Upregulation of astroglial connexin 30 impairs hippocampal synaptic activity and recognition memory.

Astrocytes crucially contribute to synaptic physiology and information processing. One of their key characteristics is to express high levels of connexins (Cxs), the gap junction-forming protein. Among them, Cx30 displays specific properties since it is postnatally expressed and dynamically upregulated by neuronal activity and modulates cognitive processes by shaping synaptic and network activities, as recently shown in knockout mice. However, it remains unknown whether local and selective upregulation of Cx30 in postnatal astrocytes within a physiological range modulates neuronal activities in the hippocampus. We here show in mice that, whereas Cx30 upregulation increases the connectivity of astroglial networks, it decreases spontaneous and evoked synaptic transmission. This effect results from a reduced neuronal excitability and translates into an alteration in the induction of synaptic plasticity and an in vivo impairment in learning processes. Altogether, these results suggest that astroglial networks have a physiologically optimized size to appropriately regulate neuronal functions.

Correction: Fast calcium transients in dendritic spines driven by extreme statistics.

[This corrects the article DOI: 10.1371/journal.pbio.2006202.].

Pannexin 1 activity in astroglia sets hippocampal neuronal network patterns.

Astroglial release of molecules is thought to actively modulate neuronal activity, but the nature, release pathway, and cellular targets of these neuroactive molecules are still unclear. Pannexin 1, expressed by neurons and astrocytes, form nonselective large pore channels that mediate extracellular exchange of molecules. The functional relevance of these channels has been mostly studied in brain tissues, without considering their specific role in different cell types, or in neurons. Thus, our knowledge of astroglial pannexin 1 regulation and its control of neuronal activity remains very limited, largely due to the lack of tools targeting these channels in a cell-specific way. We here show that astroglial pannexin 1 expression in mice is developmentally regulated and that its activation is activity-dependent. Using astrocyte-specific molecular tools, we found that astroglial-specific pannexin 1 channel activation, in contrast to pannexin 1 activation in all cell types, selectively and negatively regulates hippocampal networks, with their disruption inducing a drastic switch from bursts to paroxysmal activity. This decrease in neuronal excitability occurs via an unconventional astroglial mechanism whereby pannexin 1 channel activity drives purinergic signaling-mediated regulation of hyperpolarisation-activated cyclic nucleotide (HCN)-gated channels. Our findings suggest that astroglial pannexin 1 channel activation serves as a negative feedback mechanism crucial for the inhibition of hippocampal neuronal networks.

Astrocytic Kir4.1 channels regulate locomotion by orchestrating neuronal rhythmicity in the spinal network.

Neuronal rhythmogenesis in the spinal cord is correlated with variations in extracellular K+ levels ([K+ ]e ). Astrocytes play important role in [K+ ]e homeostasis and compute neuronal information. Yet it is unclear how neuronal oscillations are regulated by astrocytic K+ homeostasis. Here we identify the astrocytic inward-rectifying K+ channel Kir4.1 (a.k.a. Kcnj10) as a key molecular player for neuronal rhythmicity in the spinal central pattern generator (CPG). By combining two-photon calcium imaging with electrophysiology, immunohistochemistry and genetic tools, we report that astrocytes display Ca2+ transients before and during oscillations of neighboring neurons. Inhibition of astrocytic Ca2+ transients with BAPTA decreases the barium-sensitive Kir4.1 current responsible of K+ clearance. Finally, we show in mice that Kir4.1 knockdown in astrocytes progressively prevents neuronal oscillations and alters the locomotor pattern resulting in lower motor performances in challenging tasks. These data identify astroglial Kir4.1 channels as key regulators of neuronal rhythmogenesis in the CPG driving locomotion.

Versatile control of synaptic circuits by astrocytes: where, when and how?

