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Abstract Intestinal smooth muscle differentiation is a complex physico-biological process involving several different pathways. Here, we investigate the properties of Ca2+ waves in the developing intestinal mesenchyme using GCamp6f expressing mouse embryos and investigate their relationship with smooth muscle differentiation. We find that Ca2+ waves are absent in the pre-differentiation mesenchyme and start propagating immediately following α-SMA expression. Ca2+ waves are abrogated by CaV1.2 and gap-junction blockers, but are independent of the Rho pathway. The myosine light-chain kinase inhibitor ML-7 strongly disorganized or abolished Ca2+ waves, showing that perturbation of the contractile machinery at the myosine level also affected the upstream Ca2+ handling chain. Inhibiting Ca2+ waves and contractility with CaV1.2 blockers did not perturb circular smooth muscle differentiation at early stages. At later stages, CaV1.2 blockers abolished intestinal elongation and differentiation of the longitudinal smooth muscle, leading instead to the emergence of KIT-expressing interstitial cells of Cajal at the gut periphery. CaV1.2 blockers also drove apoptosis of already differentiated, CaV1.2-expressing smooth muscle and enteric neural cells. We provide fundamental new data on Ca2+ waves in the developing murine gut and their relation to myogenesis in this organ.

Lenadogene nolparvovec is a gene therapy which has been developed to treat Leber hereditary optic neuropathy (LHON) caused by a point mutation in the mitochondrial NADH dehydrogenase 4 (ND4) gene. Clinical trials have demonstrated a significant improvement of visual acuity up to 5 years after treatment by lenadogene nolparvovec but, surprisingly, unilateral treatment resulted in bilateral improvement of vision. This contralateral effect – similarly observed with other gene therapy products in development for MT-ND4-LHON – is supported by the migration of viral vector genomes and their transcripts to the contralateral eye, as reported in animals, and post-mortem samples from two patients. In this study, we used an AAV2 encoding fluorescent proteins targeting mitochondria to investigate whether these organelles themselves could transfer from the treated eye to the fellow one. We found that mitochondria travel along the visual system (optic chiasm and primary visual cortex) and reach the contralateral eye (optic nerve and retina) in physiological conditions. We also observed that, in a rotenone-induced model of retinal damage mimicking LHON, mitochondrial transfer from the healthy to the damaged eye was accelerated and enhanced. Our results thus provide a further explanation for the contralateral beneficial effect observed during clinical studies with lenadogene nolparvovec.

Abstract Fragile X syndrome (FXS) is an inherited form of intellectual disability caused by the loss of the mRNA-binding fragile X mental retardation protein (FMRP). FXS is characterized by neuronal hyperexcitability and behavioral defects, however the mechanisms underlying these critical dysfunctions remain unclear. Here, using male Fmr1 knockout mouse model of FXS, we identify abnormal extracellular potassium homeostasis, along with impaired potassium channel Kir4.1 expression and function in astrocytes. Further, we reveal that Kir4.1 mRNA is a binding target of FMRP. Finally, we show that the deficit in astroglial Kir4.1 underlies neuronal hyperexcitability and several behavioral defects in Fmr1 knockout mice. Viral delivery of Kir4.1 channels specifically to hippocampal astrocytes from Fmr1 knockout mice indeed rescues normal astrocyte potassium uptake, neuronal excitability, and cognitive and social performance. Our findings uncover an important role for astrocyte dysfunction in the pathophysiology of FXS, and identify Kir4.1 channel as a potential therapeutic target for FXS.

Summary: Astroglial networks closely interact with neuronal populations, but their functional contribution to neuronal representation of sensory information remains unexplored. The superior colliculus (SC) integrates multi-sensory information by generating distinct spatial patterns of neuronal functional responses to specific sensory stimulation. Here, we report that astrocytes from the mouse SC form extensive networks in the retinorecipient layer compared to visual cortex. This strong astroglial connectivity relies on high expression of gap-junction proteins. Genetic disruption of this connectivity functionally impairs SC retinotopic and orientation preference responses. These alterations are region specific, absent in primary visual cortex, and associated at the circuit level with a specific impairment of collicular neurons synaptic transmission. This has implications for SC-related visually induced innate behavior, as disrupting astroglial networks impairs light-evoked temporary arrest. Our results indicate that astroglial networks shape synaptic circuit activity underlying SC functional visual responses and play a crucial role in integrating visual cues to drive sensory-motor behavior.

