Physiological Perspectives on Molecular Mechanisms and Regulation of Vesicular Glutamate Transport: Lessons From Calyx of Held Synapses

Accumulation of glutamate, the primary excitatory neurotransmitter in the mammalian central nervous system, into presynaptic synaptic vesicles (SVs) depends upon three vesicular glutamate transporters (VGLUTs). Since VGLUTs are driven by a proton electrochemical gradient across the SV membrane generated by vacuolar-type H+-ATPases (V-ATPases), the rate of glutamate transport into SVs, as well as the amount of glutamate in SVs at equilibrium, are influenced by activities of both VGLUTs and V-ATPase. Despite emerging evidence that suggests various factors influencing glutamate transport by VGLUTs in vitro, little has been reported in physiological or pathological contexts to date. Historically, this was partially due to a lack of appropriate methods to monitor glutamate loading into SVs in living synapses. Furthermore, whether or not glutamate refilling of SVs can be rate-limiting for synaptic transmission is not well understood, primarily due to a lack of knowledge concerning the time required for vesicle reuse and refilling during repetitive stimulation. In this review, we first introduce a unique electrophysiological method to monitor glutamate refilling by VGLUTs in a giant model synapse from the calyx of Held in rodent brainstem slices, and we discuss the advantages and limitations of the method. We then introduce the current understanding of factors that potentially alter the amount and rate of glutamate refilling of SVs in this synapse, and discuss open questions from physiological viewpoints.

A Conditional Glutamatergic Synaptic Vesicle Marker for Drosophila

Glutamate is a principal neurotransmitter used extensively by the nervous systems of all vertebrate and invertebrate animals. It is primarily an excitatory neurotransmitter that has been implicated in nervous system development as well as a myriad of brain functions from the simple transmission of information between neurons to more complex aspects of nervous system function including synaptic plasticity, learning, and memory. Identification of glutamatergic neurons and their sites of glutamate release are thus essential for understanding the mechanisms of neural circuit function and how information is processed to generate behavior. Here we describe and characterize smFLAG-vGlut, a conditional marker of glutamatergic synaptic vesicles for the Drosophila model system. smFLAG-vGlut is validated for functionality, conditional expression, and specificity for glutamatergic neurons and synaptic vesicles. The utility of smFLAG-vGlut is demonstrated by glutamatergic neurotransmitter phenotyping of 26 different central complex neuron types of which nine were established to be glutamatergic. This illumination of glutamate neurotransmitter usage will enhance the modeling of central complex neural circuitry and thereby our understanding of information processing by this region of the fly brain. The use of smFLAG for glutamatergic neurotransmitter phenotyping and identification of glutamate release sites can be extended to any Drosophila neuron(s) represented by a binary transcription system driver.

Long-term dynamic changes of NMDA receptors following an excitotoxic challenge

Excitotoxicity is a form of neuronal death characterized by the sustained activation of N-methyl-D-aspartate receptors (NMDARs) triggered by the excitatory neurotransmitter glutamate. NADPH-diaphorase neurons [also known as nNOS (+) neurons] are a subpopulation of aspiny interneurons, largely spared following excitotoxic challenges. Unlike nNOS (-) cells, nNOS (+) neurons fail to generate reactive oxygen species in response to NMDAR activation, a key divergent step in the excitotoxic cascade. However, additional mechanisms underlying the reduced vulnerability of nNOS (+) neurons to NMDAR-driven neuronal death have not been explored. Using functional, genetic, and molecular analysis in striatal cultures, we demonstrate that nNOS (+) neurons possess distinct NMDAR properties. These specific features are primarily driven by the peculiar redox milieu of this subpopulation. In addition, we found that nNOS (+) neurons exposed to a pharmacological maneuver set to mimic chronic excitotoxicity alter their responses to NMDAR-mediated challenges. These findings suggest the presence of mechanisms providing long-term dynamic regulation of NMDARs that can have critical implications in neurotoxic settings.

