We used light-sensitive drugs to identify the brain region-specific role of mGlu5 metabotropic glutamate receptors in the control of pain. Optical activation of systemic JF-NP-26, a caged, normally inactive, negative allosteric modulator (NAM) of mGlu5 receptors, in cingulate, prelimbic, and infralimbic cortices and thalamus inhibited neuropathic pain hypersensitivity. Systemic treatment of alloswitch-1, an intrinsically active mGlu5 receptor NAM, caused analgesia, and the effect was reversed by light-induced drug inactivation in the prelimbic and infralimbic cortices, and thalamus. This demonstrates that mGlu5 receptor blockade in the medial prefrontal cortex and thalamus is both sufficient and necessary for the analgesic activity of mGlu5 receptor antagonists. Surprisingly, when the light was delivered in the basolateral amygdala, local activation of systemic JF-NP-26 reduced pain thresholds, whereas inactivation of alloswitch-1 enhanced analgesia. Electrophysiological analysis showed that alloswitch-1 increased excitatory synaptic responses in prelimbic pyramidal neurons evoked by stimulation of presumed BLA input, and decreased BLA-driven feedforward inhibition of amygdala output neurons. Both effects were reversed by optical silencing and reinstated by optical reactivation of alloswitch-1. These findings demonstrate for the first time that the action of mGlu5 receptors in the pain neuraxis is not homogenous, and suggest that blockade of mGlu5 receptors in the BLA may limit the overall analgesic activity of mGlu5 receptor antagonists. This could explain the suboptimal effect of mGlu5 NAMs on pain in human studies and validate photopharmacology as an important tool to determine ideal target sites for systemic drugs.

Mitral cells (MCs) in the main olfactory bulb relay odor information to higher-order olfactory centers by encoding the information in the form of action potentials. The firing patterns of these cells are influenced by both their intrinsic properties and their synaptic connections within the neural network. However, reports on MC firing patterns have been inconsistent, and the mechanisms underlying these patterns remain unclear. Using whole-cell patch-clamp recordings in mouse brain slices, we discovered that MCs exhibit two types of integrative behavior: regular/rhythmic firing and bursts of action potentials. These firing patterns could be transformed both spontaneously and chemically. MCs with regular firing maintained their pattern even in the presence of blockers of fast synaptic transmission, indicating this was an intrinsic property. However, regular firing could be transformed into bursting by applying GABAA receptor antagonists to block inhibitory synaptic transmission. Burst firing could be reverted to regular firing by blocking ionotropic glutamate receptors, rather than applying a GABAA receptor agonist, indicating that ionotropic glutamatergic transmission mediated this transformation. Further experiments on long-lasting currents (LLCs), which generated burst firing, also supported this mechanism. In addition, cytoplasmic Ca2+ in MCs was involved in the transformation of firing patterns mediated by glutamatergic transmission. Metabotropic glutamate receptors also played a role in LLCs in MCs. These pieces of evidence indicate that odor information can be encoded on a mitral cell (MC) platform, where it can be relayed to higher-order olfactory centers through intrinsic and dendrodendritic mechanisms in MCs.

An intricate interplay of complex spatio-temporal events underlies brain functions. Therefore, clarifying these dynamic processes is indispensable for revealing the mechanisms of brain functions. Fluorescence imaging is a powerful technique for visualizing cellular and molecular dynamics in the brain. Recent developments in fluorescent indicators and specialized optics have advanced research in the field of neuroscience. In this review, I will exemplify the power and beauty of fluorescence imaging by discussing my work focusing on the molecular dynamics of metabotropic glutamate receptor (mGluR) signaling at the synapse. By developing novel fluorescent indicators for glutamate, inositol 1,4,5-trisphosphate and Ca2+ within the endoplasmic reticulum, I succeeded in imaging the spatio-temporal dynamics of synaptic mGluR signaling, which led to the identification of novel mechanisms of mGluR-mediated glutamatergic neurotransmission. These discoveries highlight the importance of the development and application of novel fluorescence imaging techniques for the investigation of brain functions.

