Evidence for functional co-activation of N-methyl-D-aspartate receptors by glycine

The excitatory neurotransmitter glutamate acts at several receptor subtypes in the CNS, including N-methyl-D-aspartate (NMDA) receptors. The inhibitory neurotransmitter glycine is a co-agonist of NMDA receptors and functional glycinergic co-activation of NMDA receptors is theoretically possible due to the presence of background glutamate. We report the first high fidelity recording of a distinct NMDA receptor excitatory post-synaptic potential (EPSP) in the intact mammalian CNS. The EPSP was evoked by multi-shock activation of a glycinergic pathway rather than a glutamatergic pathway and had characteristics most compatible with glycine, rather than glutamate, activation of NMDA receptors.     

Preoptic rather than mediobasal hypothalamic amino acid neurotransmitter release regulates GnRH secretion during the estrogen-induced LH surge in the ovariectomized rat

Inhibitory and excitatory amino acid neurotransmitters have been suggested to participate in the feedback actions of estradiol (E2) on LH secretion. In the rat estrogen-receptive neurons have been demonstrated in the preoptic/anterior hypothalamic area (POA) and mediobasal hypothalamus/median eminence (MBH) and many of these neurons utilize gamma-aminobutyric acid (GABA) as neurotransmitter. The actions of excitatory amino acids (EAA) differ in ovariectomized (ovx) and ovx E2-substituted rats indicating that EAAs also participate in the positive feedback action of E2 on LH release. However, little information is available as to whether in vivo these transmitters exert their effects in the POA, where most of the GnRH perikarya are located, or in the MBH, i.e. at the nerve terminals. Therefore we conducted push pull cannula perfusions to compare the release rates of GABA, aspartate (ASP) and glutamate (GLU) in the MBH and POA. A subcutaneous implant of a silastic tube containing E2 resulted in LH surges in the afternoon of all treated animals. Prior to and during this LH surge the MBH release rates of neither GABA nor ASP nor GLU were significantly altered. In contrast, a conspicuous drop in preoptic GABA release occurred prior to and during the time of estrogen-induced LH surges and this was accompanied by enhanced preoptic secretion of ASP and GLU. In conclusion, we present the first data about amino acid release in the MBH during the E2-induced LH surge. Since only in the POA the LH surge is associated with changes in amino acid release, it appears that both inhibitory and excitatory amino acids act at the level of the GnRH cell bodies and/or dendrites and not on GnRH nerve terminals to mediate the feedback mechanism of E2 on LH release.     

Insect neurotransmission: neurotransmitters and their receptors

The roles of acetylcholine, dopamine, octopamine, tyramine, 5-hydroxytryptamine, histamine, glutamate, 4-aminobutanoic acid (gamma-aminobutyric acid) and a range of peptides as insect neurotransmitters are evaluated in terms of the criteria used to identify transmitters. Of the biogenic amines considered, there is good evidence that acetylcholine, dopamine, octopamine, 5-hydroxytryptamine, and histamine should be considered to be neurotransmitters, but the case for tyramine is less convincing at the moment. The evidence supporting neurotransmitter roles for glutamate and gamma-aminobutyric acid at specific insect synapses is overwhelming, but much work remains to be undertaken before the full significance of these molecules in the insect nervous system is appreciated. Attempts to characterise biogenic amine and amino acid receptors using pharmacological and molecular biological techniques have revealed considerable differences between mammalian and insect receptors. The number of insect neuropeptides isolated and identified has increased spectacularly in recent years, but genuine physiological or biochemical functions can be assigned to very few of these molecules. Of these, only proctolin fulfills the criteria expected of a neurotransmitter, and the recent discovery of proctolin receptor antagonists should enable the biology of this pentapeptide to be explored fully.     