Glutamate as a Stressoric Factor for the Ex Vivo Release of Catecholamines from the Rabbit Medial Prefrontal Cortex (mPFC)

One of the major roles of glutamic acid (Glu) is to serve as an excitatory neurotransmitter within the central nervous system (CNS). This amino acid influences the activity of several brain areas, including the thalamus, brainstem, spinal cord, basal ganglia, and pons. Catecholamines (CAs) are synthesized in the brain and adrenal medulla and by some sympathetic nerve fibers. CAs, including dopamine (DA), norepinephrine (NE), and epinephrine (E), are the principal neurotransmitters that mediate a variety of CNS functions, such as motor control, cognition, emotion, memory processing, pain, stress, and endocrine modulation. This study aims to investigate the effects of the application of various Glu concentrates (5, 50, and 200 µM) on CAs release from rabbit medial prefrontal cortex (mPFC) slices and compare any resulting correlations with CAs released from the hypothalamus during 90 min of incubation. Medial prefrontal cortex samples were dissected from decapitated, twelve-week-old female rabbits. The results demonstrated that Glu differentially influences the direct release of CAs from the mPFC and the indirect release of CAs from the hypothalamus. When under stress, the hypothalamus, a central brain structure of the HPA axis, induces and adapts such processes. Generally, there was an inhibitory effect of Glu on CAs release from mPFC slices. Our findings show that the effect arises from Glu’s action on higher-order motivational structures, which may indicate its contribution to the stress response by modulating the amount of CAs released.

Oligodendrocytes, BK channels and the preservation of myelin

Oligodendrocytes wrap multiple lamellae of their membrane, myelin, around axons of the central nervous system (CNS), to improve impulse conduction. Myelin synthesis is specialised and dynamic, responsive to local neuronal excitation. Subtle pathological insults are sufficient to cause significant neuronal metabolic impairment, so myelin preservation is necessary to safeguard neural networks. Multiple sclerosis (MS) is the most prevalent demyelinating disease of the CNS. In MS, inflammatory attacks against myelin, proposed to be autoimmune, cause myelin decay and oligodendrocyte loss, leaving neurons vulnerable. Current therapies target the prominent neuroinflammation but are mostly ineffective in protecting from neurodegeneration and the progressive neurological disability. People with MS have substantially higher levels of extracellular glutamate, the main excitatory neurotransmitter. This impairs cellular homeostasis to cause excitotoxic stress. Large conductance Ca2+-activated K+ channels (BK channels) could preserve myelin or allow its recovery by protecting cells from the resulting excessive excitability. This review evaluates the role of excitotoxic stress, myelination and BK channels in MS pathology, and explores the hypothesis that BK channel activation could be a therapeutic strategy to protect oligodendrocytes from excitotoxic stress in MS. This could reduce progression of neurological disability if used in parallel to immunomodulatory therapies.

Evolutionarily Established Palmitoylation-Dependent Regulatory Mechanisms of the Vertebrate Glutamatergic Synapse and Diseases Caused by Their Disruption

Glutamate is the major excitatory neurotransmitter in the vertebrate brain and various modifications have been established in the glutamatergic synapses. Generally, many neuronal receptors and ion channels are regulated by S-palmitoylation, a reversible post-translational protein modification. Genome sequence databases show the evolutionary acquisition and conservation concerning vertebrate-specific palmitoylation of synaptic proteins including glutamate receptors. Moreover, palmitoylation of some glutamate receptor-binding proteins is subsequently acquired only in some mammalian lineages. Recent progress in genome studies has revealed that some palmitoylation-catalyzing enzymes are the causative genes of neuropsychiatric disorders. In this review, I will summarize the evolutionary development of palmitoylation-dependent regulation of glutamatergic synapses and their dysfunctions which are caused by the disruption of palmitoylation mechanism.

Glutamate: The Master Neurotransmitter and Its Implications in Chronic Stress and Mood Disorders

This brief review article makes the argument that glutamate is deserving of its newfound attention within the neuroscience literature and that many directions of important research have yet to be explored. Glutamate is an excitatory neurotransmitter with several types of receptors found throughout the central nervous system, and its metabolism is important to maintaining optimal levels within the extracellular space. As such, it is important to memory, cognition, and mood regulation. The mechanisms by which chronic stress affect the glutamatergic system and neuroplasticity are outlined. Several implications for potential pharmacologic and non-pharmacologic interventions are discussed.

