Versatile and automated workflow for the analysis of oligodendroglial calcium signals.

Although intracellular Ca2+ signals of oligodendroglia, the myelin-forming cells of the central nervous system, regulate vital cellular processes including myelination, few studies on oligodendroglia Ca2+ signal dynamics have been carried out and existing software solutions are not adapted to the analysis of the complex Ca2+ signal characteristics of these cells. Here, we provide a comprehensive solution to analyze oligodendroglia Ca2+ imaging data at the population and single-cell levels. We describe a new analytical pipeline containing two free, open source and cross-platform software programs, Occam and post-prOccam, that enable the fully automated analysis of one- and two-photon Ca2+ imaging datasets from oligodendroglia obtained by either ex vivo or in vivo Ca2+ imaging techniques. Easily configurable, our software solution is optimized to obtain unbiased results from large datasets acquired with different imaging techniques. Compared to other recent software, our solution proved to be fast, low memory demanding and faithful in the analysis of oligodendroglial Ca2+ signals in all tested imaging conditions. Our versatile and accessible Ca2+ imaging data analysis tool will facilitate the elucidation of Ca2+-mediated mechanisms in oligodendroglia. Its configurability should also ensure its suitability with new use cases such as other glial cell types or even cells outside the CNS.

Evoked responses to single pulse electrical stimulation reveal impaired striatal excitability in a rat model of Parkinson's disease.

BACKGROUND: Sensorimotor beta oscillations are increased in Parkinson's disease (PD) due to the alteration of dopaminergic transmission. This electrophysiological read-out is reported both in patients and in animal models such as the 6-OHDA rat model obtained with unilateral nigral injection of 6-hydroxydopamine (6-OHDA). Current treatments, based on dopaminergic replacement, transiently normalize this pathological beta activity and improve patients' quality of life. OBJECTIVES: We wanted to assess in vivo whether the abnormal beta oscillations can be correlated with impaired striatal or cortical excitability of the sensorimotor system and modulated by the pharmacological manipulation of the dopaminergic system. METHODS: In the unilateral 6-OHDA rat model and control animals, we used intra-striatal and intra-cortical single-pulse electrical stimulation (SPES) and concurrent local field potentials (LFP) recordings. In the two groups, we quantified basal cortico-striatal excitability from time-resolved spectral analyses of LFP evoked responses induced remotely by intracerebral stimulations. The temporal dependance of cortico-striatal excitability to dopaminergic transmission was further tested using electrophysiological recordings combined with levodopa injection. RESULTS: LFP evoked responses after striatal stimulation showed a transient reduction of power in a large time-frequency domain in the 6-OHDA group compared to the sham group. This result was specific to the striatum, as no significant difference was observed in cortical LFP evoked responses between the two groups. This impaired striatal excitability in the 6-OHDA group was observed in the striatum at least during the first 3 months after the initial lesion. In addition, the striatum responses to SPES during a levodopa challenge showed a transient potentiation of the decrease of responsiveness in frequencies below 40 Hz. CONCLUSION: The spectral properties of striatal responses to SPES show high sensitivity to dopaminergic transmission in the unilateral 6-OHDA rat model. We thus propose that this approach could be used in preclinical models as a time-resolved biomarker of impaired dopaminergic transmission capable of monitoring progressive neurodegeneration and/or challenges to drug intake.

Optogenetics to Interrogate Neuron-Glia Interactions in Pups and Adults.

In just over 10 years, the use of optogenetic technologies in neuroscience has become widespread, having today a tremendous impact on our understanding of brain function. An extensive number of studies have implemented a variety of tools allowing for the manipulation of neurons with light, including light-activated ion channels or G protein-coupled receptors, among other innovations. In this context, the proper calibration of photostimulation in vivo remains crucial to dissect brain circuitry or investigate the effect of neuronal activity on specific subpopulations of neurons and glia. Depending on the scientific question, the design of specific stimulation protocols must consider from the choice of the animal model to the light stimulation pattern to be delivered. In this chapter, we describe a detailed framework to investigate neuron-glia interactions in both mouse pups and adults using an optogenetic approach.

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.

Neuronal activity in vivo enhances functional myelin repair.

In demyelinating diseases such as Multiple Sclerosis (MS), demyelination of neuronal fibers impairs impulse conduction and causes axon degeneration. While neuronal activity stimulates oligodendrocyte production and myelination in normal conditions, it remains unclear whether the activity of demyelinated axons restores their loss-of-function in a harmful environment. To investigate this question, we established a model to induce a moderate optogenetic stimulation of demyelinated axons in the corpus callosum at the level of the motor cortex in which cortical circuit activation and locomotor effects were reduced in adult freely moving mice. We demonstrate that a moderate activation of demyelinated axons enhances the differentiation of oligodendrocyte precursor cells onto mature oligodendrocytes, but only under a repeated stimulation paradigm. This activity-dependent increase in the oligodendrocyte pool promotes an extensive remyelination and functional restoration of conduction, as revealed by ultrastructural analyses and compound action potential recordings. Our findings reveal the need of preserving an appropriate neuronal activity in the damaged tissue to promote oligodendrocyte differentiation and remyelination, likely by enhancing axon-oligodendroglia interactions. Our results provide new perspectives for translational research using neuromodulation in demyelinating diseases.

Activity-dependent death of transient Cajal-Retzius neurons is required for functional cortical wiring.

