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We, the participants of the First Organoid Intelligence Workshop - "Forming an OI Community" (22-24 February 2022), call on the international scientific community to explore the potential of human brain-based organoid cell cultures to advance our understanding of the brain and unleash new forms of biocomputing while recognizing and addressing the associated ethical implications. The term "organoid intelligence" (OI) has been coined to describe this research and development approach (1) in a manner consistent with the term "artificial intelligence" (AI) - used to describe the enablement of computers to perform tasks normally requiring human intelligence. OI has the potential for diverse and far-reaching applications that could benefit humankind and our planet, and which urge the strategic development of OI as a collaborative scientific discipline. OI holds promise to elucidate the physiology of human cognitive functions such as memory and learning. It presents game-changing opportunities in biological and hybrid computing that could overcome significant limitations in silicon-based computing. It offers the prospect of unparalleled advances in interfaces between brains and machines. Finally, OI could allow breakthroughs in modeling and treating dementias and other neurogenerative disorders that cause an immense and growing disease burden globally. Realizing the world-changing potential of OI will require scientific breakthroughs. We need advances in human stem cell technology and bioengineering to recreate brain architectures and to model their potential for pseudo-cognitive capabilities. We need interface breakthroughs to allow us to deliver input signals to organoids, measure output signals, and employ feedback mechanisms to model learning processes. We also need novel machine learning, big data, and AI technologies to allow us to understand brain organoids.

Changes to sensory experience result in plasticity of synapses in the cortex. This experience-dependent plasticity (EDP) is a fundamental property of the brain. Yet, while much is known about neuronal roles in EDP, very little is known about the role of astrocytes. To address this issue, we used the well-described mouse whiskers-to-barrel cortex system, which expresses a number of forms of EDP. We found that all-whisker deprivation induced characteristic experience-dependent Hebbian depression (EDHD) followed by homeostatic upregulation in L2/3 barrel cortex of wild type mice. However, these changes were not seen in mutant animals (IP3R2–/–) that lack the astrocyte-expressed IP3 receptor subtype. A separate paradigm, the single-whisker experience, induced potentiation of whisker-induced response in both wild-type (WT) mice and IP3R2–/– mice. Recordings in ex vivo barrel cortex slices reflected the in vivo results so that long-term depression (LTD) could not be elicited in slices from IP3R2–/– mice, but long-term potentiation (LTP) could. Interestingly, 1 Hz stimulation inducing LTD in WT paradoxically resulted in NMDAR-dependent LTP in slices from IP3R2–/– animals. The LTD to LTP switch was mimicked by acute buffering astrocytic [Ca2+]i in WT slices. Both WT LTD and IP3R2–/– 1 Hz LTP were mediated by non-ionotropic NMDAR signaling, but only WT LTD was P38 MAPK dependent, indicating an underlying mechanistic switch. These results demonstrate a critical role for astrocytic [Ca2+]i in several EDP mechanisms in neocortex.

SummaryObjectiveHyperpolarization-activated cyclic nucleotide-gated (HCN) channels are known to be involved in the generation of absence seizures (ASs), and there is evidence that cortical and thalamic HCN channel dysfunctions may have a pro-absence role. Many HCN channel blockers are available, but their role in ASs has been investigated only by localized brain injection or in in vitro model systems due to their limited brain availability. Here, we investigated the effect on ASs of orally administered ivabradine (an HCN channel blocker approved for the treatment of heart failure in humans) following injection of the P-glycoprotein inhibitor elacridar, that is known to increase penetration into the brain of drug substrates for this efflux transporter. The action of ivabradine was also tested following in vivo microinjection in the cortical initiation network (CIN) of the somatosensory cortex and in the thalamic ventrobasal nucleus (VB) as well as on cortical and thalamocortical neurons in brain slices.MethodsWe used EEG recordings in freely moving Genetic Absence Epilepsy from Strasbourg Rats (GAERS) to assess the action of oral administration of ivabradine, with and without elacridar, on ASs. Ivabradine was also microinjected in the CIN and VB of GAERS in vivo and applied to Wistar CIN and GAERS VB slices while recording patch-clamped cortical layer 5/6 and thalamocortical neurons, respectively.ResultsOral administration of ivabradine markedly and dose-dependently reduced ASs. Ivabradine injection in CIN abolished ASs and elicited small-amplitude 4-7 Hz waves (without spikes), whereas in the VB it was less potent. Moreover, ivabradine applied to GAERS VB and Wistar CIN slices selectively decreased HCN-channel-dependent properties of cortical layer 5/6 pyramidal and thalamocortical neurons, respectively.SignificanceThese results provide the first demonstration of the anti-absence action of a systemically administered HCN channel blocker, indicating the potential of this class of drugs as a novel therapeutic avenue for ASs.

The need for human cell-based experimental platforms for the study of the brain’s most basic activity and function has never been greater. Initial progress was made through differentiation of the NT2.D1 platform, which facilitated the study of postmitotic but generic neuronal/astrocytic interactions both electrophysiologically and pharmacologically. However, induced pluripotent stem cell (iPSC) platforms can form specific neuronal and astrocytic subpopulations which are identical to those of a functioning area of the brain. Hence, not only functionality can be modeled with iPSC-derived platforms but also specific dysfunctionality in these areas, such as is seen in neurodegenerative disease. Crucially, it has become clear that the contents of the extracellular matrix (ECM) of the living brain have a disproportionate influence not only on cell development and maturity but also continued maintenance of function. For iPSC models to ascend to this level of authenticity requires new methods and technology to include a fully functional ECM in more complete models of specific brain areas. Future approaches in ECM formation, alongside more rapid iPSC-derived tissue formation through lineage reprogramming, will eventually bring human experimental platforms to a more advanced stage of development, which will be combined with the necessary flexibility and practicality for diverse applications ranging from basic research through to drug discovery.

