Glutamate Receptor Function Research Articles

Direct and indirect pathways for heterosynaptic interaction underlying developmental synapse elimination in the mouse cerebellum

Developmental synapse elimination is crucial for shaping mature neural circuits. In the neonatal mouse cerebellum, Purkinje cells (PCs) receive excitatory synaptic inputs from multiple climbing fibers (CFs) and synapses from all but one CF are eliminated by around postnatal day 20. Heterosynaptic interaction between CFs and parallel fibers (PFs), the axons of cerebellar granule cells (GCs) forming excitatory synapses onto PCs and molecular layer interneurons (MLIs), is crucial for CF synapse elimination. However, mechanisms for this heterosynaptic interaction are largely unknown. Here we show that deletion of AMPA-type glutamate receptor functions in GCs impairs CF synapse elimination mediated by metabotropic glutamate receptor 1 (mGlu1) signaling in PCs. Furthermore, CF synapse elimination is impaired by deleting NMDA-type glutamate receptors from MLIs. We propose that PF activity is crucial for CF synapse elimination by directly activating mGlu1 in PCs and indirectly enhancing the inhibition of PCs through activating NMDA receptors in MLIs.

Orb2 enables rare-codon-enriched mRNA expression during Drosophila neuron differentiation

Regulation of codon optimality is an increasingly appreciated layer of cell- and tissue-specific protein expression control. Here, we use codon-modified reporters to show that differentiation of Drosophila neural stem cells into neurons enables protein expression from rare-codon-enriched genes. From a candidate screen, we identify the cytoplasmic polyadenylation element binding (CPEB) protein Orb2 as a positive regulator of rare-codon-dependent mRNA stability in neurons. Using RNA sequencing, we reveal that Orb2-upregulated mRNAs in the brain with abundant Orb2 binding sites have a rare-codon bias. From these Orb2-regulated mRNAs, we demonstrate that rare-codon enrichment is important for mRNA stability and social behavior function of the metabotropic glutamate receptor (mGluR). Our findings reveal a molecular mechanism by which neural stem cell differentiation shifts genetic code regulation to enable critical mRNA stability and protein expression.

3-Hydroxykynurenine targets kainate receptors to promote defense against infection.

Bacterial infection involves a complex interaction between the pathogen and host where the outcome of infection is not solely determined by pathogen eradication. To identify small molecules that promote host survival by altering the host-pathogen dynamic, we conducted an in vivo chemical screen using zebrafish embryos and found that treatment with 3-hydroxykynurenine (3-HK) protects from lethal bacterial infection. 3-HK, a metabolite produced through host tryptophan metabolism, has no direct antibacterial activity but enhances host survival by restricting bacterial expansion in macrophages through a systemic mechanism that targets kainate-sensitive glutamate receptors. These findings reveal a new pathway by which tryptophan metabolism and kainate-sensitive glutamate receptors function and interact to modulate immunity, with important implications for the coordination between the immune and nervous systems in pathological conditions.

Chronic Vagotomy Blunts Cardiorespiratory Reflex Responses Through Pre- and Postsynaptic Effects in the Brainstem Nucleus Tractus Solitarii (nTS)

