Nonpeptidergic neurons suppress mast cells via glutamate to maintain skin homeostasis.

Cutaneous mast cells mediate numerous skin inflammatory processes and have anatomical and functional associations with sensory afferent neurons. We reveal that epidermal nerve endings from a subset of sensory nonpeptidergic neurons expressing MrgprD are reduced by the absence of Langerhans cells. Loss of epidermal innervation or ablation of MrgprD-expressing neurons increased expression of a mast cell gene module, including the activating receptor, Mrgprb2, resulting in increased mast cell degranulation and cutaneous inflammation in multiple disease models. Agonism of MrgprD-expressing neurons reduced expression of module genes and suppressed mast cell responses. MrgprD-expressing neurons released glutamate which was increased by MrgprD agonism. Inhibiting glutamate release or glutamate receptor binding yielded hyperresponsive mast cells with a genomic state similar to that in mice lacking MrgprD-expressing neurons. These data demonstrate that MrgprD-expressing neurons suppress mast cell hyperresponsiveness and skin inflammation via glutamate release, thereby revealing an unexpected neuroimmune mechanism maintaining cutaneous immune homeostasis.

General Anesthesia Decouples Cortical Pyramidal Neurons.

The mystery of general anesthesia is that it specifically suppresses consciousness by disrupting feedback signaling in the brain, even when feedforward signaling and basic neuronal function are left relatively unchanged. The mechanism for such selectiveness is unknown. Here we show that three different anesthetics have the same disruptive influence on signaling along apical dendrites in cortical layer 5 pyramidal neurons in mice. We found that optogenetic depolarization of the distal apical dendrites caused robust spiking at the cell body under awake conditions that was blocked by anesthesia. Moreover, we found that blocking metabotropic glutamate and cholinergic receptors had the same effect on apical dendrite decoupling as anesthesia or inactivation of the higher-order thalamus. If feedback signaling occurs predominantly through apical dendrites, the cellular mechanism we found would explain not only how anesthesia selectively blocks this signaling but also why conscious perception depends on both cortico-cortical and thalamo-cortical connectivity.

Mechanisms for Zinc and Proton Inhibition of the GluN1/GluN2A NMDA Receptor.

N-methyl-D-aspartate receptors (NMDARs) play essential roles in memory formation, neuronal plasticity, and brain development, with their dysfunction linked to a range of disorders from ischemia to schizophrenia. Zinc and pH are physiological allosteric modulators of NMDARs, with GluN2A-containing receptors inhibited by nanomolar concentrations of divalent zinc and by excursions to low pH. Despite the widespread importance of zinc and proton modulation of NMDARs, the molecular mechanism by which these ions modulate receptor activity has proven elusive. Here, we use cryoelectron microscopy to elucidate the structure of the GluN1/GluN2A NMDAR in a large ensemble of conformations under a range of physiologically relevant zinc and proton concentrations. We show how zinc binding to the amino terminal domain elicits structural changes that are transduced though the ligand-binding domain and result in constriction of the ion channel gate.

Moderate UV Exposure Enhances Learning and Memory by Promoting a Novel Glutamate Biosynthetic Pathway in the Brain.

Sunlight exposure is known to affect mood, learning, and cognition. However, the molecular and cellular mechanisms remain elusive. Here, we show that moderate UV exposure elevated blood urocanic acid (UCA), which then crossed the blood-brain barrier. Single-cell mass spectrometry and isotopic labeling revealed a novel intra-neuronal metabolic pathway converting UCA to glutamate (GLU) after UV exposure. This UV-triggered GLU synthesis promoted its packaging into synaptic vesicles and its release at glutamatergic terminals in the motor cortex and hippocampus. Related behaviors, like rotarod learning and object recognition memory, were enhanced after UV exposure. All UV-induced metabolic, electrophysiological, and behavioral effects could be reproduced by the intravenous injection of UCA and diminished by the application of inhibitor or short hairpin RNA (shRNA) against urocanase, an enzyme critical for the conversion of UCA to GLU. These findings reveal a new GLU biosynthetic pathway, which could contribute to some of the sunlight-induced neurobehavioral changes.

