NG2 cells dramatically decreases after the second postnatal week, indicating a decrease in the number of synaptic inputs onto NG2 cells during development. However, NG2 cells still receive GABAergic inputs from interneurons in the adult cortex. These inputs do not rely on the presence of functional synapses but involve a form of GABA spillover. This GABA volume transmission allows interneurons to induce phasic responses in target NG2 cells through the activation of extrasynaptic GABAA receptors. Hence, after development is complete, volume transmission allows NG2 cells to integrate neuronal activity patterns at frequencies occurring during in vivo sensory stimulation.

Functional α7-containing nicotinic receptors of NG2-expressing cells in the hippocampus

In the postnatal central nervous system, glial cells expressing the chondroitin sulfate proteoglycan NG2 (NG2‐cells) constitute a cell population exhibiting several properties of oligodendrocyte precursors such as the ability to proliferate. One particular feature of NG2‐cells is that they express several glutamatergic and GABAergic ionotropic receptors activated by synaptic neurotransmitter release. Here, we used patch‐clamp recordings, immunostaining, calcium imaging, and intracellular labeling to test for the presence of ionotropic nicotinic acetylcholine receptors (nAChRs) in NG2‐cells identified in acute hippocampal slices of mice. We demonstrated that these cells express functional nAChRs during the second postnatal week, i.e., the period in which they become the most abundant proliferative cell type of CA1 stratum radiatum. Pharmacological experiments showed that NG2‐cells express α7‐containing nAChRs. In particular, the powerful positive allosteric modulator of these receptors PNU‐120596 induced a 20‐fold increase of agonist‐induced currents and revealed rises in intracellular calcium concentration upon agonist applications. In addition, nanomolar concentrations of nicotine, which did not induce any response in these cells, largely desensitized nAChR‐mediated currents. These data indicate that the functional expression of Ca2+‐permeable α7‐containing nAChRs in hippocampal slices is not restricted to neurons and that the receptors of NG2‐cells can be desensitized by low concentrations of nicotine.

Is neuronal communication with NG2 cells synaptic or extrasynaptic

NG2‐expressing glial cells (NG2 cells) represent a major pool of progenitors able to generate myelinating oligodendrocytes, and perhaps astrocytes and neurones, in the postnatal brain. In the last decade, it has been demonstrated that NG2 cells receive functional glutamatergic and GABAergic synapses mediating fast synaptic transmission in different brain regions. However, several controversies exist in this field. While two classes of NG2 cells have been defined by the presence or absence of Na+ channels, action potential firing and neuronal input, other studies suggest that all NG2 cells possess Na+ conductances and are the target of quantal neuronal release, but are unable to trigger action potential firing. Here we bring new evidence supporting the idea that the level of expression of Na+ conductances is not a criterion to discriminate NG2 cell subpopulations in the somatosensory cortex. Surprisingly, recent reports demonstrated that NG2 cells detect quantal glutamate release from unmyelinated axons in white matter regions. Yet, it is difficult from these studies to establish whether axonal vesicular release in white matter occurs at genuine synaptic junctions or at ectopic release sites. In addition, we recently reported a new mode of extrasynaptic communication between neurones and NG2 cells that relies on pure GABA spillover and does not require GABAergic synaptic input. This review discusses the properties of quantal neuronal release onto NG2 cells and gives an extended overview of potential extrasynaptic modes of transmission, from ectopic to diffuse volume transmission, between neurones and NG2 cells in the brain.

Multiple Modes of Communication between Neurons and Oligodendrocyte Precursor Cells

The surprising discovery of bona fide synapses between neurons and oligodendrocytes precursor cells (OPCs) 15 years ago placed these progenitors as real partners of neurons in the CNS. The role of these synapses has not been established yet, but a main hypothesis is that neuron–OPC synaptic activity is a signaling pathway controlling OPC proliferation/differentiation, influencing the myelination process. However, new evidences describing non-synaptic mechanisms of communication between neurons and OPCs have revealed that neuron–OPC interactions are more complex than expected. The activation of extrasynaptic receptors by ambient neurotransmitter or local spillover and the ability of OPCs to sense neuronal activity through a potassium channel suggest that distinct modes of communication mediate different functions of OPCs in the CNS. This review discusses different mechanisms used by OPCs to interact with neurons and their potential roles during postnatal development and in brain disorders.

Postnatal Down-Regulation of the GABAA Receptor γ2 Subunit in Neocortical NG2 Cells Accompanies Synaptic-to-Extrasynaptic Switch in the GABAergic Transmission Mode

NG2 cells, a main pool of glial progenitors, express γ-aminobutyric acid A (GABA(A)) receptors (GABA(A)Rs), the functional and molecular properties of which are largely unknown. We recently reported that transmission between GABAergic interneurons and NG2 cells drastically changes during development of the somatosensory cortex, switching from synaptic to extrasynaptic communication. Since synaptic and extrasynaptic GABA(A)Rs of neurons differ in their subunit composition, we hypothesize that GABA(A)Rs of NG2 cells undergo molecular changes during cortical development accompanying the switch of transmission modes. Single-cell RT-PCR and the effects of zolpidem and α5IA on evoked GABAergic currents reveal the predominance of functional α1- and α5-containing GABA(A)Rs at interneuron-NG2 cell synapses in the second postnatal week, while the α5 expression declines later in development when responses are exclusively extrasynaptic. Importantly, pharmacological and molecular analyses demonstrate that γ2, a subunit contributing to the clustering of GABA(A)Rs at postsynaptic sites in neurons, is down-regulated in NG2 cells in a cell type-specific manner in concomitance with the decline of synaptic activity and the switch of transmission mode. In keeping with the synaptic nature of γ2 in neurons, the down-regulation of this subunit is an important molecular hallmark of the change of transmission modes between interneurons and NG2 cells during development.

Interneurons and oligodendrocyte progenitors form a structured synaptic network in the developing neocortex

NG2 cells, oligodendrocyte progenitors, receive a major synaptic input from interneurons in the developing neocortex. It is presumed that these precursors integrate cortical networks where they act as sensors of neuronal activity. We show that NG2 cells of the developing somatosensory cortex form a transient and structured synaptic network with interneurons that follows its own rules of connectivity. Fast-spiking interneurons, highly connected to NG2 cells, target proximal subcellular domains containing GABAA receptors with γ2 subunits. Conversely, non-fast-spiking interneurons, poorly connected with these progenitors, target distal sites lacking this subunit. In the network, interneuron-NG2 cell connectivity maps exhibit a local spatial arrangement reflecting innervation only by the nearest interneurons. This microcircuit architecture shows a connectivity peak at PN10, coinciding with a switch to massive oligodendrocyte differentiation. Hence, GABAergic innervation of NG2 cells is temporally and spatially regulated from the subcellular to the network level in coordination with the onset of oligodendrogenesis. DOI: http://dx.doi.org/10.7554/eLife.06953.001