Ataxia-linked SLC1A3 mutations alter EAAT1 chloride channel activity and glial regulation of CNS function

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS). Excitatory Amino Acid Transporters (EAATs) regulate extracellular glutamate by transporting it into cells, mostly glia, to terminate neurotransmission and to avoid neurotoxicity. EAATs are also chloride (Cl-) channels, but the physiological role of Cl- conductance through EAATs is poorly understood. Mutations of human EAAT1 (hEAAT1) have been identified in patients with episodic ataxia type 6 (EA6). One mutation showed increased Cl- channel activity and decreased glutamate transport, but the relative contributions of each function of hEAAT1 to mechanisms underlying the pathology of EA6 remain unclear. Here we investigated the effects of five additional EA6-related mutations on hEAAT1 function in Xenopus laevis oocytes, and on CNS function in a Drosophila melanogaster model of locomotor behavior. Our results indicate that mutations with decreased hEAAT1 Cl- channel activity and functional glutamate transport can also contribute to the pathology of EA6, highlighting the importance of Cl- homeostasis in glial cells for proper CNS function. We also identified a novel mechanism involving an ectopic sodium (Na+) leak conductance in glial cells. Together, these results strongly support the idea that EA6 is primarily an ion channelopathy of CNS glia.

An Unrecognized Fundamental Relationship between Neurotransmitters: Glutamate Protects against Catecholamine Oxidation

Neurotransmitter catecholamines (dopamine, epinephrine, and norepinephrine) are liable to undergo oxidation, which copper is deeply involved in. Catecholamine oxidation-derived neurotoxicity is recognized as a pivotal pathological mechanism in neurodegenerative diseases. Glutamate, as an excitatory neurotransmitter, is enriched in the brain at extremely high concentrations. However, the chemical biology relationship of these two classes of neurotransmitters remains largely unknown. In the present study, we assessed the influences of glutamate on the autoxidation of catecholamines, the copper- and copper-containing ceruloplasmin-mediated oxidation of catecholamines, the catecholamine-induced formation of quinoprotein, catecholamine/copper-induced hydroxyl radicals, and DNA damage in vitro. The results demonstrate that glutamate, at a physiologically achievable molar ratio of glutamate/catecholamines, has a pronounced inhibitory effect on catecholamine oxidation, catecholamine oxidation-evoked hydroxyl radicals, quinoprotein, and DNA damage. The protective mechanism of glutamate against catecholamine oxidation could be attributed to its restriction of the redox activity of copper via chelation. This previously unrecognized link between glutamate, catecholamines, and copper suggests that neurodegenerative disorders may occur and develop once the built-in equilibrium is disrupted and brings new insight into developing more effective prevention and treatment strategies for neurodegenerative diseases.

A Multiscale View of the Mechanisms Underlying Ketamine’s Antidepressant Effects: An Update on Neuronal Calcium Signaling

Major depressive disorder (MDD) is a debilitating disease characterized by depressed mood, loss of interest or pleasure, suicidal ideation, and reduced motivation or hopelessness. Despite considerable research, mechanisms underlying MDD remain poorly understood, and current advances in treatment are far from satisfactory. The antidepressant effect of ketamine is among the most important discoveries in psychiatric research over the last half-century. Neurobiological insights into the ketamine’s effects have shed light on the mechanisms underlying antidepressant efficacy. However, mechanisms underlying the rapid and sustained antidepressant effects of ketamine remain controversial. Elucidating such mechanisms is key to identifying new therapeutic targets and developing therapeutic strategies. Accumulating evidence demonstrates the contribution of the glutamatergic pathway, the major excitatory neurotransmitter system in the central nervous system, in MDD pathophysiology and antidepressant effects. The hypothesis of a connection among the calcium signaling cascade stimulated by the glutamatergic system, neural plasticity, and epigenetic regulation of gene transcription is further supported by its associations with ketamine’s antidepressant effects. This review briefly summarizes the potential mechanisms of ketamine’s effects with a specific focus on glutamatergic signaling from a multiscale perspective, including behavioral, cellular, molecular, and epigenetic aspects, to provide a valuable overview of ketamine’s antidepressant effects.