Close structural and functional interactions of astrocytes with synapses play an important role in brain function. The repertoire of ways in which astrocytes can regulate synaptic transmission is complex so that they can both promote and dampen synaptic efficacy. Such contrasting effects raise questions regarding the determinants of these divergent astroglial functions. Recent findings provide insights into where, when and how astroglial regulation of synapses takes place by revealing major molecular and functional intrinsic heterogeneity as well as switches in astrocytes occurring during development or specific patterns of neuronal activity. Astrocytes may therefore be seen as boosters or gatekeepers of synaptic circuits depending on their intrinsic and transformative properties throughout life.

Activation of the PI3K/AKT/mTOR Pathway in Cajal-Retzius Cells Leads to Their Survival and Increases Susceptibility to Kainate-Induced Seizures.

Cajal-Retzius cells (CRs) are a class of transient neurons in the mammalian cortex that play a critical role in cortical development. Neocortical CRs undergo almost complete elimination in the first two postnatal weeks in rodents and the persistence of CRs during postnatal life has been detected in pathological conditions related to epilepsy. However, it is unclear whether their persistence is a cause or consequence of these diseases. To decipher the molecular mechanisms involved in CR death, we investigated the contribution of the PI3K/AKT/mTOR pathway as it plays a critical role in cell survival. We first showed that this pathway is less active in CRs after birth before massive cell death. We also explored the spatio-temporal activation of both AKT and mTOR pathways and reveal area-specific differences along both the rostro-caudal and medio-lateral axes. Next, using genetic approaches to maintain an active pathway in CRs, we found that the removal of either PTEN or TSC1, two negative regulators of the pathway, lead to differential CR survivals, with a stronger effect in the Pten model. Persistent cells in this latter mutant are still active. They express more Reelin and their persistence is associated with an increase in the duration of kainate-induced seizures in females. Altogether, we show that the decrease in PI3K/AKT/mTOR activity in CRs primes these cells to death by possibly repressing a survival pathway, with the mTORC1 branch contributing less to the phenotype.

Fast calcium transients in dendritic spines driven by extreme statistics.

Fast calcium transients (<10 ms) remain difficult to analyse in cellular microdomains, yet they can modulate key cellular events such as trafficking, local ATP production by endoplasmic reticulum-mitochondria complex (ER-mitochondria complex), or spontaneous activity in astrocytes. In dendritic spines receiving synaptic inputs, we show here that in the presence of a spine apparatus (SA), which is an extension of the smooth ER, a calcium-induced calcium release (CICR) is triggered at the base of the spine by the fastest calcium ions arriving at a Ryanodyne receptor (RyR). The mechanism relies on the asymmetric distributions of RyRs and sarco/ER calcium-ATPase (SERCA) pumps that we predict using a computational model and further confirm experimentally in culture and slice hippocampal neurons. The present mechanism for which the statistics of the fastest particles arriving at a small target, followed by an amplification, is likely to be generic in molecular transduction across cellular microcompartments, such as thin neuronal processes, astrocytes, endfeets, or protrusions.

Modeling and Targeting Neuroglial Interactions with Human Pluripotent Stem Cell Models.

Generation of relevant and robust models for neurological disorders is of main importance for both target identification and drug discovery. The non-cell autonomous effects of glial cells on neurons have been described in a broad range of neurodegenerative and neurodevelopmental disorders, pointing to neuroglial interactions as novel alternative targets for therapeutics development. Interestingly, the recent breakthrough discovery of human induced pluripotent stem cells (hiPSCs) has opened a new road for studying neurological and neurodevelopmental disorders "in a dish". Here, we provide an overview of the generation and modeling of both neuronal and glial cells from human iPSCs and a brief synthesis of recent work investigating neuroglial interactions using hiPSCs in a pathophysiological context.

Astroglial Connexins Inactivation Increases Relapse of Depressive-like Phenotype after Antidepressant Withdrawal.