Astrocytes are now increasingly acknowledged as having fundamental and sophisticated roles in brain function and dysfunction. Unravelling the complex mechanisms that underlie human brain astrocyte-neuron interactions is therefore an essential step on the way to understanding how the brain operates. Insights into astrocyte function to date, have almost exclusively been derived from studies conducted using murine or rodent models. Whilst these have led to significant discoveries, preliminary work with human astrocytes has revealed a hitherto unknown range of astrocyte types with potentially greater functional complexity and increased neuronal interaction with respect to animal astrocytes. It is becoming apparent, therefore, that many important functions of astrocytes will only be discovered by direct physiological interrogation of human astrocytes. Recent advancements in the field of stem cell biology have provided a source of human based models. These will provide a platform to facilitate our understanding of normal astrocyte functions as well as their role in CNS pathology. A number of recent studies have demonstrated that stem cell derived astrocytes exhibit a range of properties, suggesting that they may be functionally equivalent to their in vivo counterparts. Further validation against in vivo models will ultimately confirm the future utility of these stem-cell based approaches in fulfilling the need for human- based cellular models for basic and clinical research. In this review we discuss the roles of astrocytes in the brain and highlight the extent to which human stem cell derived astrocytes have demonstrated functional activities that are equivalent to that observed in vivo.

The brain is the most complex organ in the body, controlling our highest functions, as well as regulating myriad processes which incorporate the entire physiological system. The effects of prospective therapeutic entities on the brain and central nervous system (CNS) may potentially cause significant injury, hence, CNS toxicity testing forms part of the “core battery” of safety pharmacology studies. Drug-induced seizure is a major reason for compound attrition during drug development. Currently, the rat ex vivo hippocampal slice assay is the standard option for seizure-liability studies, followed by primary rodent cultures. These models can respond to diverse agents and predict seizure outcome, yet controversy over the relevance, efficacy, and cost of these animal-based methods has led to interest in the development of human-derived models. Existing platforms often utilize rodents, and so lack human receptors and other drug targets, which may produce misleading data, with difficulties in inter-species extrapolation. Current electrophysiological approaches are typically used in a low-throughput capacity and network function may be overlooked. Human-derived induced pluripotent stem cells (iPSCs) are a promising avenue for neurotoxicity testing, increasingly utilized in drug screening and disease modeling. Furthermore, the combination of iPSC-derived models with functional techniques such as multi-electrode array (MEA) analysis can provide information on neuronal network function, with increased sensitivity to neurotoxic effects which disrupt different pathways. The use of an in vitro human iPSC-derived neural model for neurotoxicity studies, combined with high-throughput techniques such as MEA recordings, could be a suitable addition to existing pre-clinical seizure-liability testing strategies.

The molecular pathways underlying the induction and maintenance of long-term synaptic plasticity have been extensively investigated revealing various mechanisms by which neurons control their synaptic strength. The dynamic nature of neuronal connections combined with plasticity-mediated long-lasting structural and functional alterations provide valuable insights into neuronal encoding processes as molecular substrates of not only learning and memory but potentially other sensory, motor and behavioural functions that reflect previous experience. However, one key element receiving little attention in the study of synaptic plasticity is the role of neuromodulators, which are known to orchestrate neuronal activity on brain-wide, network and synaptic scales. We aim to review current evidence on the mechanisms by which certain modulators, namely dopamine, acetylcholine, noradrenaline and serotonin, control synaptic plasticity induction through corresponding metabotropic receptors in a pathway-specific manner. Lastly, we propose that neuromodulators control plasticity outcomes through steering glutamatergic transmission, thereby gating its induction and maintenance.

Astrocytes spontaneously release glutamate (Glut) as a gliotransmitter (GT), resulting in the generation of extrasynaptic NMDAR-mediated slow inward currents (SICs) in neighboring neurons, which can increase local neuronal excitability. However, there is a deficit in our knowledge of the factors that control spontaneous astrocyte GT release and the extent of its influence. We found that, in rat brain slices, increasing the supply of the physiological transmitter Glut increased the frequency and signaling charge of SICs over an extended period. This phenomenon was replicated by exogenous preexposure to the amino acid D-aspartate (D-Asp). Using D-Asp as a “false” GT, we determined the extent of local neuron excitation by GT release in ventrobasal thalamus, CA1 hippocampus, and somatosensory cortex. By analyzing synchronized neuronal NMDAR-mediated excitation, we found that the properties of the excitation were conserved in different brain areas. In the three areas, astrocyte-derived GT release synchronized groups of neurons at distances of >;200 μm. Individual neurons participated in more than one synchronized population, indicating that individual neurons can be excited by more than one astrocyte and that individual astrocytes may determine a neuron's synchronized network. The results confirm that astrocytes can act as excitatory nodes that can influence neurons over a significant range in a number of brain regions. Our findings further suggest that chronic elevation of ambient Glut levels can lead to increased GT Glut release, which may be relevant in some pathological states. SIGNIFICANCE STATEMENT Astrocytes spontaneously release glutamate (Glut) and other gliotransmitters (GTs) that can modify neuronal activity. Exposing brain slices to Glut and D-aspartate (D-Asp) before recording resulted in an increase in frequency of GT-mediated astrocyte–neuron signaling. Using D-Asp, it was possible to investigate the effects of specific GT release at neuronal NMDARs. Calcium imaging showed synchronized activity in groups of neurons in cortex, hippocampus, and thalamus. The size of these populations was similar in all areas and some neurons were involved in more than one synchronous group. The findings show that GT release is supply dependent and that the properties of the signaling and activated networks are largely conserved between different brain areas.