Summary: Throughout the brain, astrocytes form networks mediated by gap junction channels that promote the activity of neuronal ensembles. Although their inputs on neuronal information processing are well established, how molecular gap junction channels shape neuronal network patterns remains unclear. Here, using astroglial connexin-deficient mice, in which astrocytes are disconnected and neuronal bursting patterns are abnormal, we show that astrocyte networks strengthen bursting activity via dynamic regulation of extracellular potassium levels, independently of glutamate homeostasis or metabolic support. Using a facilitation-depression model, we identify neuronal afterhyperpolarization as the key parameter underlying bursting pattern regulation by extracellular potassium in mice with disconnected astrocytes. We confirm this prediction experimentally and reveal that astroglial network control of extracellular potassium sustains neuronal afterhyperpolarization via KCNQ voltage-gated K+ channels. Altogether, these data delineate how astroglial gap junctions mechanistically strengthen neuronal population bursts and point to approaches for controlling aberrant activity in neurological diseases.

Cajal-Retzius neurons number drastically decreases during postnatal life. Here, authors show that their programmed death is required for the construction of functional hippocampal circuits and memory with aberrant survival leading to gamma rhythmopathies and susceptibility to seizures.

Myotonic dystrophy type 1 (DM1) is characterized by debilitating neurological symptoms. Dinca et al. demonstrate the pronounced impact of DM1 on the morphology and RNA metabolism of astrocytes. Their findings suggest astroglial pathology in DM1 brain dysfunction.

Summary: The regulation of translation in astrocytes, the main glial cells in the brain, remains poorly characterized. We developed a high-throughput proteomics screen for polysome-associated proteins in astrocytes and focused on ribosomal protein receptor of activated protein C kinase 1 (RACK1), a critical factor in translational regulation. In astrocyte somata and perisynaptic astrocytic processes (PAPs), RACK1 preferentially binds to a number of mRNAs, including Kcnj10, encoding the inward-rectifying potassium (K+) channel Kir4.1. By developing an astrocyte-specific, conditional RACK1 knockout mouse model, we show that RACK1 represses production of Kir4.1 in hippocampal astrocytes and PAPs. Upregulation of Kir4.1 in the absence of RACK1 increases astrocytic Kir4.1-mediated K+ currents and volume. It also modifies neuronal activity attenuating burst frequency and duration. Reporter-based assays reveal that RACK1 controls Kcnj10 translation through the transcript’s 5′ untranslated region. Hence, translational regulation by RACK1 in astrocytes represses Kir4.1 expression and influences neuronal activity.

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. Upregulation of connexin 30 (Cx30) in astrocytes increases the connectivity of astroglial networks, but decreases spontaneous and evoked synaptic transmission, altering the induction of synaptic plasticity and in vivo learning processes, and suggesting that astroglial networks may have a physiologically optimized size to appropriately regulate neuronal functions.

The authors present a fluorescent probe that tracks glutamine in live cells. They demonstrate the capabilities of the probe by providing direct visual evidence of an activity-dependent glutamine supply from astroglial networks to presynaptic structures under physiological conditions.

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.

The blood-brain barrier is formed by capillary endothelial cells expressing connexin 37 (Cx37), Cx40, and Cx43 and is joined by closely apposed astrocytes expressing Cx43 and Cx30. We investigated whether connexin-targeting peptides could limit barrier leakage triggered by LPS-induced systemic inflammation in mice. Intraperitoneal LPS administration increased endothelial and astrocytic Cx43 expression; elevated TNF-α, IL-1β, IFN-γ, and IL-6 in plasma and IL-6 in the brain; and induced barrier leakage recorded over 24 hours. Barrier leakage was largely prevented by global Cx43 knockdown and Cx43/Cx30 double knockout in astrocytes, slightly diminished by endothelial Cx43 knockout, and not protected by global Cx30 knockout. Intravenous administration of Gap27 or Tat-Gap19 peptides just before LPS also prevented barrier leakage, and intravenously administered BAPTA-AM to chelate intracellular calcium was equally effective. Patch-clamp experiments demonstrated LPS-induced Cx43 hemichannel opening in endothelial cells, which was suppressed by Gap27, Gap19, and BAPTA. LPS additionally triggered astrogliosis that was prevented by intravenous Tat-Gap19 or BAPTA-AM. Cortically applied Tat-Gap19 or BAPTA-AM to primarily target astrocytes also strongly diminished barrier leakage. In vivo dye uptake and in vitro patch-clamp showed Cx43 hemichannel opening in astrocytes that was induced by IL-6 in a calcium-dependent manner. We conclude that targeting endothelial and astrocytic connexins is a powerful approach to limit barrier failure and astrogliosis.