Studies suggest that astrocytic connexins (Cx) have an important role in the regulation of high brain functions through their ability to establish fine-tuned communication with neurons within the tripartite synapse. In light of these properties, growing evidence suggests a role of Cx in psychiatric disorders such as major depression but also in the therapeutic activity of antidepressant drugs. However, the real impact of Cx on treatment response and the underlying neurobiological mechanisms remain yet to be clarified. On this ground, the present study was designed to evaluate the functional activity of Cx in a mouse model of depression based on chronic corticosterone exposure and to determine to which extent their pharmacological inactivation influences the antidepressant-like activity of venlafaxine (VENLA). On the one hand, our results indicate that depressed mice have impaired Cx-based gap-junction and hemichannel activities. On the other hand, while VENLA exerts robust antidepressant-like activity in depressed mice; this effect is abolished by the pharmacological inhibition of Cx with carbenoxolone (CBX). Interestingly, the combination of VENLA and CBX is also associated with a higher rate of relapse after treatment withdrawal. To our knowledge, this study is one of the first to develop a model of relapse, and our results reveal that Cx-mediated dynamic neuroglial interactions play a critical role in the efficacy of monoaminergic antidepressant drugs, thus providing new targets for the treatment of depression.

Distinct P2Y Receptors Mediate Extension and Retraction of Microglial Processes in Epileptic and Peritumoral Human Tissue.

Microglia exhibit multiple, phenotype-dependent motility patterns often triggered by purinergic stimuli. However, little data exist on motility of human microglia in pathological situations. Here we examine motility of microglia stained with a fluorescent lectin in tissue slices from female and male epileptic patients diagnosed with mesial temporal lobe epilepsy or cortical glioma (peritumoral cortex). Microglial shape varied from ramified to amoeboid cells predominantly in regions of high neuronal loss or closer to a tumor. Live imaging revealed unstimulated or purine-induced microglial motilities, including surveillance movements, membrane ruffling, and process extension or retraction. At different concentrations, ADP triggered opposing motilities. Low doses triggered process extension. It was suppressed by P2Y12 receptor antagonists, which also reduced process length and surveillance movements. Higher purine doses caused process retraction and membrane ruffling, which were blocked by joint application of P2Y1 and P2Y13 receptor antagonists. Purinergic effects on motility were similar for all microglia tested. Both amoeboid and ramified cells from mesial temporal lobe epilepsy or peritumoral cortex tissue expressed P2Y12 receptors. A minority of microglia expressed the adenosine A2A receptor, which has been linked with process withdrawal of rodent cells. Laser-mediated tissue damage let us test the functional significance of these effects. Moderate damage induced microglial process extension, which was blocked by P2Y12 receptor antagonists. Overall, the purine-induced motility of human microglia in epileptic tissue is similar to that of rodent microglia in that the P2Y12 receptor initiates process extension. It differs in that retraction is triggered by joint activation of P2Y1/P2Y13 receptors.SIGNIFICANCE STATEMENT Microglial cells are brain-resident immune cells with multiple functions in healthy or diseased brains. These diverse functions are associated with distinct phenotypes, including different microglial shapes. In the rodent, purinergic signaling is associated with changes in cell shape, such as process extension toward tissue damage. However, there are little data on living human microglia, especially in diseased states. We developed a reliable technique to stain microglia from epileptic and glioma patients to examine responses to purines. Low-intensity purinergic stimuli induced process extension, as in rodents. In contrast, high-intensity stimuli triggered a process withdrawal mediated by both P2Y1 and P2Y13 receptors. P2Y1/P2Y13 receptor activation has not previously been linked to microglial morphological changes.

The many ways astroglial connexins regulate neurotransmission and behavior.

Astrocytes have emerged as major players in the brain, contributing to many functions such as energy supply, neurotransmission, and behavior. They accomplish these functions in part via their capacity to form widespread intercellular networks and to release neuroactive factors, which can modulate neurotransmission at different levels, from individual synapses to neuronal networks. The extensive network communication of astrocytes is primarily mediated by gap junction channels composed of two connexins, Cx30 and Cx43, which present distinct temporal and spatial expression patterns. Yet, astroglial connexins are also involved in direct exchange with the extracellular space via hemichannels, as well as in adhesion and signaling processes via unconventional nonchannel functions. Accumulating evidence indicate that astrocytes modulate neurotransmission and behavior through these diverse connexin functions. We here review the many ways astroglial connexins regulate neuronal activity from the molecular level to behavior.