Activation and Desensitization Mechanism of AMPA Receptor-TARP Complex by Cryo-EM.

AMPA receptors mediate fast excitatory neurotransmission in the mammalian brain and transduce the binding of presynaptically released glutamate to the opening of a transmembrane cation channel. Within the postsynaptic density, however, AMPA receptors coassemble with transmembrane AMPA receptor regulatory proteins (TARPs), yielding a receptor complex with altered gating kinetics, pharmacology, and pore properties. Here, we elucidate structures of the GluA2-TARP γ2 complex in the presence of the partial agonist kainate or the full agonist quisqualate together with a positive allosteric modulator or with quisqualate alone. We show how TARPs sculpt the ligand-binding domain gating ring, enhancing kainate potency and diminishing the ensemble of desensitized states. TARPs encircle the receptor ion channel, stabilizing M2 helices and pore loops, illustrating how TARPs alter receptor pore properties. Structural and computational analysis suggests the full agonist and modulator complex harbors an ion-permeable channel gate, providing the first view of an activated AMPA receptor.

Structure of the Arabidopsis thaliana glutamate receptor-like channel GLR3.4.

Glutamate receptor-like channels (GLRs) play vital roles in various physiological processes in plants, such as wound response, stomatal aperture control, seed germination, root development, innate immune response, pollen tube growth, and morphogenesis. Despite the importance of GLRs, knowledge about their molecular organization is limited. Here we use X-ray crystallography and single-particle cryo-EM to solve structures of the Arabidopsis thaliana GLR3.4. Our structures reveal the tetrameric assembly of GLR3.4 subunits into a three-layer domain architecture, reminiscent of animal ionotropic glutamate receptors (iGluRs). However, the non-swapped arrangement between layers of GLR3.4 domains, binding of glutathione through S-glutathionylation of cysteine C205 inside the amino-terminal domain clamshell, unique symmetry, inter-domain interfaces, and ligand specificity distinguish GLR3.4 from representatives of the iGluR family and suggest distinct features of the GLR gating mechanism. Our work elaborates on the principles of GLR architecture and symmetry and provides a molecular template for deciphering GLR-dependent signaling mechanisms in plants.

Muscle cofilin alters neuromuscular junction postsynaptic development to strengthen functional neurotransmission.

Cofilin, an actin severing protein, plays key roles in muscle sarcomere addition and maintenance. Our previous work found that Drosophila cofilin (DmCFL) muscle knockdown causes progressive deterioration of muscle structure and function and produces features seen in nemaline myopathy (NM) caused by cofilin mutations. We hypothesized that disruption of actin cytoskeleton dynamics by DmCFL knockdown would impact other aspects of muscle development, and, thus, conducted an RNA sequencing analysis that unexpectedly revealed upregulated expression of numerous neuromuscular junction (NMJ) genes. We found that DmCFL is enriched in the muscle postsynaptic compartment and that DmCFL muscle knockdown causes F-actin disorganization in this subcellular domain prior to the sarcomere defects observed later in development. Despite NMJ gene expression changes, we found no significant changes in gross presynaptic Bruchpilot active zones or total postsynaptic glutamate receptor levels. However, DmCFL knockdown results in mislocalization of GluRIIA class glutamate receptors in more deteriorated muscles and strongly impaired NMJ transmission strength. These findings expand our understanding of cofilin's roles in muscle to include NMJ structural development and suggest that NMJ defects may contribute to NM pathophysiology.

Companion cell mediates wound-stimulated leaf-to-leaf electrical signaling.