Connexin 43, an astroglial gap junction protein, is enriched in perisynaptic astroglial processes and plays major roles in synaptic transmission. We have previously found that astroglial Cx43 controls synaptic glutamate levels and allows for activity-dependent glutamine release to sustain physiological synaptic transmissions and cognitiogns. However, whether Cx43 is important for the release of synaptic vesicles, which is a critical component of synaptic efficacy, remains unanswered. Here, using transgenic mice with a glial conditional knockout of Cx43 (Cx43−/−), we investigate whether and how astrocytes regulate the release of synaptic vesicles from hippocampal synapses. We report that CA1 pyramidal neurons and their synapses develop normally in the absence of astroglial Cx43. However, a significant impairment in synaptic vesicle distribution and release dynamics were observed. In particular, the FM1-43 assays performed using two-photon live imaging and combined with multi-electrode array stimulation in acute hippocampal slices, revealed a slower rate of synaptic vesicle release in Cx43−/− mice. Furthermore, paired-pulse recordings showed that synaptic vesicle release probability was also reduced and is dependent on glutamine supply via Cx43 hemichannel (HC). Taken together, we have uncovered a role for Cx43 in regulating presynaptic functions by controlling the rate and probability of synaptic vesicle release. Our findings further highlight the significance of astroglial Cx43 in synaptic transmission and efficacy.

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

Oligophrenin-1 (OPHN1) is a Rho-GTPase-activating protein (RhoGAP), whose mutations are associated with X-linked intellectual disability (XLID). OPHN1 is enriched at the synapse in both pre- and postsynaptic compartments, where it regulates the RhoA/ROCK/MLC2 signaling pathway, playing a critical role in cytoskeleton remodeling and vesicle recycling. Ophn1 knockout (KO) adult mice display some behavioral deficits in multiple tasks, reminiscent of some symptoms in the human pathology. We also previously reported a reduction in dendritic spine density in the adult hippocampus of KO mice. Yet the nature of the deficits occurring in these mice during postnatal development remains elusive. Here, we show that juvenile KO mice present normal basal synaptic transmission, but altered synaptic plasticity, with a selective impairment in long-term depression, but no change in long-term potentiation. This contrasts with the functional deficits that these mice display at the adult stage, as we found that both basal synaptic transmission and long-term potentiation are reduced at later stages, due to presynaptic alterations. In addition, the number of excitatory synapses in adult is increased, suggesting some unsuccessful compensation. Altogether, these results suggest that OPHN1 function at synapses is differentially affected during maturation of the brain, which provides some therapeutic opportunities for early intervention.

Summary: Local translation is a conserved mechanism conferring cells the ability to quickly respond to local stimuli. In the brain, it has been recently reported in astrocytes, whose fine processes contact blood vessels and synapses. Yet the specificity and regulation of astrocyte local translation remain unknown. We study hippocampal perisynaptic astrocytic processes (PAPs) and show that they contain the machinery for translation. Using a refined immunoprecipitation technique, we characterize the entire pool of ribosome-bound mRNAs in PAPs and compare it with the one expressed in the whole astrocyte. We find that a specific pool of mRNAs is highly polarized at the synaptic interface. These transcripts encode an unexpected molecular repertoire, composed of proteins involved in iron homeostasis, translation, cell cycle, and cytoskeleton. Remarkably, we observe alterations in global RNA distribution and ribosome-bound status of some PAP-enriched transcripts after fear conditioning, indicating the role of astrocytic local translation in memory and learning.