Astroglial Cx30 differentially impacts synaptic activity from hippocampal principal cells and interneurons.

Astrocytes play important roles in brain function via dynamic structural and functional interactions with neurons. Yet the underlying mechanisms remain poorly defined. A typical feature of astrocytes is the high expression of connexins, which mediate their extensive intercellular communication and regulate their structural properties. In particular, connexin 30 (Cx30), one of the two connexins abundantly expressed by astrocytes, was recently shown to be a critical regulator of excitatory synaptic transmission by controlling the astroglial coverage of synapses. However, the role of Cx30 in the regulation of inhibitory synaptic transmission and excitatory/inhibitory balance remains elusive. Here, we investigated the role of astroglial Cx30 on the electrophysiological and morphological properties of five classes of hippocampal CA1 stratum oriens and pyramidale neurons, defined by the unsupervised Ward's clustering. Using Cx30 knockout mice, we found that Cx30 alters specific properties of some subsets of CA1 interneurons, such as resting membrane potential and sag ratio, while other parameters, such as action potential threshold and saturation frequency, were more frequently altered among the different classes of neurons. The excitation-inhibition balance was also differentially and selectively modulated among the different neuron subtypes. Only slight morphological differences were observed on reconstructed neurons. Altogether, these data indicate that Cx30 differentially alters the electrophysiological and morphological properties of hippocampal cell populations, and modulates both their excitatory and inhibitory inputs. Astrocytes, via Cx30, are thus active modulators of both excitatory and inhibitory synapses in the hippocampus.

Connexin 30 controls astroglial polarization during postnatal brain development.

Astrocytes undergo intense morphological maturation during development, changing from individual sparsely branched cells to polarized and tremendously ramified cells. Connexin 30, an astroglial gap-junction channel-forming protein expressed postnatally, regulates in situ the extension and ramification of astroglial processes. However, the involvement of connexin 30 in astroglial polarization, which is known to control cell morphology, remains unexplored. We found that connexin 30, independently of gap-junction-mediated intercellular biochemical coupling, alters the orientation of astrocyte protrusion, centrosome and Golgi apparatus during polarized migration in an in vitro wound-healing assay. Connexin 30 sets the orientation of astroglial motile protrusions via modulation of the laminin/ß1 integrin/Cdc42 polarity pathway. Connexin 30 indeed reduces laminin levels, inhibits the redistribution of the ß1-integrin extracellular matrix receptors, and inhibits the recruitment and activation of the small Rho GTPase Cdc42 at the leading edge of migrating astrocytes. In vivo, connexin 30, the expression of which is developmentally regulated, also contributes to the establishment of hippocampal astrocyte polarity during postnatal maturation. This study thus reveals that connexin 30 controls astroglial polarity during development.

Pannexin 1 channels and ATP release in epilepsy: two sides of the same coin : The contribution of pannexin-1, connexins, and CALHM ATP-release channels to purinergic signaling.

Purinergic signaling mediated by ATP and its metabolites contributes to various brain physiological processes as well as to several pathological conditions, including neurodegenerative and neurological disorders, such as epilepsy. Among the different ATP release pathways, pannexin 1 channels represent one of the major conduits being primarily activated in pathological contexts. Investigations on in vitro and in vivo models of epileptiform activity and seizures in mice and human tissues revealed pannexin 1 involvement in aberrant network activity and epilepsy, and highlighted that pannexin 1 exerts a complex role. Pannexin 1 can indeed either sustain seizures through release of ATP that can directly activate purinergic receptors, or tune down epileptic activity via ATP-derived adenosine that decreases neuronal excitability. Interestingly, in-depth analysis of the literature unveils that this dichotomy is only apparent, as it depends on the model of seizure induction and the type of evoked epileptiform activity, two factors that can differentially activate pannexin 1 channels and trigger distinct intracellular signaling cascades. Here, we review the general properties and ATP permeability of pannexin 1 channels, and discuss their impact on acute epileptiform activity and chronic epilepsy according to the regime of activity and disease state. These data pave the way for the development of new antiepileptic strategies selectively targeting pannexin 1 channels in a context-dependent manner.