An enhanced tonic GABA-A inhibition in the thalamus plays a crucial role in experimental absence seizures, and has been attributed, on the basis of indirect evidence, to a dysfunction of the astrocytic GABA transporter-1 (GAT-1). Here, the GABA transporter current was directly investigated in thalamic astrocytes from a well-established genetic model of absence seizures, the Genetic Absence Epilepsy Rats from Strasbourg (GAERS), and its non-epileptic control (NEC) strain. We also characterized the novel form of GABAergic and glutamatergic astrocyte-to-neuron signalling by recording slow outward currents (SOCs) and slow inward currents (SICs), respectively, in thalamocortical (TC) neurons of both strains. In patch-clamped astrocytes, the GABA transporter current was abolished by combined application of the selective GAT-1 and GAT-3 blocker, NO711 (30µM) and SNAP5114 (60µM), respectively, to GAERS and NEC thalamic slices. NO711 alone significantly reduced (41%) the transporter current in NEC, but had no effect in GAERS. SNAP5114 alone reduced by half the GABA transporter current in NEC, whilst it abolished it in GAERS. SIC properties did not differ between GAERS and NEC TC neurons, whilst moderate changes in SOC amplitude and kinetics were observed. These data provide the first direct demonstration of a malfunction of the astrocytic thalamic GAT-1 transporter in absence epilepsy and support an abnormal astrocytic modulation of thalamic ambient GABA levels. Moreover, while the glutamatergic astrocyte-neuron signalling is unaltered in the GAERS thalamus, the changes in some properties of the GABAergic astrocyte-neuron signaling in this epileptic strain may contribute to the generation of absence seizures.

The control of cellular water flow is mediated by the aquaporin (AQP) family of membrane proteins. The structural features of the family and the mechanism of selective water passage through the AQP pore are established, but there remains a gap in our knowledge of how water transport is regulated. Two broad possibilities exist. One is controlling the passage of water through the AQP pore, but this only has been observed as a phenomenon in some plant and microbial AQPs. An alternative is controlling the number of AQPs in the cell membrane. Here, we describe a novel pathway in mammalian cells whereby a hypotonic stimulus directly induces intracellular calcium elevations through transient receptor potential channels, which trigger AQP1 translocation. This translocation, which has a direct role in cell volume regulation, occurs within 30 s and is dependent on calmodulin activation and phosphorylation of AQP1 at two threonine residues by protein kinase C. This direct mechanism provides a rationale for the changes in water transport that are required in response to constantly changing local cellular water availability. Moreover, because calcium is a pluripotent and ubiquitous second messenger in biological systems, the discovery of its role in the regulation of AQP translocation has ramifications for diverse physiological and pathophysiological processes, as well as providing an explanation for the rapid regulation of water flow that is necessary for cell homeostasis.

The rodent ventrobasal (VB) thalamus contains a relatively uniform population of thalamocortical (TC) neurons that receive glutamatergic input from the vibrissae and the somatosensory cortex, and inhibitory input from the nucleus reticularis thalami (nRT). In this study we describe ?-aminobutyric acid (GABA)(A) receptor-dependent slow outward currents (SOCs) in TC neurons that are distinct from fast inhibitory postsynaptic currents (IPSCs) and tonic currents. SOCs occurred spontaneously or could be evoked by hypo-osmotic stimulus, and were not blocked by tetrodotoxin, removal of extracellular Ca(2+) or bafilomycin A1, indicating a non-synaptic, non-vesicular GABA origin. SOCs were more common in TC neurons of the VB compared with the dorsal lateral geniculate nucleus, and were rarely observed in nRT neurons, whilst SOC frequency in the VB increased with age. Application of THIP, a selective agonist at d-subunit-containing GABA(A) receptors, occluded SOCs, whereas the benzodiazepine site inverse agonist s-CCB had no effect, but did inhibit spontaneous and evoked IPSCs. In addition, the occurrence of SOCs was reduced in mice lacking the d-subunit, and their kinetics were also altered. The anti-epileptic drug vigabatrin increased SOC frequency in a time-dependent manner, but this effect was not due to reversal of GABA transporters. Together, these data indicate that SOCs in TC neurons arise from astrocytic GABA release, and are mediated by d-subunit-containing GABA(A) receptors. Furthermore, these findings suggest that the therapeutic action of vigabatrin may occur through the augmentation of this astrocyte-neuron interaction, and highlight the importance of glial cells in CNS (patho) physiology.

The rodent ventrobasal (VB) thalamus receives sensory inputs from the whiskers and projects to the cortex, from which it receives reciprocal excitatory afferents. Much is known about the properties and functional roles of these glutamatergic inputs to thalamocortical neurons in the VB, but no data are available on how these afferents can affect thalamic glial cells. In this study, we used combined electrophysiological recordings and intracellular calcium ([Ca(2+)](i)) imaging to investigate glial cell responses to synaptic afferent stimulation. VB thalamus glial cells can be divided into two groups based on their [Ca(2+)](i) and electrophysiological responses to sensory and corticothalamic stimulation. One group consists of astrocytes, which stain positively for S100B and preferentially load with SR101, have linear current-voltage relations and low input resistance, show no voltage-dependent [Ca(2+)](i) responses, but express mGluR5-dependent [Ca(2+)](i) transients following stimulation of the sensory and/or corticothalamic excitatory afferent pathways. Cells of the other glial group, by contrast, stain positively for NG2, and are characterized by high input resistance, the presence of voltage-dependent [Ca(2+)](i) elevations and voltage-gated inward currents. There were no synaptically induced [Ca(2+)](i) elevations in these cells under control conditions. These results show that thalamic glial cell responses to synaptic input exhibit different properties to those of thalamocortical neurons. As VB astrocytes can respond to synaptic stimulation and signal to neighbouring neurons, this glial cell organization may have functional implications for the processing of somatosensory information and modulation of behavioural state-dependent thalamocortical network activities.