Leaf wounding triggers rapid long-range electrical signaling that initiates systemic defense responses to protect the plants from further attack. In Arabidopsis, this process largely depends on clade three GLUTAMATE RECEPTOR-LIKE (GLR) genes GLR3.3 and GLR3.6. In the cellular context, phloem sieve elements and xylem contact cells where GLRs were mostly present are implicated in the signaling events. In spite of that, the spatial requirements of different leaf cell types for leaf-to-leaf signaling remain poorly investigated. In this study, we dissected cell-type-specific long-distance wound signaling mediated by GLR3s and showed that phloem companion cells are critical in shaping the functions of GLR3.3 and GLR3.6 in the signaling pathway. GLR3.3-mediated response is phloem-specific, during which, GLR3.3 has to be renewed from companion cells to allow its function in sieve elements. GLR3.6 functions dually in ectopic phloem companion cells, in addition to xylem contact cells. Furthermore, the action of GLR3.6 in phloem is independent of its paralog GLR3.3 and probably requires synthesis of GLR3.6 from xylem contact cells. Overall, our work highlights that the phloem companion cell is crucial for both GLRs in controlling leaf-to-leaf electrical signaling.

News about amino acid metabolism in plant-microbe interactions.

Plants constantly come into contact with a diverse mix of pathogenic and beneficial microbes. The ability to distinguish between them and to respond appropriately is essential for plant health. Here we review recent progress in understanding the role of amino acid sensing, signaling, transport, and metabolism during plant-microbe interactions. Biochemical pathways converting individual amino acids into active compounds have recently been elucidated, and comprehensive large-scale approaches have brought amino acid sensors and transporters into focus. These findings show that plant central amino acid metabolism is closely interwoven with stress signaling and defense responses at various levels. The individual biochemical mechanisms and the interconnections between the different processes are just beginning to emerge and might serve as a foundation for new plant protection strategies.

The Neuroscience of Drug Reward and Addiction.

Drug consumption is driven by a drug's pharmacological effects, which are experienced as rewarding, and is influenced by genetic, developmental, and psychosocial factors that mediate drug accessibility, norms, and social support systems or lack thereof. The reinforcing effects of drugs mostly depend on dopamine signaling in the nucleus accumbens, and chronic drug exposure triggers glutamatergic-mediated neuroadaptations in dopamine striato-thalamo-cortical (predominantly in prefrontal cortical regions including orbitofrontal cortex and anterior cingulate cortex) and limbic pathways (amygdala and hippocampus) that, in vulnerable individuals, can result in addiction. In parallel, changes in the extended amygdala result in negative emotional states that perpetuate drug taking as an attempt to temporarily alleviate them. Counterintuitively, in the addicted person, the actual drug consumption is associated with an attenuated dopamine increase in brain reward regions, which might contribute to drug-taking behavior to compensate for the difference between the magnitude of the expected reward triggered by the conditioning to drug cues and the actual experience of it. Combined, these effects result in an enhanced motivation to "seek the drug" (energized by dopamine increases triggered by drug cues) and an impaired prefrontal top-down self-regulation that favors compulsive drug-taking against the backdrop of negative emotionality and an enhanced interoceptive awareness of "drug hunger." Treatment interventions intended to reverse these neuroadaptations show promise as therapeutic approaches for addiction.

Single nuclei RNA-seq reveals a medium spiny neuron glutamate excitotoxicity signature prior to the onset of neuronal death in an ovine Huntington's disease model.

Huntington's disease (HD) is a neurodegenerative genetic disorder caused by an expansion in the CAG repeat tract of the huntingtin (HTT) gene resulting in behavioural, cognitive, and motor defects. Current knowledge of disease pathogenesis remains incomplete, and no disease course-modifying interventions are in clinical use. We have previously reported the development and characterisation of the OVT73 transgenic sheep model of HD. The 73 polyglutamine repeat is somatically stable and therefore likely captures a prodromal phase of the disease with an absence of motor symptomatology even at 5-years of age and no detectable striatal cell loss. To better understand the disease-initiating events we have undertaken a single nuclei transcriptome study of the striatum of an extensively studied cohort of 5-year-old OVT73 HD sheep and age matched wild-type controls. We have identified transcriptional upregulation of genes encoding N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors in medium spiny neurons, the cell type preferentially lost early in HD. Further, we observed an upregulation of astrocytic glutamate uptake transporters and medium spiny neuron GABAA receptors, which may maintain glutamate homeostasis. Taken together, these observations support the glutamate excitotoxicity hypothesis as an early neurodegeneration cascade-initiating process but the threshold of toxicity may be regulated by several protective mechanisms. Addressing this biochemical defect early may prevent neuronal loss and avoid the more complex secondary consequences precipitated by cell death.