Cloning and functional expression of a Drosophila metabotropic glutamate receptor expressed in the embryonic CNS

The excitatory neurotransmitter glutamate plays important roles in the mammalian brain, ranging from synaptic plasticity to memory. To mediate these functions, glutamate activates two types of receptors: ligand-gated channels and metabotropic receptors coupled to G-proteins. Both families of glutamate receptors share no sequence homology and possess original structural features compared with other ligand-gated channels and G-protein-coupled receptors, respectively. Glutamate-gated receptor-channel subunits have already been characterized in invertebrates. Here we report the cloning and functional characterization of an invertebrate metabotropic glutamate receptor (DmGluRA) isolated from Drosophila melanogaster. This receptor displays 45 and 43% amino acid sequence identity with its mammalian homologs mGluR3 and mGluR2, respectively. Moreover, its pharmacology and transduction mechanisms are surprisingly similar to those of mGluR2 and mGluR3. DmGluRA is expressed in the CNS of the late embryo. These results indicate that the original structural features of both glutamate receptor types are conserved from insects to mammals and suggest that the functions of these receptors have been highly conserved during evolution.     

P19 cells differentiate into glutamatergic and glutamate-responsive neurons in vitro

The neurotransmitter L-glutamate has been associated with a number of developmental events within the central nervous system including synaptogenesis and the refinement of topographically ordered neural maps. As a model for studying such events at the molecular level, we have examined the expression of glutamate and glutamate receptors in neurons that develop from P19 cells in response to retinoids. We report here that many P19-derived neurons do contain glutamate in secretory vesicles and that this glutamate appears to function as a neurotransmitter. The neurotransmitter GABA is also present in these cultures and both glutamate and GABA appeared to co-localize in some neuronal processes. Both neurotransmitters were released from the neurons in response to membrane depolarization. These neurons also express various glutamate receptor subunits including GluR1, GluR4 and NMDAR1 as detected by immunological methods. Using whole-cell patch-clamping, we have recorded spontaneous postsynaptic potentials which increase in both amplitude and frequency with time in culture and which are sensitive to the glutamate antagonist kynurenic acid Thus, P19-derived neurons mature in culture and form electrically active neural networks involving glutamate and glutamate receptors.     

Acquired hemophilia secondary to factor VIII inhibitors after pregnancy

According to current concepts, the excitatory amino acid glutamate is involved in the pathogenesis of Parkinson's disease (PD). Overactivity of glutamatergic projection neurons and beneficial effect of antiglutamatergic substances in animal experiments suggest that excess supply of glutamate might contribute to the pathophysiology of PD. Reduced activity of the glutamate metabolizing enzyme glutamine synthetase (GS) leads to decreased uptake of glutamate and thus abundant glutamate. Here we report that PD patients and age-matched controls are comparable with respect to GS activity in peripheral blood mononuclear cells (PBMC). These results imply no systemic dysregulation of the enzyme GS in patients with PD.     

Excitatory amino acid levels in cerebrospinal fluid of patients with infantile spasms

Infantile spasm is an age-specific epileptic encephalopathy. Long-term intellectual outcome of affected infants remains poor. The pathogenesis of infantile spasms, as well as the development of mental retardation, remains unclear. Increased excitatory amino acid neurotransmission may play a role in neuronal dysfunction and epilepsy. To study the significance of cerebrospinal fluid excitatory amino acids in infantile spasms, we determined glutamate and aspartate concentrations in cerebrospinal fluid of 13 patients with infantile spasms and 13 controls. The aspartate level in cerebrospinal fluid of the patients with infantile spasms (968 +/- 416 nmol/l) was higher than the control group (426 +/- 272 nmol/l). No difference in the mean glutamate levels was found between the patients (966 +/- 395 nmol/l) and the controls (1135 +/- 594 nmol/l). The elevated aspartate levels in cerebrospinal fluid of the patients with infantile spasms might be secondary to change in metabolism of aspartate. Aspartate is an excitatory and neurotoxic neurotransmitter, which might have a role in triggering the spasms and the development of neuronal dysfunctions in the patients with infantile spasms.     

Identification of the amino acid subsets accounting for the ligand binding specificity of a glutamate receptor

In a situation so far unique among neurotransmitter receptors, glutamate receptors share amino acid sequence similarities with the bacterial periplasmic binding proteins (PBPs). On the basis of the primary structure similarity of two bacterial periplasmic proteins (lysine/arginine/ornithine- and phosphate-binding proteins) with the chick cerebellar kainate-binding protein (KBP), a member of the ionotropic glutamate receptor family, we have generated a three-dimensional model structure of the KBP extracellular domain. By an interplay between homology modeling and site-directed mutagenesis, we have investigated the kainate binding properties of 55 different mutants (corresponding to 43 positions) and studied the interactions of some of these mutants with various glutamatergic ligands. As a result, we present here the subsets of amino acids accounting for the binding free energies and specificities of KBP for kainate, glutamate, and CNQX and propose a three-dimensional model, at the microarchitectural level, of the glutamatergic binding domain.     