Abstract Epilepsy is a neurological condition that affects 1% of the world population. Conventional treatments of epilepsy use drugs targeting neuronal excitability, inhibitory or excitatory transmission. Yet, one third of patients presents an intractable form of epilepsy and fails to respond to pharmacological anti-epileptic strategies. The ketogenic diet is a well-established non-pharmacological treatment that has been proven to be effective in reducing seizure frequency in the pharmaco-resistant patients. This dietary solution is however extremely restrictive and can be associated with complications caused by the high [fat]:[carbohydrate + protein] ratio. Recent advances suggest that the traditional 4:1 ratio of the ketogenic diet is not a requisite for its therapeutic effect. We show here that combining nutritional strategies targeting specific amino-acids, carbohydrates and fatty acids with a low [fat]:[proteins + carbohydrates] ratio also reduces excitatory drive and protects against seizures to the same extent as the ketogenic diet. Similarly, the morphological and molecular correlates of temporal lobe seizures were reduced in animals fed with the combined diet. These results provide evidence that low-fat dietary strategies more palatable than the ketogenic diet could be useful in epilepsy.

Fast calcium transients (

The metabolic syndrome, which comprises obesity and diabetes, is a major public health problem and the awareness of energy homeostasis control remains an important worldwide issue. The energy balance is finely regulated by the central nervous system (CNS), notably through neuronal networks, located in the hypothalamus and the dorsal vagal complex (DVC), which integrate nutritional, humoral and nervous information from the periphery. The glial cells’ contribution to these processes emerged few year ago. However, its underlying mechanism remains unclear. Glial connexin 43 hemichannels (Cx43 HCs) enable direct exchange with the extracellular space and can regulate neuronal network activity. In the present study, we sought to determine the possible involvement of glial Cx43 HCs in energy balance regulation. We here show that Cx43 is strongly expressed in the hypothalamus and DVC and is associated with glial cells. Remarkably, we observed a close apposition of Cx43 with synaptic elements in both the hypothalamus and DVC. Moreover, the expression of hypothalamic Cx43 mRNA and protein is modulated in response to fasting and diet-induced obesity. Functionally, we found that Cx43 HCs are largely open in the arcuate nucleus (ARC) from acute mice hypothalamic slices under basal condition, and significantly inhibited by TAT-GAP19, a mimetic peptide that specifically blocks Cx43 HCs activity. Moreover, intracerebroventricular (i.c.v.) TAT-GAP19 injection strongly decreased food intake, without further alteration of glycaemia, energy expenditures or locomotor activity. Using the immediate early gene c-Fos expression, we found that i.c.v. TAT-GAP19 injection induced neuronal activation in hypothalamic and brainstem nuclei dedicated to food intake regulation. Altogether, these results suggest a tonic delivery of orexigenic molecules associated with glial Cx43 HCs activity and a possible modulation of this tonus during fasting and obesity.

The plasticity of the cytoskeleton architecture and membrane properties is important for the establishment of cell polarity, adhesion and migration. Here, we present a method which combines stimulated emission depletion (STED) super-resolution imaging and atomic force microscopy (AFM) to correlate cytoskeletal structural information with membrane physical properties in live astrocytes. Using STED compatible dyes for live cell imaging of the cytoskeleton, and simultaneously mapping the cell surface topology with AFM, we obtain unprecedented detail of highly organized networks of actin and microtubules in astrocytes. Combining mechanical data from AFM with optical imaging of actin and tubulin further reveals links between cytoskeleton organization and membrane properties. Using this methodology we illustrate that scratch-induced migration induces cytoskeleton remodeling. The latter is caused by a polarization of actin and microtubule elements within astroglial cell processes, which correlates strongly with changes in cell stiffness. The method opens new avenues for the dynamic probing of the membrane structural and functional plasticity of living brain cells. It is a powerful tool for providing new insights into mechanisms of cell structural remodeling during physiological or pathological processes, such as brain development or tumorigenesis.