Objective. Patients with rheumatoid arthritis (RA) have increased concentrations of the amino acid glutamate in synovial fluid. This study was undertaken to determine whether glutamate receptors are expressed in the synovial joint, and to determine whether activation of glutamate receptors on human synoviocytes contributes to RA disease pathology. Methods. Glutamate receptor expression was examined in tissue samples from rat knee joints and in human fibroblast-like synoviocytes (FLS). FLS from 5 RA patients and 1 normal control were used to determine whether a range of glutamate receptor antagonists influenced expression of the proinflammatory cytokine interleukin-6 (IL-6), enzymes involved in matrix degradation and cytokine processing (matrix metalloproteinase 2 [MMP-2] and MMP-9), and the inhibitors of these enzymes (tissue inhibitor of metalloproteinases 1 [TIMP-1] and TIMP-2). IL-6 concentrations were determined by enzyme-linked immunosorbent assay, MMP activity was measured by gelatin zymography, and TIMP activity was determined by reverse zymography. Fluorescence imaging of intracellular calcium concentrations in live RA FLS stimulated with specific antagonists was used to reveal functional activation of glutamate receptors that modulated IL-6 or MMP-2. Results. Ionotropic and metabotropic glutamate receptor subunit mRNA were expressed in the patella, fat pad, and meniscus of the rat knee and in human articular cartilage. Inhibition of N-methyl-D-aspartate (NMDA) receptors in RA FLS increased proMMP-2 release, whereas non-NMDA ionotropic glutamate receptor antagonists reduced IL-6 production by these cells. Stimulation with glutamate, NMDA, or kainate (KA) increased intracellular calcium concentrations in RA FLS, demonstrating functional activation of specific ionotropic glutamate receptors. Conclusion. Our findings indicate that activation of NMDA and KA glutamate receptors on human synoviocytes may contribute to joint destruction by increasing IL-6 expression. © 2007, American College of Rheumatology.

Astrocytes are now increasingly acknowledged as having fundamental and sophisticated roles in brain function and dysfunction. Unravelling the complex mechanisms that underlie human brain astrocyte–neuron interactions is therefore an essential step on the way to understanding how the brain operates. Insights into astrocyte function to date have almost exclusively been derived from studies conducted using murine or rodent models. Whilst these have led to significant discoveries, preliminary work with human astrocytes has revealed a hitherto unknown range of astrocyte types with potentially greater functional complexity and increased neuronal interaction with respect to animal astrocytes. It is becoming apparent, therefore, that many important functions of astrocytes will only be discovered by direct physiological interrogation of human astrocytes. Recent advancements in the field of stem cell biology have provided a source of human‐based models. These will provide a platform to facilitate our understanding of normal astrocyte functions as well as their role in CNS pathology. A number of recent studies have demonstrated that stem cell‐derived astrocytes exhibit a range of properties, suggesting that they may be functionally equivalent to their in vivo counterparts. Further validation against in vivo models will ultimately confirm the future utility of these stem cell‐based approaches in fulfilling the need for human‐based cellular models for basic and clinical research. In this review we discuss the roles of astrocytes in the brain and highlight the extent to which human stem cell‐derived astrocytes have demonstrated functional activities that are equivalent to those observed in vivo.

Changing the whisker complement on a rodent’s snout can lead to two forms of experience-dependent plasticity (EDP) in the neurons of the barrel cortex, where whiskers are somatotopically represented. One form, termed coding plasticity, concerns changes in synaptic transmission and connectivity between neurons. This is thought to underlie learning and memory processes and so adaptation to a changing environment. The second, called homeostatic plasticity, serves to maintain a restricted dynamic range of neuronal activity thus preventing its saturation or total downregulation. Current explanatory models of cortical EDP are almost exclusively neurocentric. However, in recent years, increasing evidence has emerged on the role of astrocytes in brain function, including plasticity. Indeed, astrocytes appear as necessary partners of neurons at the core of the mechanisms of coding and homeostatic plasticity recorded in neurons. In addition to neuronal plasticity, several different forms of astrocytic plasticity have recently been discovered. They extend from changes in receptor expression and dynamic changes in morphology to alteration in gliotransmitter release. It is however unclear how astrocytic plasticity contributes to the neuronal EDP. Here, we review the known and possible roles for astrocytes in the barrel cortex, including its plasticity.

In relaxed wakefulness, the EEG exhibits robust rhythms in the alpha band (8-13 Hz), which decelerate to theta (approximately 2-7 Hz) frequencies during early sleep. In animal models, these rhythms occur coherently with synchronized activity in the thalamus. However, the mechanisms of this thalamic activity are unknown. Here we show that, in slices of the lateral geniculate nucleus maintained in vitro, activation of the metabotropic glutamate receptor (mGluR) mGluR1a induces synchronized oscillations at alpha and theta frequencies that share similarities with thalamic alpha and theta rhythms recorded in vivo. These in vitro oscillations are driven by an unusual form of burst firing that is present in a subset of thalamocortical neurons and are synchronized by gap junctions. We propose that mGluR1a-induced oscillations are a potential mechanism whereby the thalamus promotes EEG alpha and theta rhythms in the intact brain.

In this review, we summarize three sets of findings that have recently been observed in thalamic astrocytes and neurons, and discuss their significance for thalamocortical loop dynamics. (i) A physiologically relevant ‘window’ component of the low–voltage–activated, T–type Ca 2+ current ( I Twindow ) plays an essential part in the slow (less than 1 Hz) sleep oscillation in adult thalamocortical (TC) neurons, indicating that the expression of this fundamental sleep rhythm in these neurons is not a simple reflection of cortical network activity. It is also likely that I Twindow underlies one of the cellular mechanisms enabling TC neurons to produce burst firing in response to novel sensory stimuli. (ii) Both electrophysiological and dye–injection experiments support the existence of gap junction–mediated coupling among young and adult TC neurons. This finding indicates that electrical coupling–mediated synchronization might be implicated in the high and low frequency oscillatory activities expressed by this type of thalamic neuron. (iii) Spontaneous intracellular Ca 2+ ([Ca 2+ ] i ) waves propagating among thalamic astrocytes are able to elicit large and long–lasting N –methyl–D–aspartate–mediated currents in TC neurons. The peculiar developmental profile within the first two postnatal weeks of these astrocytic [Ca 2+ ] i transients and the selective activation of these glutamate receptors point to a role for this astrocyte–to–neuron signalling mechanism in the topographic wiring of the thalamocortical loop. As some of these novel cellular and intracellular properties are not restricted to thalamic astrocytes and neurons, their significance may well apply to (patho)physiological functions of glial and neuronal elements in other brain areas.