Recent advances in the pharmacology of the vestibulo-ocular reflex system

Recent advances in the pharmacology of the vestibulo-ocular reflex have had a major impact on our understanding of the vestibular system, the sensory system primarily concerned with the stabilization of gaze and posture during head movement. Increasing evidence suggests that afferent transmission from the receptor hair cells in the vestibular labyrinth to the vestibular nerve probably involves glutamate acting on a number of excitatory amino acid receptor subtypes. Furthermore, hair-cell sensitivity appears to be regulated by cholinergic, GABA-mediated and, possibly, peptide-mediated efferent feedback from the CNS. Likewise, it seems clear that an excitatory amino acid, probably glutamate, is the major transmitter used by the vestibular nerve in its synapses with neurones of the brainstem vestibular nucleus. In this review, Paul Smith and Cynthia Darlington discuss the large number of receptor subtypes that have been identified in the vestibular nucleus, including receptors for several peptides that may have a role in co-transmission.     

Neurotransmission and the ontogeny of human brain

The early appearance of neurotransmitters in brain tissue refers to their regulative functions on the neuronal circuits. Many neurotransmitters have direct effects on neuronal outgrowth and differentiation during brain development, which precede their role in synaptic information coding. Both the neurotrophic and neurotoxic properties of excitatory amino acids (EAAs) have focused special interest on glutamatergic neurotransmission during brain development. Therefore, this work intends to review and discuss developmental alterations of the EAA neurotransmitter system in the human brain, their relation to human brain maturation and implications for pathological processes during early human brain development.     

beta-Amyloid neurotoxicity in human cortical culture is not mediated by excitotoxins

beta-Amyloid is a metabolic product of the amyloid precursor protein, which accumulates abnormally in senile plaques in the brains of patients with Alzheimer's disease. The neurotoxicity of beta-amyloid has been observed in cell culture and in vivo, but the mechanism of this effect is unclear. In this report, we describe the direct neurotoxicity of beta-amyloid in high-density primary cultures of human fetal cortex. In 36-day-old cortical cultures, beta-amyloid neurotoxicity was not inhibited by the broad-spectrum excitatory amino acid receptor antagonist kynurenate or the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid under conditions that inhibited glutamate and NMDA neurotoxicity. In 8-day-old cortical cultures, neurons were resistant to glutamate and NMDA toxicity but were still susceptible to beta-amyloid neurotoxicity, which was unaffected by excitatory amino acid receptor antagonists. Treatment with beta-amyloid caused chronic neurodegenerative changes, including neuronal clumping and dystrophic neurites, whereas glutamate treatment caused rapid neuronal swelling and neurite fragmentation. These results suggest that beta-amyloid is directly neurotoxic to primary human cortical neurons by a mechanism that does not involve excitatory amino acid receptors.     

Effects of acetylcholine and glutamate on isolated neurons of locomotory network of Clione

In Clione limacina, locomotory rhythm is produced in the central pattern generator by reciprocal activity of two groups of interneurons. Dorsal (D) and ventral (V) phase interneurons activate neurons of the same phase and inhibit neurons of the opposite phase. Which neurotransmitters are used by these interneurons is not clear. In this study, identified follower neurons to V and D interneurons were isolated, and their responses to the local application of potential neurotransmitters were examined. Acetylcholine exerted inhibitory action on the isolated D-phase neurons and excitatory action on V-phase neurons. Glutamate produced excitation in D-phase neurons, and inhibition in V-phase neurons. These results suggest that acetylcholine is the neurotransmitter of D-phase interneurons, while glutamate might be the neurotransmitter of V-phase interneurons.     