Metabotropic glutamate receptors (mGluRs) play a pivotal role in the neuroendocrine-immune regulation. In this study, eight mGluRs were identified in the Pacific Oyster Crassostrea gigas, which were classified into three subfamilies based on genetic similarity. All CgmGluRs harbor variable numbers of PBP1 domains at the N-terminus. The sequence and structural features of CgmGluRs are highly similar to mGluRs in other species. A uniformly upregulated expression of CgmGluRs was observed during D-shaped larval stage compared to early D-shaped larval stage. The transcripts of CgmGluRs were detectable in various tissues of oyster. Different CgmGluR exhibited diverse expression patterns response against different PAMP stimulations, among which CgmGluR5 was significantly downregulated under these stimulations, reflecting its sensitivity and broad-spectrum responsiveness to microbes. Following LPS stimulation, the mRNA expression of CgmGluR5 and CgCALM1 in haemocytes was suppressed within 6 h and returned to normal levels by 12 h. Inhibition of CgmGluR5 activity resulted in a significant reduction in CgCALM1 expression after 12 h. Further KEGG enrichment analysis suggested that CgmGluR5 might modulate calcium ion homeostasis and metabolic pathways by regulating CgCALM1. This research delivers the systematic analysis of mGluR in the Pacific Oyster, offering insights into evolutionary characteristics and immunoregulatory function of mGluR in mollusks.

In the nervous system, G protein-coupled receptors (GPCRs) control neuronal excitability, synaptic transmission, synaptic plasticity, and, ultimately, behavior through spatiotemporally precise initiation of a variety of signaling pathways. However, despite their critical importance, there is incomplete understanding of how these receptors are regulated to tune their signaling to specific neurophysiological contexts. A deeper mechanistic picture of neuromodulatory GPCR function is needed to fully decipher their biological roles and effectively harness them for the treatment of neurological and psychiatric disorders. In this review, we highlight recent progress in identifying novel modes of regulation of neuromodulatory GPCRs, including G protein- and receptor-targeting mechanisms, receptor-receptor crosstalk, and unique features that emerge in the context of chemical synapses. These emerging principles of neuromodulatory GPCR tuning raise critical questions to be tackled at the molecular, cellular, synaptic, and neural circuit levels in the future.

Short- and long-term forms of N-methyl-d-aspartate receptor (NMDAR)-dependent potentiation (most commonly termed short-term potentiation (STP) and long-term potentiation (LTP)) are co-induced in hippocampal slices by theta-burst stimulation, which mimics naturally occurring patterns of neuronal activity. While NMDAR-dependent LTP (NMDAR-LTP) is said to be the cellular correlate of long-term memory storage, NMDAR-dependent STP (NMDAR-STP) is thought to underlie the encoding of shorter-lasting memories. The mechanisms of NMDAR-LTP have been researched much more extensively than those of NMDAR-STP, which is characterized by its extreme stimulation dependence. Thus, in the absence of low-frequency test stimulation, which is used to test the magnitude of potentiation, NMDAR-STP does not decline until the stimulation is resumed. NMDAR-STP represents, therefore, an inverse variant of Hebbian synaptic plasticity, illustrating that inactive synapses can retain their strength unchanged until they become active again. The mechanisms, by which NMDAR-STP is stored in synapses without a decrement, are unknown and we report here that activation of metabotropic glutamate receptors may be critical in maintaining the potentiated state of synaptic transmission. This article is part of a discussion meeting issue \'Long-term potentiation: 50 years on\'.

In the human body, the majority of tryptophan is metabolized through the kynurenine pathway. This consists of several metabolites collectively called the kynurenines and includes, among others, kynurenic acid, L-kynurenine, or quinolinic acid. The wealth of metabolites, as well as the associated molecular targets and biological pathways, bring about a situation wherein even a slight imbalance in the kynurenine levels, both in the periphery and central nervous system, have broad consequences regarding general health. Cinnabarinic acid (CA) is the least known trace kynurenine, and its physiological and pathological roles are not widely understood. Some studies, however, indicate that it might be neuroprotective. Information on its hepatoprotective properties have also emerged, although these are pioneering studies and need to be replicated. Therefore, in this review, I aim to present and critically discuss the current knowledge on CA and its role in physiological and pathological settings to guide future studies.

