Tonic NMDAR Currents in the Brain: Regulation and Cognitive Functions.

Synaptically localized N-methyl-D-aspartate receptors (NMDARs) play a crucial role in important cognitive functions by mediating synaptic transmission and plasticity. In contrast, a tonic NMDAR current exists, thought to be mediated by extrasynaptic NMDARs, with a less clear function. This review provides a comprehensive overview of tonic NMDAR currents, focusing on their roles in synaptic transmission/plasticity and their impact on cognitive functions and psychiatric disorders. We discuss the roles of three endogenous ligands (i.e. glutamate, glycine, and D-serine) and receptors in mediating tonic NMDAR currents and explore the diverse mechanisms that regulate tonic NMDAR currents. In light of recent controversies surrounding the source of D-serine, we highlight the recent findings suggesting that astrocytes release D-serine to modulate tonic NMDAR currents and control cognitive flexibility. Furthermore, we propose the distinct roles of neuronal and astrocytic D-serine in different locations and their implications on synaptic regulation and cognitive functions. The potential roles of tonic NMDAR currents in various psychiatric disorders, such as schizophrenia and autism spectrum disorders (ASD), are discussed in the context of the NMDAR hypofunction hypothesis. By presenting the mechanisms in which various cells, particularly astrocytes, regulate tonic NMDAR currents, we aim to stimulate future research in NMDAR hypofunction- or hyperfunction-related psychiatric disorders. This review will not only provide a better understanding of the complex interplay between tonic NMDAR currents and cognitive functions but also shed light on its potential therapeutic target for the treatment of various psychiatric disorders.

Hypothalamic astrocyte NAD+ salvage pathway mediates the coupling of dietary fat overconsumption in a mouse model of obesity.

Nicotinamide adenine dinucleotide (NAD)+ serves as a crucial coenzyme in numerous essential biological reactions, and its cellular availability relies on the activity of the nicotinamide phosphoribosyltransferase (NAMPT)-catalyzed salvage pathway. Here we show that treatment with saturated fatty acids activates the NAD+ salvage pathway in hypothalamic astrocytes. Furthermore, inhibition of this pathway mitigates hypothalamic inflammation and attenuates the development of obesity in male mice fed a high-fat diet (HFD). Mechanistically, CD38 functions downstream of the NAD+ salvage pathway in hypothalamic astrocytes burdened with excess fat. The activation of the astrocytic NAMPT-NAD+-CD38 axis in response to fat overload induces proinflammatory responses in the hypothalamus. It also leads to aberrantly activated basal Ca2+ signals and compromised Ca2+ responses to metabolic hormones such as insulin, leptin, and glucagon-like peptide 1, ultimately resulting in dysfunctional hypothalamic astrocytes. Our findings highlight the significant contribution of the hypothalamic astrocytic NAD+ salvage pathway, along with its downstream CD38, to HFD-induced obesity.

Hypothalamic GABRA5-positive neurons control obesity via astrocytic GABA.

The lateral hypothalamic area (LHA) regulates food intake and energy balance. Although LHA neurons innervate adipose tissues, the identity of neurons that regulate fat is undefined. Here we show that GABRA5-positive neurons in LHA (GABRA5LHA) polysynaptically project to brown and white adipose tissues in the periphery. GABRA5LHA are a distinct subpopulation of GABAergic neurons and show decreased pacemaker firing in diet-induced obesity mouse models in males. Chemogenetic inhibition of GABRA5LHA suppresses fat thermogenesis and increases weight gain, whereas gene silencing of GABRA5 in LHA decreases weight gain. In the diet-induced obesity mouse model, GABRA5LHA are tonically inhibited by nearby reactive astrocytes releasing GABA, which is synthesized by monoamine oxidase B (Maob). Gene silencing of astrocytic Maob in LHA facilitates fat thermogenesis and reduces weight gain significantly without affecting food intake, which is recapitulated by administration of a Maob inhibitor, KDS2010. We propose that firing of GABRA5LHA suppresses fat accumulation and selective inhibition of astrocytic GABA is a molecular target for treating obesity.