Sensory information from the heart, lungs, and gastrointestinal tract travels to the brain via afferents of the paired vagus nerves, where it is first integrated in the brainstem nTS that critically contributes to cardiorespiratory function. We have shown that reducing input to the nTS by unilaterally transecting the vagus nerve (vagotomy) induces astrocyte reactivity and microglial activation, and reduces sighs in hypoxia. However, the cardiorespiratory effects of vagotomy and contributing mechanisms are not well understood. We hypothesized that chronic vagotomy blunts cardiorespiratory reflex responses to vagal afferent stimulation via reduced nTS neuronal activity. Male Sprague-Dawley rats (6-wk) underwent right cervical vagotomy (or sham surgery) caudal to the nodose ganglion. After 7 days rats were anesthetized, ventilated, and instrumented to record mean arterial pressure (MAP), heart rate (HR), splanchnic sympathetic and phrenic nerve activity (SSNA and PhrNA, respectively), which decreased in response to vagal afferent stimulation in sham animals. Vagotomy blunted these decreases in MAP, SSNA, and PhrNA compared to shams. In rats expressing the calcium indicator GCaMP8m in nTS neurons, vagotomy also reduced neuronal Ca2+ responses to afferent stimulation both in vivo (photometry) and in vitro (live slice imaging). We then hypothesized that these changes were due to altered signaling at the level of the tripartite synapse, where astrocytes enwrap the sensory afferent-nTS soma synapse. We found that there is likely a vagal afferent contribution to the altered reflex responses, as vagotomy reduced presynaptic Ca2+ responses in vagal afferent terminals to solitary tract stimulation in the brainstem slice. Glutamate agonists, which induce similar cardiorespiratory responses as vagal stimulation, were nanoinjected into the nTS to determine if there was a postsynaptic contribution to the blunted responses as well. Responses to glutamate injection were also blunted following vagotomy, indicating that astrocytic glutamate uptake or nTS soma glutamate receptor function may be altered. Blocking astrocytic excitatory amino acid transporters, which remove extracellular glutamate in the nTS induced similar baseline effects and did not restore cardiorespiratory responses to glutamate in vagotomy. However, nanoinjection of NMDA into the nTS to activate ionotropic glutamate receptors mimicked the blunted responses to glutamate following vagotomy, with no difference in responses to AMPA nanoinjection. Likewise, immunohistochemistry and Western blot showed that vagotomy reduced NMDA receptor subunit expression in the nTS. Together these results indicate that chronic vagotomy blunts cardiorespiratory responses to vagal afferent stimulation, likely through reduced presynaptic activation and decreased NMDA receptor expression and function. These studies will help improve our understanding of how afferent input induces neuroplasticity within the nTS, and the resulting impacts on cardiorespiratory function in health and disease. NIH HL132836, HL128454, HL98602, T32OD011126. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibiting Hippo pathway kinases releases WWC1 to promote AMPAR-dependent synaptic plasticity and long-term memory in mice.

The localization, number, and function of postsynaptic AMPA-type glutamate receptors (AMPARs) are crucial for synaptic plasticity, a cellular correlate for learning and memory. The Hippo pathway member WWC1 is an important component of AMPAR-containing protein complexes. However, the availability of WWC1 is constrained by its interaction with the Hippo pathway kinases LATS1 and LATS2 (LATS1/2). Here, we explored the biochemical regulation of this interaction and found that it is pharmacologically targetable in vivo. In primary hippocampal neurons, phosphorylation of LATS1/2 by the upstream kinases MST1 and MST2 (MST1/2) enhanced the interaction between WWC1 and LATS1/2, which sequestered WWC1. Pharmacologically inhibiting MST1/2 in male mice and in human brain-derived organoids promoted the dissociation of WWC1 from LATS1/2, leading to an increase in WWC1 in AMPAR-containing complexes. MST1/2 inhibition enhanced synaptic transmission in mouse hippocampal brain slices and improved cognition in healthy male mice and in male mouse models of Alzheimer's disease and aging. Thus, compounds that disrupt the interaction between WWC1 and LATS1/2 might be explored for development as cognitive enhancers.

Measuring Presynaptic Calcium Influx at the Drosophila Larval Neuromuscular Junction.

Synaptic transmission plays a critical role in information processing and storage within the nervous system. The triggering of action potentials activates voltage-gated calcium channels at presynaptic active zones, facilitating the calcium-dependent release of synaptic vesicles. Homeostatic mechanisms are crucial in stabilizing synaptic function. At the Drosophila neuromuscular junction, a compensatory increase in presynaptic neurotransmitter release occurs when postsynaptic glutamate receptor function is pharmacologically or genetically impaired, thereby stabilizing synaptic output. This adaptation is known as presynaptic homeostatic potentiation (PHP). Recent advancements, including confocal and super-resolution imaging techniques, have demonstrated an increase in presynaptic calcium influx during both the rapid induction and long-term maintenance of PHP. These observations indicate that the abundance and structural organization of presynaptic calcium channels, along with various active zone components, undergo modifications following the suppression of postsynaptic glutamate receptors. Such findings underscore the critical roles of trafficking and stabilization of presynaptic calcium channels and active zone proteins in homeostatic plasticity. This protocol describes using calcium indicators and confocal imaging methods to measure single-action potential-evoked presynaptic calcium influx during PHP.