Programmed cell death and early activity contribute to the emergence of functional cortical circuits. While most neuronal populations are scaled-down by death, some subpopulations are entirely eliminated, raising the question of the importance of such demise for cortical wiring. Here, we addressed this issue by focusing on Cajal-Retzius neurons (CRs), key players in cortical development that are eliminated in postnatal mice in part via Bax-dependent apoptosis. Using Bax-conditional mutants and CR hyperpolarization, we show that the survival of electrically active subsets of CRs triggers an increase in both dendrite complexity and spine density of upper layer pyramidal neurons, leading to an excitation/inhibition imbalance. The survival of these CRs is induced by hyperpolarization, highlighting an interplay between early activity and neuronal elimination. Taken together, our study reveals a novel activity-dependent programmed cell death process required for the removal of transient immature neurons and the proper wiring of functional cortical circuits.

Photo-switchable tweezers illuminate pore-opening motions of an ATP-gated P2X ion channel.

P2X receptors function by opening a transmembrane pore in response to extracellular ATP. Recent crystal structures solved in apo and ATP-bound states revealed molecular motions of the extracellular domain following agonist binding. However, the mechanism of pore opening still remains controversial. Here we use photo-switchable cross-linkers as 'molecular tweezers' to monitor a series of inter-residue distances in the transmembrane domain of the P2X2 receptor during activation. These experimentally based structural constraints combined with computational studies provide high-resolution models of the channel in the open and closed states. We show that the extent of the outer pore expansion is significantly reduced compared to the ATP-bound structure. Our data further reveal that the inner and outer ends of adjacent pore-lining helices come closer during opening, likely through a hinge-bending motion. These results provide new insight into the gating mechanism of P2X receptors and establish a versatile strategy applicable to other membrane proteins.

Molecular structure and function of P2X receptors.

ATP-gated P2X receptors are trimeric ion channels selective to cations. Recent progress in the molecular biophysics of these channels enables a better understanding of their function. In particular, data obtained from biochemical, electrophysiogical and molecular engineering in the light of recent X-ray structures now allow delineation of the principles of ligand binding, channel opening and allosteric modulation. However, although a picture emerges as to how ATP triggers channel opening, there are a number of intriguing questions that remain to be answered, in particular how the pore itself opens in response to ATP and how the intracellular domain, for which structural information is limited, moves during activation. In this review, we provide a summary of functional studies in the context of the post-structure era, aiming to clarify our understanding of the way in which P2X receptors function in response to ATP binding, as well as the mechanism by which allosteric modulators are able to regulate receptor function. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'.

Optical control of an ion channel gate.

The powerful optogenetic pharmacology method allows the optical control of neuronal activity by photoswitchable ligands tethered to channels and receptors. However, this approach is technically demanding, as it requires the design of pharmacologically active ligands. The development of versatile technologies therefore represents a challenging issue. Here, we present optogating, a method in which the gating machinery of an ATP-activated P2X channel was reprogrammed to respond to light. We found that channels covalently modified by azobenzene-containing reagents at the transmembrane segments could be reversibly turned on and off by light, without the need of ATP, thus revealing an agonist-independent, light-induced gating mechanism. We demonstrate photocontrol of neuronal activity by a light-gated, ATP-insensitive P2X receptor, providing an original tool devoid of endogenous sensitivity to delineate P2X signaling in normal and pathological states. These findings open new avenues to specifically activate other ion channels independently of their natural stimulus.

Novel glycolipid TLR2 ligands of the type Pam2Cys-α-Gal: synthesis and biological properties.

A more complete understanding of the mechanism of action of TLR agonists has fueled the investigation of new synthetic immunoadjuvants. In this context, we designed and synthesized glycolipids of the type Pam(2)Cys-α-Galactose as novel immunoadjuvants. Their synthesis required modifying a hydrophobic tBoc-[2,3-bispalmitoyloxy-(2R)-propyl]-R-cysteinyl moiety, i.e. the minimal structure required for TLR2 agonist activity, by addition of a hydrophilic head, either an α-Galactosylpyranose or an α-Galactosylfuranose to gain respectively Pam(2)CGalp and Pam(2)CGalf. While preparing a carbohydrate building block, an unexpected stereoselectivity was observed during a halide ion-catalytic process on a protected galactofuranose: the alpha anomer was obtained with surprisingly high selectivity (α/ß ratio>9) and with good isolated yield (51%). The TLR2 binding properties of Pam(2)CGalp and Pam(2)CGalf were then fully evaluated. Their efficiency in triggering the proliferation of BALB/c mouse splenocytes was also compared to that of Pam(2)CAG and Pam(3)CAG, two well-established ligands of TLRs. Moreover, the maturation state of murine dendritic cells previously incubated with either Pam(2)CGalp or Pam(2)CGalf was monitored by flow cytometry and compared to that induced by lipopolysaccharide. Pam(2)CGalp and Pam(2)CGalf were found to be equivalent TLR2 agonists, and induced splenocyte proliferation and DC maturation. With very similar activity, Pam(2)CGalp and Pam(2)CGalf were also 10-fold to 100-fold better than Pam(2)CAG and Pam(3)CAG at inducing B cell proliferation. This represents the first time a glucidic head has been added to the tBoc-[2,3-bispalmitoyloxy-(2R)-propyl]-R-cysteinyl moiety whilst maintaining the immunomodulating activity. This should greatly enrich the data available on Pam(2)C structure/activity relationships.