GABA tone regulation and its cognitive functions in the brain.

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter released at GABAergic synapses, mediating fast-acting phasic inhibition. Emerging lines of evidence unequivocally indicate that a small amount of extracellular GABA - GABA tone - exists in the brain and induces a tonic GABA current that controls neuronal activity on a slow timescale relative to that of phasic inhibition. Surprisingly, studies indicate that glial cells that synthesize GABA, such as astrocytes, release GABA through non-vesicular mechanisms, such as channel-mediated release, and thereby act as the source of GABA tone in the brain. In this Review, we first provide an overview of major advances in our understanding of the cell-specific molecular and cellular mechanisms of GABA synthesis, release and clearance that regulate GABA tone in various brain regions. We next examine the diverse ways in which the tonic GABA current regulates synaptic transmission and synaptic plasticity through extrasynaptic GABAA-receptor-mediated mechanisms. Last, we discuss the physiological mechanisms through which tonic inhibition modulates cognitive function on a slow timescale. In this Review, we emphasize that the cognitive functions of tonic GABA current extend beyond mere inhibition, laying a foundation for future research on the physiological and pathophysiological roles of GABA tone regulation in normal and abnormal psychiatric conditions.

Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice.

Autism spectrum disorders (ASD) represent neurodevelopmental disorders characterized by social deficits, repetitive behaviors, and various comorbidities, including epilepsy. ANK2, which encodes a neuronal scaffolding protein, is frequently mutated in ASD, but its in vivo functions and disease-related mechanisms are largely unknown. Here, we report that mice with Ank2 knockout restricted to cortical and hippocampal excitatory neurons (Ank2-cKO mice) show ASD-related behavioral abnormalities and juvenile seizure-related death. Ank2-cKO cortical neurons show abnormally increased excitability and firing rate. These changes accompanied decreases in the total level and function of the Kv7.2/KCNQ2 and Kv7.3/KCNQ3 potassium channels and the density of these channels in the enlengthened axon initial segment. Importantly, the Kv7 agonist, retigabine, rescued neuronal excitability, juvenile seizure-related death, and hyperactivity in Ank2-cKO mice. These results suggest that Ank2 regulates neuronal excitability by regulating the length of and Kv7 density in the AIS and that Kv7 channelopathy is involved in Ank2-related brain dysfunctions.

Unaltered Tonic Inhibition in the Arcuate Nucleus of Diet-induced Obese Mice.

The principal inhibitory transmitter, γ-aminobutyric acid (GABA), is critical for maintaining hypothalamic homeostasis and released from neurons phasically, as well as from astrocytes tonically. Although astrocytes in the arcuate nucleus (ARC) of the hypothalamus are shown to transform into reactive astrocytes, the tonic inhibition by astrocytic GABA has not been adequately investigated in diet-induced obesity (DIO). Here, we investigated the expression of monoamine oxidase-B (MAOB), a GABA-synthesizing enzyme, in reactive astrocytes in obese mice. We observed that a chronic high-fat diet (HFD) significantly increased astrocytic MAOB and cellular GABA content, along with enhanced hypertrophy of astrocytes in the ARC. Unexpectedly, we found that the level of tonic GABA was unaltered in chronic HFD mice using whole-cell patch-clamp recordings in the ARC. Furthermore, the GABA-induced current was increased with elevated GABAA receptor α5 (GABRA5) expression. Surprisingly, we found that a nonselective GABA transporter (GAT) inhibitor, nipecotic acid (NPA)-induced current was significantly increased in chronic HFD mice. We observed that GAT1 inhibitor, NO711-induced current was significantly increased, whereas GAT3 inhibitor, SNAP5114-induced current was not altered. The unexpected unaltered tonic inhibition was due to an increase of GABA clearance in the ARC by neuronal GAT1 rather than astrocytic GAT3. These results imply that increased astrocytic GABA synthesis and neuronal GABAA receptor were compensated by GABA clearance, resulting in unaltered tonic GABA inhibition in the ARC of the hypothalamus in obese mice. Taken together, GABA-related molecular pathways in the ARC dynamically regulate the tonic inhibition to maintain hypothalamic homeostasis against the HFD challenge.