Recent studies in the ventrobasal (VB) thalamus have shown that astrocytes display spontaneous intracellular calcium [Ca 2+ ] i oscillations early postnatally. [Ca 2+ ] i oscillations are correlated in groups of up to five astrocytes, and propagate between cells. NMDA receptor-mediated, long lasting inward currents in thalamocortical (TC) neurons of the VB complex are correlated to [Ca 2+ ] i increases in neighbouring astrocytes, and stimulation of astrocytic [Ca 2+ ] i increases also lead to inward currents in neurons. These findings suggest that astrocytes are spontaneously active and can induce neuronal activity, a reversal of the previously held view of neuron–glia interactions in the central nervous system. This activity occurs at an important period in the development of the thalamus and therefore suggests a potential functional role in a variety of processes. Along with data on the neurotransmitter receptor repertoire of thalamic astrocytes these findings enlarge the body of knowledge on astrocytes in the thalamus, and further contribute to the emerging field of astrocyte–neuron and neuron–astrocyte interactions in the central nervous system.

The link between off-target anticholinergic effects of medications and acute cognitive impairment in older adults requires urgent investigation. We aimed to determine whether a relevant in vitro model may aid the identification of anticholinergic responses to drugs and the prediction of anticholinergic risk during polypharmacy. In this preliminary study we employed a co-culture of human-derived neurons and astrocytes (NT2.N/A) derived from the NT2 cell line. NT2.N/A cells possess much of the functionality of mature neurons and astrocytes, key cholinergic phenotypic markers and muscarinic acetylcholine receptors (mAChRs). The cholinergic response of NT2 astrocytes to the mAChR agonist oxotremorine was examined using the fluorescent dye fluo-4 to quantitate increases in intracellular calcium [Ca2+]i. Inhibition of this response by drugs classified as severe (dicycloverine, amitriptyline), moderate (cyclobenzaprine) and possible (cimetidine) on the Anticholinergic Cognitive Burden (ACB) scale, was examined after exposure to individual and pairs of compounds. Individually, dicycloverine had the most significant effect regarding inhibition of the astrocytic cholinergic response to oxotremorine, followed by amitriptyline then cyclobenzaprine and cimetidine, in agreement with the ACB scale. In combination, dicycloverine with cyclobenzaprine had the most significant effect, followed by dicycloverine with amitriptyline. The order of potency of the drugs in combination frequently disagreed with predicted ACB scores derived from summation of the individual drug scores, suggesting current scales may underestimate the effect of polypharmacy. Overall, this NT2.N/A model may be appropriate for further investigation of adverse anticholinergic effects of multiple medications, in order to inform clinical choices of suitable drug use in the elderly.

The gamma-aminobutyric acid (GABA) metabolite gamma-hydroxybutyric acid (GHB) shows a variety of behavioural effects when administered to animals and humans, including reward/addiction properties and absence seizures. At the cellular level, these actions of GHB are mediated by activation of neuronal GABA(B) receptors (GABA(B)Rs) where it acts as a weak agonist. Because astrocytes respond to endogenous and exogenously applied GABA by activation of both GABA(A) and GABA(B)Rs, here we investigated the action of GHB on astrocytes on the ventral tegmental area (VTA) and the ventrobasal (VB) thalamic nucleus, two brain areas involved in the reward and proepileptic action of GHB, respectively, and compared it with that of the potent GABA(B)R agonist baclofen. We found that GHB and baclofen elicited dose-dependent (ED50: 1.6 mM and 1.3 µM, respectively) transient increases in intracellular Ca(2+) in VTA and VB astrocytes of young mice and rats, which were accounted for by activation of their GABA(B)Rs and mediated by Ca(2+) release from intracellular store release. In contrast, prolonged GHB and baclofen exposure caused a reduction in spontaneous astrocyte activity and glutamate release from VTA astrocytes. These findings have key (patho)physiological implications for our understanding of the addictive and proepileptic actions of GHB.

Epilepsy is generally viewed as resulting from an unbalanced excitatory/inhibitory drive, where either excitatory transmission is enhanced and/or inhibitory transmission is decreased. However, studies in genetic and pharmacological models of non-convulsive typical absence seizures have revealed that an increased activation of extrasynaptic γ-aminobutyric acidA (GABAA) receptors (eGABAARs), and the resulting enhanced tonic GABAA inhibition in thalamocortical (TC) neurons, is a necessary and sufficient condition for the expression of these seizures. Importantly, in genetic absence models, the mechanism underlying eGABAAR gain of function is non-neuronal in nature as it results from a malfunction in the thalamic astrocytic GABA transporter, GAT-1. These results challenge the existing view that typical absence seizures are underpinned by a widespread loss of GABAergic function in TC circuits, and are supported by the evidence that drugs that increase GABAergic signalling elicit or aggravate absence seizures in animal model and humans. Furthermore, by highlighting a vital role for astrocytes and eGABAARs in the pathophysiology of typical absence epilepsy, these new findings offer novel targets for the development of more effective anti-absence drugs.

Astrocytes modulate synaptic strength. This effect occurs, reports a new paper, because ATP-dependent vesicular release of astrocytic glutamate acts on presynaptic neuronal NMDA receptors to increase synaptic efficacy.