Neto proteins differentially modulate the gating properties of Drosophila NMJ glutamate receptors.

The formation of functional synapses requires co-assembly of ion channels with their accessory proteins which controls where, when, and how neurotransmitter receptors function. The auxiliary protein Neto modulates the function of kainate-type glutamate receptors in vertebrates as well as at the Drosophila neuromuscular junction (NMJ), a glutamatergic synapse widely used for genetic studies on synapse development. We previously reported that Neto is essential for the synaptic recruitment and function of glutamate receptors. Here, using outside-out patch-clamp recordings and fast ligand application, we examine for the first time the biophysical properties of recombinant Drosophila NMJ receptors expressed in HEK293T cells and compare them with native receptor complexes of genetically controlled composition. The two Neto isoforms, Neto-α and Neto-β, differentially modulate the gating properties of NMJ receptors. Surprisingly, we found that deactivation is extremely fast and that the decay of synaptic currents resembles the rate of iGluR desensitization. The functional analyses of recombinant iGluRs that we report here should greatly facilitate the interpretation of compound in vivo phenotypes of mutant animals.

Excitotoxic Storms of Ischemic Stroke: A Non-neuronal Perspective.

Numerous neurological disorders share a fatal pathologic process known as glutamate excitotoxicity. Among which, ischemic stroke is the major cause of mortality and disability worldwide. For a long time, the main idea of developing anti-excitotoxic neuroprotective agents was to block glutamate receptors. Despite this, there has been little successful clinical translation to date. After decades of "neuron-centered" views, a growing number of studies have recently revealed the importance of non-neuronal cells. Glial cells, cerebral microvascular endothelial cells, blood cells, and so forth are extensively engaged in glutamate synthesis, release, reuptake, and metabolism. They also express functional glutamate receptors and can listen and respond for fast synaptic transmission. This broadens the thoughts of developing excitotoxicity antagonists. In this review, the critical contribution of non-neuronal cells in glutamate excitotoxicity during ischemic stroke will be emphasized in detail, and the latest research progress as well as corresponding therapeutic strategies will be updated at length, aiming to reconceptualize glutamate excitotoxicity in a non-neuronal perspective.

Integrin α3 is required for high-frequency repetitive transcranial magnetic stimulation-induced glutamatergic synaptic transmission in mice with ischemia.

Repetitive transcranial magnetic stimulation (rTMS) is an effective therapy in post-stroke motor recovery. However, the underlying mechanisms of rTMS regulates long-lasting changes with synaptic transmission and glutamate receptors function (including AMPARs or NMDARs) remains unclear. Mice were received 10-Hz rTMS treatment once daily on the third day after photothrombotic (PT) stroke for 18 days. Motor behaviors and the Western blot were used to evaluate the therapeutic efficacy of 10-Hz rTMS in the mice with PT model. Moreover, we used wild-type (WT) and NEX-α3-/- mice to further explore the 10-Hz rTMS effect. We found that 10-Hz rTMS improved the post-stroke motor performance in the PT mice. Moreover, the levels of AMPAR, vGlut1, and integrin α3 in the peri-infarct were significantly increased in the rTMS group. In contrast, 10-Hz rTMS did not induce these aforementioned effects in NEX-α3-/- mice. The amplitude of AMPAR-mediated miniature excitatory postsynaptic currents (EPSCs) and evoked EPSCs was increased in the WT + rTMS group, but did not change in NEX-α3-/- mice with rTMS. In this study, 10-Hz rTMS improved the glutamatergic synaptic transmission in the peri-infract cortex through effects on integrin α3 and AMPARs, which resulted in motor function recovery after stroke.

Discovery and characterization of a specific inhibitor of serine-threonine kinase cyclin-dependent kinase-like 5 (CDKL5) demonstrates role in hippocampal CA1 physiology.