Astrocytic urea cycle detoxifies Aß-derived ammonia while impairing memory in Alzheimer's disease.

Alzheimer's disease (AD) is one of the foremost neurodegenerative diseases, characterized by beta-amyloid (Aß) plaques and significant progressive memory loss. In AD, astrocytes are proposed to take up and clear Aß plaques. However, how Aß induces pathogenesis and memory impairment in AD remains elusive. We report that normal astrocytes show non-cyclic urea metabolism, whereas Aß-treated astrocytes show switched-on urea cycle with upregulated enzymes and accumulated entering-metabolite aspartate, starting-substrate ammonia, end-product urea, and side-product putrescine. Gene silencing of astrocytic ornithine decarboxylase-1 (ODC1), facilitating ornithine-to-putrescine conversion, boosts urea cycle and eliminates aberrant putrescine and its toxic byproducts ammonia and H2O2 and its end product GABA to recover from reactive astrogliosis and memory impairment in AD. Our findings implicate that astrocytic urea cycle exerts opposing roles of beneficial Aß detoxification and detrimental memory impairment in AD. We propose ODC1 inhibition as a promising therapeutic strategy for AD to facilitate removal of toxic molecules and prevent memory loss.

Astrocytes Render Memory Flexible by Releasing D-Serine and Regulating NMDA Receptor Tone in the Hippocampus.

BACKGROUND: NMDA receptor (NMDAR) hypofunction has been implicated in several psychiatric disorders with impairment of cognitive flexibility. However, the molecular mechanism of how NMDAR hypofunction with decreased NMDAR tone causes the impairment of cognitive flexibility has been minimally understood. Furthermore, it has been unclear whether hippocampal astrocytes regulate NMDAR tone and cognitive flexibility. METHODS: We employed cell type-specific genetic manipulations, ex vivo electrophysiological recordings, sniffer patch recordings, cutting-edge biosensor for norepinephrine, and behavioral assays to investigate whether astrocytes can regulate NMDAR tone by releasing D-serine and glutamate. Subsequently, we further investigated the role of NMDAR tone in heterosynaptic long-term depression, metaplasticity, and cognitive flexibility. RESULTS: We found that hippocampal astrocytes regulate NMDAR tone via BEST1-mediated corelease of D-serine and glutamate. Best1 knockout mice exhibited reduced NMDAR tone and impairments of homosynaptic and α1 adrenergic receptor-dependent heterosynaptic long-term depression, which leads to defects in metaplasticity and cognitive flexibility. These impairments in Best1 knockout mice can be rescued by hippocampal astrocyte-specific BEST1 expression or enhanced NMDAR tone through D-serine supplement. D-serine injection in Best1 knockout mice during initial learning rescues subsequent reversal learning. CONCLUSIONS: These findings indicate that NMDAR tone during initial learning is important for subsequent learning, and hippocampal NMDAR tone regulated by astrocytic BEST1 is critical for heterosynaptic long-term depression, metaplasticity, and cognitive flexibility.

The tripartite glutamatergic synapse.