Neuronal excitability relies on inward sodium and outward potassium fluxes during action potentials. To prevent neuronal hyperexcitability, potassium ions have to be taken up quickly. However, the dynamics of the activity-dependent potassium fluxes and the molecular pathways underlying extracellular potassium homeostasis remain elusive. To decipher the specific and acute contribution of astroglial Kir4.1 channels in controlling potassium homeostasis and the moment to moment neurotransmission, we built a tri-compartment model accounting for potassium dynamics between neurons, astrocytes and the extracellular space. We here demonstrate that astroglial Kir4.1 channels are sufficient to account for the slow membrane depolarization of hippocampal astrocytes and crucially contribute to extracellular potassium clearance during basal and high activity. By quantifying the dynamics of potassium levels in neuron-glia-extracellular space compartments, we show that astrocytes buffer within 6 to 9 seconds more than 80% of the potassium released by neurons in response to basal, repetitive and tetanic stimulations. Astroglial Kir4.1 channels directly lead to recovery of basal extracellular potassium levels and neuronal excitability, especially during repetitive stimulation, thereby preventing the generation of epileptiform activity. Remarkably, we also show that Kir4.1 channels strongly regulate neuronal excitability for slow 3 to 10 Hz rhythmic activity resulting from probabilistic firing activity induced by sub-firing stimulation coupled to Brownian noise. Altogether, these data suggest that astroglial Kir4.1 channels are crucially involved in extracellular potassium homeostasis regulating theta rhythmic activity.

Neuronal networks can generate complex patterns of activity that depend on membrane properties of individual neurons as well as on functional synapses. To decipher the impact of synaptic properties and connectivity on neuronal network behavior, we investigate the responses of neuronal ensembles from small (5-30 cells in a restricted sphere) and large (acute hippocampal slice) networks to single electrical stimulation: in both cases, a single stimulus generated a synchronous long-lasting bursting activity. While an initial spike triggered a reverberating network activity that lasted 2-5 seconds for small networks, we found here that it lasted only up to 300 milliseconds in slices. To explain this phenomena present at different scales, we generalize the depression-facilitation model and extracted the network time constants. The model predicts that the reverberation time has a bell shaped relation with the synaptic density, revealing that the bursting time cannot exceed a maximum value. Furthermore, before reaching its maximum, the reverberation time increases sub-linearly with the synaptic density of the network. We conclude that synaptic dynamics and connectivity shape the mean burst duration, a property present at various scales of the networks. Thus bursting reverberation is a property of sufficiently connected neural networks, and can be generated by collective depression and facilitation of underlying functional synapses.

Recent evidence has shown that astrocytes play essential roles in synaptic transmission and plasticity. Nevertheless, how neuronal activity alters astroglial functional properties and whether such properties also display specific forms of plasticity still remain elusive. Here, we review research findings supporting this aspect of astrocytes, focusing on their roles in the clearance of extracellular potassium and glutamate, two neuroactive substances promptly released during excitatory synaptic transmission. Their subsequent removal, which is primarily carried out by glial potassium channels and glutamate transporters, is essential for proper functioning of the brain. Similar to neurons, different forms of short- and long-term plasticity in astroglial uptake have been reported. In addition, we also present novel findings showing robust potentiation of astrocytic inward currents in response to repetitive stimulations at mild frequencies, as low as 0.75 Hz, in acute hippocampal slices. Interestingly, neurotransmission was hardly affected at this frequency range, suggesting that astrocytes may be more sensitive to low frequency stimulation and may exhibit stronger plasticity than neurons to prevent hyperexcitability. Taken together, these important findings strongly indicate that astrocytes display both short- and long-term plasticity in their clearance of excess neuroactive substances from the extracellular space, thereby regulating neuronal activity and brain homeostasis.

Synaptic transmission relies on several processes, such as the location of a released vesicle, the number and type of receptors, trafficking between the postsynaptic density (PSD) and extrasynaptic compartment, as well as the synapse organization. To study the impact of these parameters on excitatory synaptic transmission, we present a computational model for the fast AMPA-receptor mediated synaptic current. We show that in addition to the vesicular release probability, due to variations in their release locations and the AMPAR distribution, the postsynaptic current amplitude has a large variance, making a synapse an intrinsic unreliable device. We use our model to examine our experimental data recorded from CA1 mice hippocampal slices to study the differences between mEPSC and evoked EPSC variance. The synaptic current but not the coefficient of variation is maximal when the active zone where vesicles are released is apposed to the PSD. Moreover, we find that for certain type of synapses, receptor trafficking can affect the magnitude of synaptic depression. Finally, we demonstrate that perisynaptic microdomains located outside the PSD impacts synaptic transmission by regulating the number of desensitized receptors and their trafficking to the PSD. We conclude that geometrical modifications, reorganization of the PSD or perisynaptic microdomains modulate synaptic strength, as the mechanisms underlying long-term plasticity.