Synaptic transmission from retinal photoreceptors to downstream ON-type bipolar cells (BCs) depends on the postsynaptic metabotropic glutamate receptor mGluR6, located at the BC dendritic tips. Glutamate binding to mGluR6 initiates G-protein signaling that ultimately leads to BC depolarization in response to light. The mGluR6 receptor also engages in trans-synaptic interactions with presynaptic ELFN adhesion proteins. The roles of post-translational modifications in mGluR6 trafficking and function are unknown. Treatment with glycosidase enzymes PNGase F and Endo H demonstrated that both endogenous and heterologously expressed mGluR6 contain complex N-glycosylation acquired in the Golgi. Pull-down experiments with ELFN1 and ELFN2 extracellular domains revealed that these proteins interact exclusively with the complex glycosylated form of mGluR6. Mutation of the four predicted N-glycosylation sites, either singly or in combination, revealed that all four sites are glycosylated. Single mutations partially reduced, but did not abolish, surface expression in heterologous cells, while triple mutants had little or no surface expression, indicating that no single glycosylation site is necessary or sufficient for plasma membrane trafficking. Mutation at N445 severely impaired both ELFN1 and ELFN2 binding. All single mutants exhibited dendritic tip enrichment in rod BCs, as did the triple mutant with N445 as the sole N-glycosylation site, demonstrating that glycosylation at N445 is sufficient but not necessary for dendritic tip localization. The quadruple mutant was completely mislocalized. These results reveal a key role for complex N-glycosylation in regulating mGluR6 trafficking and ELFN binding, and by extension, function of the photoreceptor synapses.

Acting as GTPase activating proteins promoting the silencing of activated G-proteins, regulators of G protein signaling (RGSs) are generally considered negative modulators of cell signaling. In the CNS, the expression of RGS4 is altered in diverse pathologies and its upregulation was reported in astrocytes exposed to an inflammatory environment. In a model of cultured cortical astrocytes, we herein investigate the influence of RGS4 on intracellular calcium signaling mediated by type 5 metabotropic glutamate receptor (mGluR5), which is known to support the bidirectional communication between neurons and glial cells. RGS4 activity was manipulated by exposure to the inhibitor CCG 63802 or by infecting the cells with lentiviruses designed to achieve the silencing or overexpression of RGS4. The pharmacological inhibition or silencing of RGS4 resulted in a decrease in the percentage of cells responding to the mGluR5 agonist DHPG and in the proportion of cells showing typical calcium oscillations. Conversely, RGS4-lentivirus infection increased the percentage of cells showing calcium oscillations. While the physiological implication of cytosolic calcium oscillations in astrocytes is still under investigation, the fine-tuning of calcium signaling likely determines the coding of diverse biological events. Indirect signaling modulators such as RGS4 inhibitors, used in combination with receptor ligands, could pave the way for new therapeutic approaches for diverse neurological disorders with improved efficacy and selectivity.

Glutamate (Glu) toxicity has been an important research topic in toxicology and neuroscience studies. In vitro and in vivo studies have shown that Group II metabotropic Glu2 (mGlu2) activators have cell viability effects. This study aims to determine a candidate ligand with high mGlu2 allosteric region activity among cytotoxicity-safe molecules using the in silico positioning method and to evaluate its cell viability effect in vitro. We investigated the candidate molecule\'s cell viability effect on the SH-SY5Y human neuroblastoma cell line by MTT analysis. In the study, LY 379268 (agonist) and JNJ-46281222 (positive allosteric modulator; PAM) were used as control reference molecules. Drug bank screening yielded THRX-195518 (docking score being -12.4 kcal/mol) as a potential novel drug candidate that has a high docking score and has not been mentioned in the literature so far. The orthosteric agonist LY 379268 exhibited a robust protective effect in our study. Additionally, our findings demonstrate that JNJ-46281222 and THRX-195518, identified as activating the mGlu2 allosteric region through in silico methods, preserve cell viability against Glu toxicity. Therefore, our study not only emphasizes the positive effects of this compound on cell viability against Glu toxicity but also sheds light on the potential of THRX-195518, acting as a mGlu2 PAM, based on in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) data, as a candidate drug molecule. These findings underscore the potential utility of THRX-195518 against both neurotoxicity and Central Nervous System (CNS) disorders, providing valuable insights.