Astroglial cells were long considered as structural and metabolic supporting cells are which do not directly participate in information processing in the brain. Discoveries of responsiveness of astrocytes to synaptically-released glutamate and their capability to release agonists of glutamate receptors awakened extensive studies of glia-neuron communications and led to the revolutionary changes in our understanding of brain cellular networks. Nowadays, astrocytes are widely acknowledged as inseparable element of glutamatergic synapses and role for glutamatergic astrocyte-neuron interactions in the brain computation is emerging. Astroglial glutamate receptors, in particular of NMDA, mGluR3 and mGluR5 types, can activate a variety of molecular cascades leading astroglial-driven modulation of extracellular levels of glutamate and activity of neuronal glutamate receptors. Their preferential location to the astroglial perisynaptic processes facilitates interaction of astrocytes with individual excitatory synapses. Bi-directional glutamatergic communication between astrocytes and neurons underpins a complex, spatially-distributed modulation of synaptic signalling thus contributing to the enrichment of information processing by the neuronal networks. Still, further research is needed to bridge the substantial gaps in our understanding of mechanisms and physiological relevance of astrocyte-neuron glutamatergic interactions, in particular ability of astrocytes directly activate neuronal glutamate receptors by releasing glutamate and, arguably, d-Serine. An emerging roles for aberrant changes in glutamatergic astroglial signalling, both neuroprotective and pathogenic, in neurological and neurodegenerative diseases also require further investigation. This article is part of the special Issue on 'Glutamate Receptors - The Glutamatergic Synapse'.

Differential Proximity of Perisynaptic Astrocytic Best1 at the Excitatory and Inhibitory Tripartite Synapses in APP/PS1 and MAOB-KO Mice Revealed by Lattice Structured Illumination Microscopy.

Bestrophin-1 (Best1) is a GABA- and glutamate-permeable, Ca2+-activated Cl- channel, which is mainly expressed in astrocytes and localized at the microdomain or perisynaptic junction of the tripartite synapse. Distribution of Best1 is dramatically changed in pathological conditions such as Alzheimer's disease. However, it is still unknown whether Best1 is located at the glutamatergic or GABAergic tripartite synapses. Here, we utilized the Lattice structured illumination microscopy (Lattice SIM) to visualize Best1 expression at the perisynaptic junctions of the tripartite synapses in CA1 of mouse hippocampus. We performed co-labeling with antibodies against 1) Best1 and vesicular glutamate transporter-2 (vGLUT2) or 2) Best1 and vesicular GABA transporter (vGAT) to measure the proximity of Best1-containing perisynapse to glutamatergic or GABAergic presynapse, respectively. In addition, we examined two transgenic mouse lines of 1) APP/PS1 mouse showing high astrocytic MAOB activity and cytosolic GABA and 2) MAOB-KO mouse showing low astrocytic GABA. Lattice SIM images were further processed by Imaris, which allowed 3D-rendering and spot identification. We found that astrocytic Best1 was distributed closer to the glutamatergic synapses than GABAergic synapses in the wild-type mice. In APP/PS1 mice, Best1 distribution was significantly changed by moving away from the glutamatergic synapses while moving closer to the GABAergic synapses. On the contrary, in MAOB-KO mice, the Best1 distribution was dramatically changed by moving closer to the glutamatergic synapses and moving far away from the GABAergic synapses. Our findings propose that the proximity of Best1-containing perisynapses to presynapses dynamically changes according to the level of astrocytic cytosolic GABA.

Astrocytes Control Sensory Acuity via Tonic Inhibition in the Thalamus.

Sensory discrimination is essential for survival. However, how sensory information is finely controlled in the brain is not well defined. Here, we show that astrocytes control tactile acuity via tonic inhibition in the thalamus. Mechanistically, diamine oxidase (DAO) and the subsequent aldehyde dehydrogenase 1a1 (Aldh1a1) convert putrescine into GABA, which is released via Best1. The GABA from astrocytes inhibits synaptically evoked firing at the lemniscal synapses to fine-tune the dynamic range of the stimulation-response relationship, the precision of spike timing, and tactile discrimination. Our findings reveal a novel role of astrocytes in the control of sensory acuity through tonic GABA release.