The action of ethosuximide (ETX) on Na+, K+, and Ca2+currents and on tonic and burst-firing patterns was investigated in rat and cat thalamic neuronsin vitroby using patch and sharp microelectrode recordings. In thalamocortical (TC) neurons of the rat dorsal lateral geniculate nucleus (LGN), ETX (0.75–1 mm) decreased the noninactivating Na+current,INaP, by 60% but had no effect on the transient Na+current. In TC neurons of the rat and cat LGN, the whole-cell transient outward current was not affected by ETX (up to 1 mm), but the sustained outward current was decreased by 39% at 20 mV in the presence of ETX (0.25–0.5 mm): this reduction was not observed in a low Ca2+(0.5 mm) and high Mg2+(8 mm) medium or in the presence of Ni2+(1 mm) and Cd2+(100 μm). In addition, ETX (up to 1 mm) had no effect on the low-threshold Ca2+current,IT, of TC neurons of the rat ventrobasal (VB) thalamus and LGN and in neurons of the rat nucleus reticularis thalami nor on the high-threshold Ca2+current in TC neurons of the rat LGN.Sharp microelectrode recordings in TC neurons of the rat and cat LGN and VB showed that ETX did not change the resting membrane potential but increased the apparent input resistance at potentials greater than −60 mV, resulting in an increase in tonic firing. In contrast, ETX decreased the number of action potentials in the burst evoked by a low-threshold Ca2+potential. The frequency of the remaining action potentials in a burst also was decreased, whereas the latency of the first action potential was increased. Similar effects were observed on the burst firing evoked during intrinsic δ oscillations.These results indicate an action of ETX onINaPand on the Ca2+-activated K+current, which explains the decrease in burst firing and the increase in tonic firing, and, together with the lack of action on low- and high-threshold Ca2+currents, the results cast doubts on the hypothesis that a reduction ofITin thalamic neurons underlies the therapeutic action of this anti-absence medicine.

The properties of the Na+current present in thalamocortical neurons of the dorsal lateral geniculate nucleus were investigated in dissociated neonate rat and cat neurons and in neurons from slices of neonate and adult rats using patch and sharp electrode recordings.The steady-state activation and inactivation of the transient Na+current (INa) was well fitted with a Boltzmann curve (voltage of half-maximal activation and inactivation,V1/2, −29.84 mV and −70.04 mV, respectively). Steady-state activation and inactivation curves showed a small region of overlap, indicating the occurrence of aINawindow current.INadecay could be fitted with a single exponential function, consistent with the presence of only one channel type.Voltage ramp and step protocols showed the presence of a noninactivating component of the Na+current (INaP) that activated at potentials more negative (V1/2= −56.93 mV) than those ofINa. The maximal amplitude ofINaPwas ∼2.5% ofINa, thus significantly greater than the calculated contribution (0.2%) of theINawindow component. Comparison of results from dissociated neurons and neurons in slices suggested a dendritic as well as a somatic localization ofINaP. Inclusion of papain in the patch electrode removed the fast inactivation ofINaand induced a current with voltage-dependence (V1/2= −56.92) and activation parameters similar to those ofINaP.Current-clamp recordings with sharp electrodes showed thatINaPcontributed to depolarizations evoked from potentials of approximately −60 mV and unexpectedly to the amplitude and latency of low-threshold Ca2+potentials, suggesting that this noninactivating component of the Na+channel population plays an important role in the integrative properties of thalamocortical neurons during both tonic and burst-firing patterns.

Astrocytes are increasingly implicated in a range of functions in the brain, many of which were previously ascribed to neurons. Much of the prevailing interest centers on the role of astrocytes in the modulation of synaptic transmission and their involvement in the induction of forms of plasticity such as long-term potentiation and long-term depression. However, there is also an increasing realization that astrocytes themselves can undergo plasticity. This plasticity may be manifest as changes in protein expression which may modify calcium activity within the cells, changes in morphology that affect the environment of the synapse and the extracellular space, or changes in gap junction astrocyte coupling that modify the transfer of ions and metabolites through astrocyte networks. Plasticity in the way that astrocytes release gliotransmitters can also have direct effects on synaptic activity and neuronal excitability. Astrocyte plasticity can potentially have profound effects on neuronal network activity and be recruited in pathological conditions. An emerging principle of astrocyte plasticity is that it is often induced by neuronal activity, reinforcing our emerging understanding of the working brain as a constant interaction between neurons and glial cells.