Pathological loss-of-function mutations in cyclin-dependent kinase-like 5 (CDKL5) cause CDKL5 deficiency disorder (CDD), a rare and severe neurodevelopmental disorder associated with severe and medically refractory early-life epilepsy, motor, cognitive, visual, and autonomic disturbances in the absence of any structural brain pathology. Analysis of genetic variants in CDD has indicated that CDKL5 kinase function is central to disease pathology. CDKL5 encodes a serine-threonine kinase with significant homology to GSK3β, which has also been linked to synaptic function. Further, Cdkl5 knock-out rodents have increased GSK3β activity and often increased long-term potentiation (LTP). Thus, development of a specific CDKL5 inhibitor must be careful to exclude cross-talk with GSK3β activity. We synthesized and characterized specific, high-affinity inhibitors of CDKL5 that do not have detectable activity for GSK3β. These compounds are very soluble in water but blood-brain barrier penetration is low. In rat hippocampal brain slices, acute inhibition of CDKL5 selectively reduces postsynaptic function of AMPA-type glutamate receptors in a dose-dependent manner. Acute inhibition of CDKL5 reduces hippocampal LTP. These studies provide new tools and insights into the role of CDKL5 as a newly appreciated key kinase necessary for synaptic plasticity. Comparisons to rodent knock-out studies suggest that compensatory changes have limited the understanding of the roles of CDKL5 in synaptic physiology, plasticity, and human neuropathology.

Potentiation of neuronal activity by tonic GluD1 current in brain slices

Ion channel function of native delta glutamate receptors (GluDR) is incompletely understood. Previously, we and others have shown that activation of Gαq protein‐coupled receptors (GqPCR) produces a slow inward current carried by GluD1R. GluD1R also carries a tonic cation current of unknown cause. Here, using voltage‐clamp electrophysiological recordings from adult mouse brain slices containing the dorsal raphe nucleus, we find no role of ongoing G‐protein‐coupled receptor activity in generating or sustaining tonic GluD1R currents. Neither augmentation nor disruption of G protein activity affects tonic GluD1R currents, suggesting that ongoing G‐protein‐coupled receptor activity does not give rise to tonic GluD1R currents. Further, the tonic GluD1R current is unaffected by the addition of external glycine or D‐serine, which influences GluD2R current at millimolar concentrations. Instead, GqPCR‐stimulated and tonic GluD1R currents are regulated by physiological levels of external calcium. In current‐clamp recordings, block of GluD1R channels hyperpolarizes the membrane by ~7 mV at subthreshold potentials, reducing excitability. Thus, GluD1R carries a G‐protein‐independent tonic current that contributes to subthreshold neuronal excitation in the dorsal raphe nucleus.

Neuronal SNAP-23 is critical for synaptic plasticity and spatial memory independently of NMDA receptor regulation

SNARE-mediated membrane fusion plays a crucial role in presynaptic vesicle exocytosis and also in postsynaptic receptor delivery. The latter is considered particularly important for synaptic plasticity and learning and memory, yet the identity of the key SNARE proteins remains elusive. Here, we investigate the role of neuronal synaptosomal-associated protein-23 (SNAP-23) by analyzing pyramidal-neuron specific SNAP-23 conditional knockout (cKO) mice. Electrophysiological analysis of SNAP-23 deficient neurons using acute hippocampal slices showed normal basal neurotransmission in CA3-CA1 synapses with unchanged AMPA and NMDA currents. Nevertheless, we found theta-burst stimulation-induced long-term potentiation (LTP) was vastly diminished in SNAP-23 cKO slices. Moreover, unlike syntaxin-4 cKO mice where both basal neurotransmission and LTP decrease manifested changes in a broad set of behavioral tasks, deficits of SNAP-23 cKO are more limited to spatial memory. Our data reveal that neuronal SNAP-23 is selectively crucial for synaptic plasticity and spatial memory without affecting basal glutamate receptor function.