The molecular mechanism of synaptic activity-induced astrocytic volume transient.

KEY POINTS: Neuronal activity causes astrocytic volume change via K+ uptake through TREK-1 containing two-pore domain potassium channels. The volume transient is terminated by Cl- efflux through the Ca2+ -activated anion channel BEST1. The source of the Ca2+ required to open BEST1 appears to be the stretch-activated TRPA1 channel. Intense neuronal activity is synaptically coupled with a physical change in astrocytes via volume transients. ABSTRACT: The brain volume changes dynamically and transiently upon intense neuronal activity through a tight regulation of ion concentrations and water movement across the plasma membrane of astrocytes. We have recently demonstrated that an intense neuronal activity and subsequent astrocytic AQP4-dependent volume transient are critical for synaptic plasticity and memory. We have also pharmacologically demonstrated a functional coupling between synaptic activity and the astrocytic volume transient. However, the precise molecular mechanisms of how intense neuronal activity and the astrocytic volume transient are coupled remain unclear. Here we utilized an intrinsic optical signal imaging technique combined with fluorescence imaging using ion sensitive dyes and molecular probes and electrophysiology to investigate the detailed molecular mechanisms in genetically modified mice. We report that a brief synaptic activity induced by a train stimulation (20 Hz, 1 s) causes a prolonged astrocytic volume transient (80 s) via K+ uptake through TREK-1 containing two-pore domain potassium (K2P) channels, but not Kir4.1 or NKCC1. This volume change is terminated by Cl- efflux through the Ca2+ -activated anion channel BEST1, but not the volume-regulated anion channel TTYH. The source of the Ca2+ required to open BEST1 appears to be the stretch-activated TRPA1 channel in astrocytes, but not IP3 R2. In summary, our study identifies several important astrocytic ion channels (AQP4, TREK-1, BEST1, TRPA1) as the key molecules leading to the neuronal activity-dependent volume transient in astrocytes. Our findings reveal new molecular and cellular mechanisms for the synaptic coupling of intense neuronal activity with a physical change in astrocytes via volume transients.

Ultrasonic Neuromodulation via Astrocytic TRPA1.

Low-intensity, low-frequency ultrasound (LILFU) is the next-generation, non-invasive brain stimulation technology for treating various neurological and psychiatric disorders. However, the underlying cellular and molecular mechanism of LILFU-induced neuromodulation has remained unknown. Here, we report that LILFU-induced neuromodulation is initiated by opening of TRPA1 channels in astrocytes. The Ca2+ entry through TRPA1 causes a release of gliotransmitters including glutamate through Best1 channels in astrocytes. The released glutamate activates NMDA receptors in neighboring neurons to elicit action potential firing. Our results reveal an unprecedented mechanism of LILFU-induced neuromodulation, involving TRPA1 as a unique sensor for LILFU and glutamate-releasing Best1 as a mediator of glia-neuron interaction. These discoveries should prove to be useful for optimization of human brain stimulation and ultrasonogenetic manipulations of TRPA1.

Activation of Astrocytic µ-Opioid Receptor Causes Conditioned Place Preference.

The underlying mechanisms of how positive emotional valence (e.g., pleasure) causes preference of an associated context is poorly understood. Here, we show that activation of astrocytic µ-opioid receptor (MOR) drives conditioned place preference (CPP) by means of specific modulation of astrocytic MOR, an exemplar endogenous Gi protein-coupled receptor (Gi-GPCR), in the CA1 hippocampus. Long-term potentiation (LTP) induced by a subthreshold stimulation with the activation of astrocytic MOR at the Schaffer collateral pathway accounts for the memory acquisition to induce CPP. This astrocytic MOR-mediated LTP induction is dependent on astrocytic glutamate released upon activation of the astrocytic MOR and the consequent activation of the presynaptic mGluR1. The astrocytic MOR-dependent LTP and CPP were recapitulated by a chemogenetic activation of astrocyte-specifically expressed Gi-DREADD hM4Di. Our study reveals that the transduction of inhibitory Gi-signaling into augmented excitatory synaptic transmission through astrocytic glutamate is critical for the acquisition of contextual memory for CPP.