Neurons in the brain signal to each other by the release of chemical neurotransmitters. These are concentrated in vesicles at the presynaptic terminal and following a calcium influx the vesicles release the transmitter to act at postsynaptic receptors. In this respect adenosine is unusual, as it can be released from presynaptic vesicles, but an increase in extracellular adenosine may also result from other mechanisms. Adenosine produced in a cell can efflux passively by membrane transport proteins called equilibrative nucleoside transporters (ENTs) and extracellular adenosine can be produced from the hydrolysis of ATP by extracellularly localised enzymes called ectonucleotidases. To further complicate matters, ATP itself can be released from cells by vesicular release or through membrane channels formed of connexin. Understanding the mechanisms and function of adenosine in the brain is, therefore of great interest due to its widespread presence and diverse roles as a modulatory neurotransmitter. A major action of adenosine is the inhibition of excitatory transmission via its action at presynaptic A1 receptors. Indeed, in pathological conditions such as stroke and epilepsy, an increase in adenosine release acts to reduce neuronal activity, resulting in a neuro-protective effect. (Boison, 2008). Another physiological role of adenosine seems to be as a somnogen, whereby an increase in extracellular levels of this neurotransmitter during wakefulness leads to the promotion of sleep associated neuronal network oscillations (Brown et al. 2012). Different adenosine release mechanisms have been known and described for some time (Latini & Pedata, 2001), and it is also well known that release characteristics vary by preparation, brain area and stimulus; release may occur in response to glutamatergic agonists, ischaemia or electrical stimulation. While the situation appears complicated, past efforts to discriminate the contribution of different components were also made more difficult by the limitations of contemporaneous techniques; these included indirectly inferring adenosine levels by determining the degree of induced presynaptic inhibition, or by the use of microdialysis or radiolabelling assays. Recently, however, the development of new techniques such as transgenic mouse models, multiphoton imaging and detection methods, together with an increased acceptance of a significant role for non-neuronal cells in brain function, has made it a potentially exciting and fruitful time to be studying adenosine in the brain. Using a transgenic dnSNARE mouse mutant where astrocytic vesicular release is selectively impaired, Pascual et al. (2005) demonstrated that astrocytic vesicular ATP release was a source of adenosine that induced hippocampal heterosynaptic depression. Another study, using calcium imaging and selective neuronal patch clamp methods, concluded a predominant role for adenosine release via ENTs underlying synaptic depression (Lovatt et al. 2012). In this issue of The Journal of PhysiologyWall & Dale (2013) used combined electrophysiological recordings with adenosine- sensitive microelectrode biosensors in a hippocampal slice preparation. These biosensors have a tip diameter of 50 μm or less and can be inserted into the slice preparation to electrochemically detect adenosine, and permit real-time measurements with a high temporal resolution. They found that adenosine was released following Schaffer collateral stimulation and that this release was stimulation frequency-dependent with a peak at the relatively low frequency of 5 Hz. Extracellular adenosine concentration increase was dependent on ionotropic glutamate receptor activation and was also calcium dependent. The adenosine increase was inhibited by pharmacological inhibition of ectonucleotidase, indicating a contribution from ATP hydrolysis. However, in hippocampal slices from a transgenic mouse model lacking ectonucleotidase, adenosine release could still be measured following synaptic stimulation. This release was inhibited by pharmacological block of ENTs, indicating two parallel release mechanisms. To investigate whether adenosine also came from different cellular sources, the authors used the astrocytic metabolic poison fluoroacetate. Inhibiting the contribution of astrocytes resulted in a change in kinetics in the measured adenosine increases following synaptic stimulation so that rise and decay were more rapid. To further define the astrocytic mechanism, experiments were then conducted in dnSNARE mice where vesicular release, including that of ATP, is inhibited. Confirming the effect seen with fluoroacetate, the kinetics of adenosine release were more rapid compared to wild-type mice, indicating the loss of a slow astrocytic vesicular release component. Figure 1 Glutamate released by Schaffer collateral stimulation activates pyramidal neurones to release adenosine via equilibrative nucleotide transporters (ENT), and astrocytes to release ATP, which is hydrolysed by ectonucleotidase (ENase) to adenosine. Combining pharmacological, electrophysiological and microelectrode biosensor techniques therefore shows that, in the hippocampal slice preparation, stimulation of the Shaffer collateral induces an increase in adenosine, which results from a contribution from both neurones and astrocytes, which in turn display different temporal characteristics. This is, therefore, another example of astrocyte–neuron interaction in the brain, where both cell types act to increase the levels of the same transmitter, with the astrocytic component ostensibly extending the time period in which adenosine levels are elevated. The study provides answers to many questions about hippocampal adenosine release, yet, despite the small diameter of the biosensors, these results cannot tell us about the spatial cellular and subcellular specificity of the release. The difference in kinetics of neuronal and astrocytic adenosine hints at different physiological targets and roles; for example, one source might preferentially target synaptic or extrasynaptic receptors, or primarily target either neuronal or astrocytic networks. Addressing such questions may therefore require the further refinement and development of new techniques to detect and monitor the effects of adenosine.

The NT2.D1 cell line is one of the most well-documented embryocarcinoma cell lines, and can be differentiated into neurons and astrocytes. Great focus has also been placed on defining the electrophysiological properties of the neuronal cells, and more recently we have investigated the functional properties of their associated astrocytes. We now show for the first time that human stem cell-derived astrocytes produce glycogen and that co-cultures of these cells demonstrate a functional astrocyte-neuron lactate shuttle (ANLS). The ANLS hypothesis proposes that during neuronal activity, glutamate released into the synaptic cleft is taken up by astrocytes and triggers glucose uptake, which is converted into lactate and released via monocarboxylate transporters for neuronal use. Using mixed cultures of NT2-derived neurons and astrocytes, we have shown that these cells modulate their glucose uptake in response to glutamate. Additionally, we demonstrate that in response to increased neuronal activity and under hypoglycaemic conditions, co-cultures modulate glycogen turnover and increase lactate production. Similar results were also shown after treatment with glutamate, potassium, isoproterenol, and dbcAMP. Together, these results demonstrate for the first time a functional ANLS in a human stem cell-derived co-culture.

Background: Aquaporin channels ensure appropriate membrane permeability to water in all cells. Results: Following a hypotonic stimulus, subcellular localization of aquaporin 1 occurs via a mechanism dependent on transient receptor potential channels, extracellular calcium influx, calmodulin, and the phosphorylation of two threonines (157 and 239) of aquaporin 1. Conclusion: Rapid translocation of aquaporin 1 regulates membrane water permeability. Significance: This mechanism may serve as a prototype for the rapid regulation of aquaporin function., The control of cellular water flow is mediated by the aquaporin (AQP) family of membrane proteins. The structural features of the family and the mechanism of selective water passage through the AQP pore are established, but there remains a gap in our knowledge of how water transport is regulated. Two broad possibilities exist. One is controlling the passage of water through the AQP pore, but this only has been observed as a phenomenon in some plant and microbial AQPs. An alternative is controlling the number of AQPs in the cell membrane. Here, we describe a novel pathway in mammalian cells whereby a hypotonic stimulus directly induces intracellular calcium elevations through transient receptor potential channels, which trigger AQP1 translocation. This translocation, which has a direct role in cell volume regulation, occurs within 30 s and is dependent on calmodulin activation and phosphorylation of AQP1 at two threonine residues by protein kinase C. This direct mechanism provides a rationale for the changes in water transport that are required in response to constantly changing local cellular water availability. Moreover, because calcium is a pluripotent and ubiquitous second messenger in biological systems, the discovery of its role in the regulation of AQP translocation has ramifications for diverse physiological and pathophysiological processes, as well as providing an explanation for the rapid regulation of water flow that is necessary for cell homeostasis.