Cognitive Impairment Associated with Schizophrenia: From Pathophysiology to Treatment.

Cognitive impairment is a core feature of schizophrenia and a major contributor to poor functional outcomes. Methods for assessment of cognitive dysfunction in schizophrenia are now well established. In addition, there has been increasing appreciation in recent years of the additional role of social cognitive impairment in driving functional outcomes and of the contributions of sensory-level dysfunction to higher-order impairments. At the neurochemical level, acute administration of N-methyl-d-aspartate receptor (NMDAR) antagonists reproduces the pattern of neurocognitive dysfunction associated with schizophrenia, encouraging the development of treatments targeted at both NMDAR and its interactome. At the local-circuit level, an auditory neurophysiological measure, mismatch negativity, has emerged both as a veridical index of NMDAR dysfunction and excitatory/inhibitory imbalance in schizophrenia and as a critical biomarker for early-stage translational drug development. Although no compounds have yet been approved for treatment of cognitive impairment associated with schizophrenia, several candidates are showing promise in early-phase testing.

Necl2/3-mediated mechanism for tripartite synapse formation.

Ramified, polarized protoplasmic astrocytes interact with synapses via perisynaptic astrocyte processes (PAPs) to form tripartite synapses. These astrocyte-synapse interactions mutually regulate their structures and functions. However, molecular mechanisms for tripartite synapse formation remain elusive. We developed an in vitro co-culture system for mouse astrocytes and neurons that induced astrocyte ramifications and PAP formation. Co-cultured neurons were required for astrocyte ramifications in a neuronal activity-dependent manner, and synaptically-released glutamate and activation of astrocytic mGluR5 metabotropic glutamate receptor were likely involved in astrocyte ramifications. Astrocytic Necl2 trans-interacted with axonal Necl3, inducing astrocyte-synapse interactions and astrocyte functional polarization by recruiting EAAT1/2 glutamate transporters and Kir4.1 K+ channel to the PAPs, without affecting astrocyte ramifications. This Necl2/3 trans-interaction increased functional synapse number. Thus, astrocytic Necl2, synaptically-released glutamate and axonal Necl3 cooperatively formed tripartite glutamatergic synapses in vitro. Studies on hippocampal mossy fiber synapses in Necl3 knockout and Necl2/3 double knockout mice confirmed these previously unreported mechanisms for astrocyte-synapse interactions and astrocyte functional polarization in vivo.

Glucose-derived glutamate drives neuronal terminal differentiation in vitro.

Neuronal maturation is the phase during which neurons acquire their final characteristics in terms of morphology, electrical activity, and metabolism. However, little is known about the metabolic pathways governing neuronal maturation. Here, we investigate the contribution of the main metabolic pathways, namely glucose, glutamine, and fatty acid oxidation, during the maturation of primary rat hippocampal neurons. Blunting glucose oxidation through the genetic and chemical inhibition of the mitochondrial pyruvate transporter reveals that this protein is critical for the production of glutamate, which is required for neuronal arborization, proper dendritic elongation, and spine formation. Glutamate supplementation in the early phase of differentiation restores morphological defects and synaptic function in mitochondrial pyruvate transporter-inhibited cells. Furthermore, the selective activation of metabotropic glutamate receptors restores the impairment of neuronal differentiation due to the reduced generation of glucose-derived glutamate and rescues synaptic local translation. Fatty acid oxidation does not impact neuronal maturation. Whereas glutamine metabolism is important for mitochondria, it is not for endogenous glutamate production. Our results provide insights into the role of glucose-derived glutamate as a key player in neuronal terminal differentiation.

How astrocytic chloride modulates brain states.