Neuronal Activity Drives Astroglial Connexin 30 in Perisynaptic Processes and Shapes Its Functions.

Astrocytes play key roles in brain functions through dynamic interactions with neurons. One of their typical features is to express high levels of connexins (Cxs), Cx43 and Cx30, the gap junction (GJ)-forming proteins. Cx30 is involved in basic cognitive processes and shapes synaptic and network activities, as shown by recent studies in transgenic animals. Yet it remains unknown whether astroglial Cx30 expression, localization, and functions are endogenously and dynamically regulated by neuronal activity and could therefore play physiological roles in neurotransmission. We here show that neuronal activity increased hippocampal Cx30 protein levels via a posttranslational mechanism regulating lysosomal degradation. Neuronal activity also increased Cx30 protein levels at membranes and perisynaptic processes, as revealed by superresolution imaging. This translated at the functional level in the activation of Cx30 hemichannels and in Cx30-mediated remodeling of astrocyte morphology independently of GJ biochemical coupling. Altogether, these data show activity-dependent dynamics of Cx30 expression, perisynaptic localization, and functions.

The intellectual disability protein Oligophrenin-1 controls astrocyte morphology and migration.

Astrocytes are involved in several aspects of neuronal development and properties which are altered in intellectual disability (ID). Oligophrenin-1 is a RhoGAP protein implicated in actin cytoskeleton regulation, and whose mutations are associated with X-linked ID. Oligophrenin-1 is expressed in neurons, where its functions have been widely reported at the synapse, as well as in glial cells. However, its roles in astrocytes are still largely unexplored. Using in vitro and in vivo models of oligophrenin1 disruption in astrocytes, we found that oligophrenin1 regulates at the molecular level the RhoA/ROCK/MLC2 pathway in astroglial cells. We also showed at the cellular level that oligophrenin1 modulates astrocyte morphology and migration both in vitro and in vivo, and is involved in glial scar formation. Altogether, these data suggest that oligophrenin1 deficiency alters not only neuronal but also astrocytic functions, which might contribute to the development of ID.

Gamma-aminobutyric acidergic transmission underlies interictal epileptogenicity in pediatric focal cortical dysplasia.

OBJECTIVE: Dysregulation of γ-aminobutyric acidergic (GABAergic) transmission has been reported in lesional acquired epilepsies (gliomas, hippocampal sclerosis). We investigated its involvement in a developmental disorder, human focal cortical dysplasia (FCD), focusing on chloride regulation driving GABAergic signals. METHODS: In vitro recordings of 47 human cortical acute slices from 11 pediatric patients who received operations for FCD were performed on multielectrode arrays. GABAergic receptors and chloride regulators were pharmacologically modulated. Immunostaining for chloride cotransporter KCC2 and interneurons were performed on recorded slices to correlate electrophysiology and expression patterns. RESULTS: FCD slices retain intrinsic epileptogenicity. Thirty-six of 47 slices displayed spontaneous interictal discharges, along with a pattern specific to the histological subtypes. Ictal discharges were induced in proepileptic conditions in 6 of 8 slices in the areas generating spontaneous interictal discharges, with a transition to seizure involving the emergence of preictal discharges. Interictal discharges were sustained by GABAergic signaling, as a GABAA receptor blocker stopped them in 2 of 3 slices. Blockade of NKCC1 Cl- cotransporters further controlled interictal discharges in 9 of 12 cases, revealing a Cl- dysregulation affecting actions of GABA. Immunohistochemistry highlighted decreased expression and changes in KCC2 subcellular localization and a decrease in the number of GAD67-positive interneurons in regions generating interictal discharges. INTERPRETATION: Altered chloride cotransporter expression and changes in interneuron density in FCD may lead to paradoxical depolarization of pyramidal cells. Spontaneous interictal discharges are consequently mediated by GABAergic signals, and targeting chloride regulation in neurons may be considered for the development of new antiepileptic drugs. Ann Neurol 2019; 1-14 ANN NEUROL 2019;85:204-217.