Pharmacological Dissection of Intrinsic Optical Signal Reveals a Functional Coupling between Synaptic Activity and Astrocytic Volume Transient.

The neuronal activity-dependent change in the manner in which light is absorbed or scattered in brain tissue is called the intrinsic optical signal (IOS), and provides label-free, minimally invasive, and high spatial (~100 µm) resolution imaging for visualizing neuronal activity patterns. IOS imaging in isolated brain slices measured at an infrared wavelength (>700 nm) has recently been attributed to the changes in light scattering and transmittance due to aquaporin-4 (AQP4)-dependent astrocytic swelling. The complexity of functional interactions between neurons and astrocytes, however, has prevented the elucidation of the series of molecular mechanisms leading to the generation of IOS. Here, we pharmacologically dissected the IOS in the acutely prepared brain slices of the stratum radiatum of the hippocampus, induced by 1 s/20 Hz electrical stimulation of Schaffer-collateral pathway with simultaneous measurement of the activity of the neuronal population by field potential recordings. We found that 55% of IOSs peak upon stimulation and originate from postsynaptic AMPA and NMDA receptors. The remaining originated from presynaptic action potentials and vesicle fusion. Mechanistically, the elevated extracellular glutamate and K+ during synaptic transmission were taken up by astrocytes via a glutamate transporter and quinine-sensitive K2P channel, followed by an influx of water via AQP-4. We also found that the decay of IOS is mediated by the DCPIB- and NPPB-sensitive anion channels in astrocytes. Altogether, our results demonstrate that the functional coupling between synaptic activity and astrocytic transient volume change during excitatory synaptic transmission is the major source of IOS.

AAV-Mediated Astrocyte-Specific Gene Expression under Human ALDH1L1 Promoter in Mouse Thalamus.

Adeno-associated virus (AAV)-mediated gene delivery has been proposed to be an essential tool of gene therapy for various brain diseases. Among several cell types in the brain, astrocyte has become a promising therapeutic target for brain diseases, as more and more contribution of astrocytes in pathophysiology has been revealed. Until now, genetically targeting astrocytes has been possible by utilizing the glial fibrillary acidic protein (GFAP) promoter. In some brain areas including thalamus, however, the GFAP expression in astrocytes is reported to be low, making it difficult to genetically target astrocytes using GFAP promoter. To study the function of astrocytes in thalamus, which serves as a relay station, there is a great need for identifying an alternative astrocyte-specific promoter in thalamus. Recently, a new astrocyte-specific promoter of ALDH1L1 has been identified. However, it has not been examined in thalamus. Here we developed and characterized an AAV vector expressing Cre recombinase under the human ALDH1L1 promoter, AAV-hALDH1L1-Cre. To test the cell-type specific expression of AAV-hALDH1L1-Cre, AAV virus was injected into several brain regions of Ai14 (RCL-tdTomato) mouse, which reports Cre activity by tdTomato expression. In thalamus, we observed that tdTomato was found mostly in astrocytes (91.71%), with minimal occurrence in neurons (2.67%). In contrast, tdTomato signal was observed in both neurons and astrocytes of the amygdala (neuron: 68.13%, astrocyte: 28.35%) and hippocampus (neuron: 76.25%, astrocyte: 18.00%), which is consistent with the previous report showing neuronal gene expression under rat ALDH1L1 promoter. Unexpectedly, tdTomato was found mostly in neurons (91.98%) with minimal occurrence in astrocytes (6.66%) of the medial prefrontal cortex. In conclusion, hALDH1L1 promoter shows astrocyte-specificity in thalamus and may prove to be useful for targeting thalamic astrocytes in mouse.