The way the central nervous system (CNS) responds to diverse stimuli is contingent upon the specific brain state of the individual, including sleep and wakefulness. Despite the wealth of readout parameters and data delineating the brain states, the primary mechanisms are yet to be identified. Here we highlight the role of astrocytes, with a specific emphasis on chloride (Cl-) homeostasis as a modulator of brain states. Neuronal activity is regulated by the concentration of ions that determine excitability. Astrocytes, as the CNS homeostatic cells, are recognised for their proficiency in maintaining dynamic homeostasis of ions, known as ionostasis. Nevertheless, the contribution of astrocyte-driven ionostasis to the genesis of brain states or their response to sleep-inducing pharmacological agents has been overlooked. Our objective is to underscore the significance of astrocytic Cl- homeostasis, elucidating how it may underlie the modulation of brain states. We endeavour to contribute to a comprehensive understanding of the interplay between astrocytes and brain states.

Synaptic and cellular endocannabinoid signaling mechanisms regulate stress-induced plasticity of nucleus accumbens somatostatin neurons.

Interneuron populations within the nucleus accumbens (NAc) orchestrate excitatory-inhibitory balance, undergo experience-dependent plasticity, and gate-motivated behavior, all biobehavioral processes heavily modulated by endogenous cannabinoid (eCB) signaling. While eCBs are well known to regulate synaptic plasticity onto NAc medium spiny neurons and modulate NAc function at the behavioral level, how eCBs regulate NAc interneuron function is less well understood. Here, we show that eCB signaling differentially regulates glutamatergic and feedforward GABAergic transmission onto NAc somatostatin-expressing interneurons (NAcSOM+) in an input-specific manner, while simultaneously increasing postsynaptic excitability of NAcSOM+ neurons, ultimately biasing toward vHPC (ventral hippocampal), and away from BLA (basolateral amygdalalar), activation of NAcSOM+ neurons. We further demonstrate that NAcSOM+ are activated by stress in vivo and undergo stress-dependent plasticity, evident as a global increase in intrinsic excitability and an increase in excitation-inhibition balance specifically at vHPC, but not BLA, inputs onto NAcSOM+ neurons. Importantly, both forms of stress-induced plasticity are dependent on eCB signaling at cannabinoid type 1 receptors. These findings reveal eCB-dependent mechanisms that sculpt afferent input and excitability of NAcSOM+ neurons and demonstrate a key role for eCB signaling in stress-induced plasticity of NAcSOM+-associated circuits.

Glutamate-GABA imbalance mediated by miR-8-5p and its STTM regulates phase-related behavior of locusts.

The aggregation of locusts from solitary to gregarious phases is crucial for the formation of devastating locust plagues. Locust management requires research on the prevention of aggregation or alternative and greener solutions to replace insecticide use, and insect-derived microRNAs (miRNAs) show the potential for application in pest control. Here, we performed a genome-wide screen of the differential expression of miRNAs between solitary and gregarious locusts and showed that miR-8-5p controls the γ-aminobutyric acid (GABA)/glutamate functional balance by directly targeting glutamate decarboxylase (Gad). Blocking glutamate-GABA neurotransmission by miR-8-5p overexpression or Gad RNAi in solitary locusts decreased GABA production, resulting in locust aggregation behavior. Conversely, activating this pathway by miR-8-5p knockdown in gregarious locusts induced GABA production to eliminate aggregation behavior. Further results demonstrated that ionotropic glutamate/GABA receptors tuned glutamate/GABA to trigger/hamper the aggregation behavior of locusts. Finally, we successfully established a transgenic rice line expressing the miR-8-5p inhibitor by short tandem target mimic (STTM). When locusts fed on transgenic rice plants, Gad transcript levels in the brain increased greatly, and aggregation behavior was lost. This study provided insights into different regulatory pathways in the phase change of locusts and a potential control approach through behavioral regulation in insect pests.

Nanoelectrochemistry reveals how soluble Aß42 oligomers alter vesicular storage and release of glutamate.