A major focus of stem cell research is the generation of neurons that may then be implanted to treat neurodegenerative diseases. However, a picture is emerging where astrocytes are partners to neurons in sustaining and modulating brain function. We therefore investigated the functional properties of NT2 derived astrocytes and neurons using electrophysiological and calcium imaging approaches. NT2 neurons (NT2Ns) expressed sodium dependent action potentials, as well as responses to depolarisation and the neurotransmitter glutamate. NT2Ns exhibited spontaneous and coordinated calcium elevations in clusters and in extended processes, indicating local and long distance signalling. Tetrodotoxin sensitive network activity could also be evoked by electrical stimulation. Similarly, NT2 astrocytes (NT2As) exhibited morphology and functional properties consistent with this glial cell type. NT2As responded to neuronal activity and to exogenously applied neurotransmitters with calcium elevations, and in contrast to neurons, also exhibited spontaneous rhythmic calcium oscillations. NT2As also generated propagating calcium waves that were gap junction and purinergic signalling dependent. Our results show that NT2 derived astrocytes exhibit appropriate functionality and that NT2N networks interact with NT2A networks in co-culture. These findings underline the utility of such cultures to investigate human brain cell type signalling under controlled conditions. Furthermore, since stem cell derived neuron function and survival is of great importance therapeutically, our findings suggest that the presence of complementary astrocytes may be valuable in supporting stem cell derived neuronal networks. Indeed, this also supports the intriguing possibility of selective therapeutic replacement of astrocytes in diseases where these cells are either lost or lose functionality.

Astrocytes release gliotransmitters, notably glutamate, that can affect neuronal and synaptic activity. In particular, astrocytic glutamate release results in the generation of N-methyl D-aspartate receptor (NMDA-R) mediated slow inward currents (SICs) in neurons. However, factors underlying the emergence of SICs, and their physiological roles are largely unknown. Here we show that, in acute slices of rat somatosensory thalamus, stimulation of Lemniscal or cortical afferents results in a sustained increase of SICs in thalamocortical (TC) neurons that outlasts the duration of the stimulus by an hour. This long term enhancement (LTE) of astrocytic glutamate release is induced by group I metabotropic glutamate receptors (mGluRs), and is dependent on astrocytic intracellular calcium ([Ca2+]i). Neuronal SICs are mediated by extrasynaptic NR2B subunit-containing NMDA-Rs and are capable of eliciting bursts. These are distinct from T-type Ca2+ channel dependent bursts of action potentials, and are synchronized in neighboring TC neurons. These findings describe a previously unrecognized form of excitatory, non-synaptic plasticity in the central nervous system (CNS) that feeds forward to generate local neuronal firing long after stimulus termination.

Aberrant amyloid-s peptide (As) accumulation along with altered expression and function of nicotinic acetylcholine receptors (nAChRs) stand prominently in the etiology of Alzheimer's disease (AD). Since the discovery that As is bound to a7 nAChRs under many experimental settings, including post-mortem AD brain, much effort has been expended to understand the implications of this interaction in the disease milieu. This research update will review the current literature on the a7 nAChR-As interaction in vitro and in vivo, the functional consequences of this interaction from sub-cellular to cognitive levels, and discuss the implications these relationships might have for AD therapies.

International audience; Gamma-hydroxybutyric acid (GHB) is an endogenous brain substance that has diverse neuropharmacological actions, including rewarding properties in different animal species and in humans. As other drugs of abuse, GHB affects the firing of ventral tegmental neurons (VTA) in anaesthetized animals and hyperpolarizes dopaminergic neurons in VTA slices. However, no direct behavioural data on the effects of GHB applied in the VTA or in the target regions of its dopaminergic neurons, e.g. the nucleus accumbens (NAc), are available. Here, we investigated the effects of various doses of intravenous GHB in maintaining self-administration (from 0.001 to 10 mg/kg per infusion), and its ability to induce conditioned place preference (CPP) in rats when given orally (175-350 mg/kg) or injected directly either in the VTA or NAc (from 10 to 300 mug/0.5 mul per side). Our results indicate that while only 0.01 mg/kg per infusion GHB maintained self-administration, although not on every test day, 350 mg/kg GHB given orally induced CPP. CPP was also observed when GHB was injected in the VTA (30-100 mug/0.5 mul per side) but not in the NAc. Together with recent in-vitro findings, these results suggest that the rewarding properties of GHB mainly occur via disinhibition of VTA dopaminergic neurons.

Astrocytes respond to chemical, electrical and mechanical stimuli with transient increases in intracellular calcium concentration ([Ca2+]i). We now show that astrocytes in situ display intrinsic [Ca2+]i oscillations that are not driven by neuronal activity. These spontaneous astrocytic oscillations can propagate as waves to neighboring astrocytes and trigger slowly decaying NMDA receptor-mediated inward currents in neurons located along the wave path. These findings show that astrocytes in situ can act as a primary source for generating neuronal activity in the mammalian central nervous system.

It is now recognized that astrocytes participate in synaptic communication through intimate interactions with neurons. A principal mechanism is through the release of gliotransmitters (GTs) such as ATP, D-serine and most notably, glutamate, in response to astrocytic calcium elevations. We and others have shown that amyloid-β (Aβ), the toxic trigger for Alzheimer's disease (AD), interacts with hippocampal α7 nicotinic acetylcholine receptors (nAChRs). Since α7nAChRs are highly permeable to calcium and are expressed on hippocampal astrocytes, we investigated whether Aβ could activate astrocytic α7nAChRs in hippocampal slices and induce GT glutamate release. We found that biologically-relevant concentrations of Aβ1-42 elicited α7nAChR-dependent calcium elevations in hippocampal CA1 astrocytes and induced NMDAR-mediated slow inward currents (SICs) in CA1 neurons. In the Tg2576 AD mouse model for Aβ over-production and accumulation, we found that spontaneous astrocytic calcium elevations were of higher frequency compared to wildtype (WT). The frequency and kinetic parameters of AD mice SICs indicated enhanced gliotransmission, possibly due to increased endogenous Aβ observed in this model. Activation of α7nAChRs on WT astrocytes increased spontaneous inward currents on pyramidal neurons while α7nAChRs on astrocytes of AD mice were abrogated. These findings suggest that, at an age that far precedes the emergence of cognitive deficits and plaque deposition, this mouse model for AD-like amyloidosis exhibits augmented astrocytic activity and glutamate GT release suggesting possible repercussions for preclinical AD hippocampal neural networks that contribute to subsequent cognitive decline.

Neural activity locally increases cerebral blood flow to deliver oxygen to working neurons. A new paper shows that astrocytes can detect activity at glutamatergic synapses via calcium changes in astrocytic endfeet and directly couple it to blood flow in vitro and in vivo.