Synaptic scaling of corticostriatal circuits underlies hyperactivity in GABA Transporter-1 deficient mice

Homeostatic synaptic scaling entails adjustment of synaptic strength on a cell to prolonged changes of neuronal activity, which is postulated to participate in neuropsychiatric disorders invivo. Here, we find that sustained elevation in ambient GABA levels, by either genetic deletion or pharmacological blockade of GABA transporter-1 (GAT1), leads to synaptic scaling up of corticostriatal pathways, which underlies locomotor hyperactivity. Meanwhile, medium spinyneurons of the dorsal striatum exhibit an aberrant increase in excitatory synaptic transmission and corresponding structural changes in dendritic spines. Mechanistically, GAT1 deficiency dampens the expression and function of metabotropic glutamate receptors (mGluRs) and endocannabinoid (eCB)-dependent long-term depression of excitatory transmission. Conversely, restoring mGluR function in GAT1 deficient mice rescues excitatory transmission. Lastly, pharmacological potentiation of mGluR-eCB signaling or inhibition of homomeric-GluA1 AMPA receptors eliminates locomotor hyperactivity in the GAT1 deficient mice. Together, these results reveal a synaptic scaling mechanism in corticostriatal pathways that regulate locomotor activity.

Glutamatergic neurotransmission: A potential pharmacotherapeutic target for the treatment of cognitive disorders.

In the mammalian brain, glutamate is regarded to be the primary excitatory neurotransmitter due to its widespread distribution and wide range of metabolic functions. Glutamate plays key roles in regulating neurogenesis, synaptogenesis, neurite outgrowth, and neuron survival in the brain. Ionotropic and metabotropic glutamate receptors, neurotransmitters, neurotensin, neurosteroids, and others co-ordinately formulate a complex glutamatergic network in the brain that maintains optimal excitatory neurotransmission. Cognitive activities are potentially synchronized by the glutamatergic activities in the brain via restoring synaptic plasticity. Dysfunctional glutamate receptors and other glutamatergic components are responsible for the aberrant glutamatergic activity in the brain that cause cognitive impairments, loss of synaptic plasticity, and neuronal damage. Thus, controlling the brain's glutamatergic transmission and modifying glutamate receptor function could be a potential therapeutic strategy for cognitive disorders. Certain drugs that regulate glutamate receptor activities have shown therapeutic promise in improving cognitive functions in preclinical and clinical studies. However, several issues regarding precise functional information of glutamatergic activity are yet to be comprehensively understood. The present article discusses the scope of developing glutamatergic systems as prospective pharmacotherapeutic targets to treat cognitive disorders. Special attention has been given to recent developments, challenges, and future prospects.

Metabotropic glutamate receptor 1 alpha: a unique receptor variant with variable implications for Alzheimer's disease pathogenesis.

Metabotropic glutamate receptor 1 alpha: a unique receptor variant with variable implications for Alzheimer's disease pathogenesis.

Metabotropic glutamate receptor function and regulation of sleep-wake cycles.

Metabotropic glutamate (mGlu) receptors are the most abundant family of G-protein coupled receptors and are widely expressed throughout the central nervous system (CNS). Alterations in glutamate homeostasis, including dysregulations in mGlu receptor function, have been indicated as key contributors to multiple CNS disorders. Fluctuations in mGlu receptor expression and function also occur across diurnal sleep-wake cycles. Sleep disturbances including insomnia are frequently comorbid with neuropsychiatric, neurodevelopmental, and neurodegenerative conditions. These often precede behavioral symptoms and/or correlate with symptom severity and relapse. Chronic sleep disturbances may also be a consequence of primary symptom progression and can exacerbate neurodegeneration in disorders including Alzheimer's disease (AD). Thus, there is a bidirectional relationship between sleep disturbances and CNS disorders; disrupted sleep may serve as both a cause and a consequence of the disorder. Importantly, comorbid sleep disturbances are rarely a direct target of primary pharmacological treatments for neuropsychiatric disorders even though improving sleep can positively impact other symptom clusters. This chapter details known roles of mGlu receptor subtypes in both sleep-wake regulation and CNS disorders focusing on schizophrenia, major depressive disorder, post-traumatic stress disorder, AD, and substance use disorder (cocaine and opioid). In this chapter, preclinical electrophysiological, genetic, and pharmacological studies are described, and, when possible, human genetic, imaging, and post-mortem studies are also discussed. In addition to reviewing the important relationships between sleep, mGlu receptors, and CNS disorders, this chapter highlights the development of selective mGlu receptor ligands that hold promise for improving both primary symptoms and sleep disturbances.