Glutamate (Glu) is the major excitatory transmitter in the nervous system. Impairment of its vesicular release by ß-amyloid (Aß) oligomers is thought to participate in pathological processes leading to Alzheimer's disease. However, it remains unclear whether soluble Aß42 oligomers affect intravesicular amounts of Glu or their release in the brain, or both. Measurements made in this work on single Glu varicosities with an amperometric nanowire Glu biosensor revealed that soluble Aß42 oligomers first caused a dramatic increase in vesicular Glu storage and stimulation-induced release, accompanied by a high level of parallel spontaneous exocytosis, ultimately resulting in the depletion of intravesicular Glu content and greatly reduced release. Molecular biology tools and mouse models of Aß amyloidosis have further established that the transient hyperexcitation observed during the primary pathological stage is mediated by an altered behavior of VGLUT1 responsible for transporting Glu into synaptic vesicles. Thereafter, an overexpression of Vps10p-tail-interactor-1a, a protein that maintains spontaneous release of neurotransmitters by selective interaction with t-SNAREs, resulted in a depletion of intravesicular Glu content, triggering advanced-stage neuronal malfunction. These findings are expected to open perspectives for remediating Aß42-induced neuronal hyperactivity and neuronal degeneration.

Serotonin Strengthens a Developing Glutamatergic Synapse through a PI3K-Dependent Mechanism.

It is well established that, during neural circuit development, glutamatergic synapses become strengthened via NMDA receptor (NMDAR)-dependent upregulation of AMPA receptor (AMPAR)-mediated currents. In addition, however, it is known that the neuromodulator serotonin is present throughout most regions of the vertebrate brain while synapses are forming and being shaped by activity-dependent processes. This suggests that serotonin may modulate or contribute to these processes. Here, we investigate the role of serotonin in the developing retinotectal projection of the Xenopus tadpole. We altered endogenous serotonin transmission in stage 48/49 (∼10-21 days postfertilization) Xenopus tadpoles and then carried out a set of whole-cell electrophysiological recordings from tectal neurons to assess retinotectal synaptic transmission. Because tadpole sex is indeterminate at these early stages of development, experimental groups were composed of randomly chosen tadpoles. We found that pharmacologically enhancing and reducing serotonin transmission for 24 h up- and downregulates, respectively, AMPAR-mediated currents at individual retinotectal synapses. Inhibiting 5-HT2 receptors also significantly weakened AMPAR-mediated currents and abolished the synapse strengthening effect seen with enhanced serotonin transmission, indicating a 5-HT2 receptor-dependent effect. We also determine that the serotonin-dependent upregulation of synaptic AMPAR currents was mediated via an NMDAR-independent, PI3K-dependent mechanism. Altogether, these findings indicate that serotonin regulates AMPAR currents at developing synapses independent of NMDA transmission, which may explain its role as an enabler of activity-dependent plasticity.

Activity-Dependent Stabilization of Nascent Dendritic Spines Requires Nonenzymatic CaMKIIα Function.

The outgrowth and stabilization of nascent dendritic spines are crucial processes underlying learning and memory. Most new spines retract shortly after growth; only a small subset is stabilized and integrated into the new circuit connections that support learning. New spine stabilization has been shown to rely upon activity-dependent molecular mechanisms that also contribute to long-term potentiation (LTP) of synaptic strength. Indeed, disruption of the activity-dependent targeting of the kinase CaMKIIα to the GluN2B subunit of the NMDA-type glutamate receptor disrupts both LTP and activity-dependent stabilization of new spines. Yet it is not known which of CaMKIIα's many enzymatic and structural functions are important for new spine stabilization. Here, we used two-photon imaging and photolysis of caged glutamate to monitor the activity-dependent stabilization of new dendritic spines on hippocampal CA1 neurons from mice of both sexes in conditions where CaMKIIα functional and structural interactions were altered. Surprisingly, we found that inhibiting CaMKIIα kinase activity either genetically or pharmacologically did not impair activity-dependent new spine stabilization. In contrast, shRNA knockdown of CaMKIIα abolished activity-dependent new spine stabilization, which was rescued by co-expressing shRNA-resistant full-length CaMKIIα, but not by a truncated monomeric CaMKIIα. Notably, overexpression of phospho-mimetic CaMKIIα-T286D, which exhibits activity-independent targeting to GluN2B, enhanced basal new spine survivorship in the absence of additional glutamatergic stimulation, even when kinase activity was disrupted. Together, our results support a model in which nascent dendritic spine stabilization requires structural and scaffolding interactions mediated by dodecameric CaMKIIα that are independent of its enzymatic activities.