Astroglial Cx30 sustains neuronal population bursts independently of gap-junction mediated biochemical coupling.

Astroglial networks mediated by gap junction channels contribute to neurotransmission and promote neuronal coordination. Connexin 30, one of the two main astroglial gap junction forming protein, alters at the behavioral level the reactivity of mice to novel environment and at the synaptic level excitatory transmission. However, the role and function of Cx30 at the neuronal network level remain unclear. We thus investigated whether Cx30 regulates neuronal population bursts and associated convulsive behavior. We found in vivo that Cx30 is upregulated by kainate-induced seizures and that it regulates in turn the severity of associated behavioral seizures. Using electrophysiology ex vivo, we report that Cx30 regulates aberrant network activity via control of astroglial glutamate clearance independently of gap-junction mediated biochemical coupling. Altogether, our results indicate that astroglial Cx30 is an important player in orchestrating neuronal network activity.

New Insights on Astrocyte Ion Channels: Critical for Homeostasis and Neuron-Glia Signaling.

Initial biophysical studies on glial cells nearly 50 years ago identified these cells as being electrically silent. These first studies also demonstrated a large K(+) conductance, which led to the notion that glia may regulate extracellular K(+) levels homeostatically. This view has now gained critical support from the study of multiple disease models discussed herein. Dysfunction of a major astrocyte K(+) channel, Kir4.1, appears as an early pathological event underlying neuronal phenotypes in several neurodevelopmental and neurodegenerative diseases. An expanding list of other astrocyte ion channels, including the calcium-activated ion channel BEST-1, hemichannels, and two-pore domain K(+) channels, all contribute to astrocyte biology and CNS function and underpin new forms of crosstalk between neurons and glia. Once considered merely the glue that holds the brain together, it is now increasingly recognized that astrocytes contribute in several fundamental ways to neuronal function. Emerging new insights and future perspectives of this active research area are highlighted within. SIGNIFICANCE STATEMENT: The critical role of astrocyte potassium channels in CNS homeostasis has been reemphasized by recent studies conducted in animal disease models. Emerging evidence also supports the signaling role mediated by astrocyte ion channels such as BEST1, hemichannels, and two-pore channels, which enable astrocytes to interact with neurons and regulate synaptic transmission and plasticity. This minisymposium highlights recent developments and future perspectives of these research areas.

A Highly Selective Potassium Sensor for the Detection of Potassium in Living Tissues.

The development of highly selective sensors for potassium is of great interest in biology. Two new hydrosoluble potassium sensors (Calix-COU-Alkyne and Calix-COU-Am) based on a calix[4]arene bis(crown-6) and an extended coumarin were synthesized and characterized. The photophysical properties and complexation studies of these compounds have been investigated and show high molar extinction coefficients and high fluorescence quantum yields. Upon complexation with potassium in the millimolar concentration range, an increase of one- and two-photon fluorescence emission is detected. A twofold fluorescence enhancement is observed upon excitation at λ=405 nm. The ligands present excellent selectivity for potassium in the presence of various competitive cations in water and in a physiological medium. The photophysical properties are not affected by the presence of a large amount of competing cations (Na+ , Ca2+ , Mg2+ , etc.). Ex vivo measurements on mouse hippocampal slices show that Calix-COU-Alkyne accumulates extracellularly and does not alter the neuronal activity. Furthermore, the sensor can be utilized to monitor slow extracellular K+ increase induced by inhibition of K+ entry into the cells.