Sex- and age-dependent effects of chronic corticosterone exposure on depressive-like, anxiety-like, and fear-related behavior: Role of amygdala glutamate receptors in the rat.

Persistent glucocorticoid elevation consistent with chronic stress exposure can lead to psychopathology, including mood and anxiety disorders. Women and stress-exposed adolescents are more likely to be diagnosed with mood disorders, suggesting that sex and age are important factors in determining vulnerability, though much remains to be determined regarding the mechanisms underlying this risk. Thus, the aim of the present experiments was to use the chronic corticosterone (CORT) exposure paradigm, a model of depression-like behavior that has previously been established primarily in adult males, to determine the mood-related effects of CORT in female and adolescent rats. Depression- and anxiety-like effects in adulthood were determined using the sucrose preference (SPT), the forced swim test (FST), the elevated plus maze, and fear conditioning. Basolateral amygdala (BLA) and medial prefrontal cortex (mPFC) glutamate receptor subunit levels were then measured. In a subsequent experiment, adult male and female rats were tested for the effects of pharmacological activation (via AMPA) or inhibition (via NBQX) of AMPA receptors in the BLA on behavior in the FST. Overall, females showed reduced anxiety- and depressive-like behaviors relative to males. However, females treated with CORT in adolescence, but not adulthood, had increased immobility in the FST, indicative of depression-like behavior. In contrast, CORT did not alter behavior in adolescent-treated males, though the previously reported depression-like effect of adult CORT exposure was observed. Control females had higher expression of the AMPA receptor subunits GluA1 and GluA2/3 selectively in the BLA relative to males. Adolescent CORT treatment, however, decreased BLA GluA1 and GluA2/3 expression in females, but increased expression in males, consistent with the direction of depression-like behavioral effects. Male and female rats also demonstrated opposing patterns of response to BLA AMPA receptor modulation in the FST, with AMPA infusion magnifying the sex difference of decreased immobility in females. Overall, these experiments show that increased glutamate receptor function in the BLA may decrease the risk of developing depressive-like behavior, further supporting efforts to target glutamatergic receptors for the treatment of stress-related psychiatric disorders. These findings also support further focus on sex as a biological variable in neuropsychiatric research.

Structural Insights into Function of Ionotropic Glutamate Receptors

Structural Insights into Function of Ionotropic Glutamate Receptors

A Silent Allosteric Modulator

Deterioration in cognitive dysfunction in Alzheimer’s disease (AD) patients is associated with microglia-mediated synaptic destruction. Using AD mouse models Spurrier and colleagues discerned the function of the metabotropic glutamate receptor 5 (mGluR5) on synaptic loss and demonstrated that a silent allosteric modulator (SAM) of mGLuR5 when given through the oral route successfully restored synaptic density and reduced phosphorylated TAU accumulation. The treatment in AD mice with SAM reversed the induced changes as a result of altered gene expression and blocked the synaptic confinement of the C1q complement protein. The findings of this study suggest that targeting mGluR5 with SAMs could be a powerful strategy for restricting AD-related synaptic loss. Sci. Transl. Med.14, eabi8593 (2022).

Role of glutamate receptor complex in the organism. Ligands of NMDA receptors in neurodegenerative processes – a modern state of the problem

(S)-glutamic acid (glutamate) is the main excitatory mediator in the central nervous system, responsible for regulating of many physiological functions. Dysfunction of the glutamatergic system characterizes of many pathological conditions in neurology and psychiatry, and the aberrant function of glutamate receptors plays a key role in the development of neurodegenerative processes. Glutamate is crucial for many aspects of normal brain function, including memory, learning, and motor planning. In addition, glutamate is involved in the regulation of the peripheral nervous and endocrine systems. Glutamate receptors are critically important molecules necessary for the physiological functioning of the brain: they modulate neurotransmission and regulate the strength of excitatory and inhibitory transmission in the nervous system. In this regard, the use of drugs that affect glutamatergic transmission has an impact on the most important processes of neuronal transmission. The research and development of pharmacological agents involved in the processes of glutamate transmission is a relevant task of modern neuropsychopharmacology and has a purpose to improve the effectiveness and safety of available glutamatergic molecules.