Sites of volatile anesthetic action on kainate (Glutamate receptor 6) receptors

Molecular mechanisms of anesthetic action on neurotransmitter receptors are poorly understood. The major excitatory neurotransmitter in the central nervous system is glutamate, and recent studies found that volatile anesthetics inhibit the function of the alpha-amino-3-hydroxyisoxazolepropionic acid subtype of glutamate receptors (e.g. glutamate receptor 3 (GluR3)), but enhance kainate (GluR6) receptor function. We used this dissimilar pharmacology to identify sites of anesthetic action on the kainate GluR6 receptor by constructing chimeric GluR3/GluR6 receptors. Results with chimeric receptors implicated a transmembrane region (TM4) of GluR6 in the action of halothane. Site-directed mutagenesis subsequently showed that a specific amino acid, glycine 819 in TM4, is important for enhancement of receptor function by halothane (0. 2-2 mM). Mutations of Gly-819 also markedly decreased the response to isoflurane (0.2-2 mM), enflurane (0.2-2 mM), and 1-chloro-1,2, 2-trifluorocyclobutane (0.2-2 mM). The nonanesthetics 1, 2-dichlorohexafluorocyclobutane and 2,3-dichlorooctafluorobutane had no effect on the functions of either wild-type GluR6 or receptors mutated at Gly-819. Ethanol and pentobarbital inhibited the function of both wild-type and mutant receptors. These results suggest that a specific amino acid, Gly-819, is critical for the action of volatile anesthetics, but not of ethanol or pentobarbital, on the GluR6 receptor.     

GABA- and glutamate-immunoreactive synapses on sympathetic preganglionic neurons projecting to the superior cervical ganglion

Our previous work suggests that virtually all of the synapses on sympathetic preganglionic neurons projecting to the rat adrenal medulla are immunoreactive for either the inhibitory amino acid, gamma-aminobutyric acid (GABA) or the excitatory amino acid, L-glutamate. To investigate whether or not this is true for other groups of sympathetic preganglionic neurons, and to determine whether or not the proportion of inputs containing each type of amino acid neurotransmitter is the same for different groups of sympathetic preganglionic neurons, we retrogradely labelled rat and rabbit sympathetic preganglionic neurons projecting to the superior cervical ganglion and used post-embedding immunogold on ultrathin sections to localise GABA- and glutamate-immunoreactivity. The cell bodies and dendrites of both rat and rabbit sympathetic preganglionic neurons projecting to the superior cervical ganglion received synapses and direct contacts from nerve fibres immunoreactive for GABA and from nerve fibres immunoreactive for glutamate. In the rat, GABA was present in 48.9% of the inputs to sympathetic preganglionic neurons projecting to the superior cervical ganglion, and glutamate was present in 51.7% of inputs. Double immunogold labelling for glutamate and GABA on the same section, as well as labelling of consecutive serial sections for the two antigens, indicated that GABA and glutamate occur in separate populations of nerve fibres that provide input to rat sympathetic preganglionic neurons projecting to the superior cervical ganglion. We now have shown that GABA or glutamate is present in virtually all of the inputs to sympathetic preganglionic neurons projecting to the superior cervical ganglion and in essentially all of the inputs to sympathetic preganglionic neurons supplying the adrenal medulla. These findings are consistent with the hypothesis that all fast synaptic transmission in central autonomic pathways may be mediated by either excitatory or inhibitory amino acids. Furthermore, we showed a statistically significant difference in the proportion of glutamate-immunoreactive inputs between sympathetic preganglionic neurons projecting to the superior cervical ganglion and sympathoadrenal neurons (data from Llewellyn-Smith et al. [Llewellyn-Smith, I.J., Phend, K.D., Minson, J.B., Pilowsky, P.M., Chalmers, J.P., 1992. Glutamate immunoreactive synapses on retrogradely labelled sympathetic neurons in rat thoracic spinal cord. Brain Res. 581, 67-80]), with preganglionics supplying the adrenal medulla receiving more excitatory inputs than those supplying the superior cervical ganglion. This increased excitatory input to sympathoadrenal neurons may explain the predominant activation of these neurons following baroreceptor unloading.     

Alterations in the Ha-ras-1 and the p53 pathway genes in the progression of N-methyl-N-nitrosourea-induced rat mammary tumors

Glutamate is the most prominent excitatory neurotransmitter in the retina and brain. It has become clear that the physiology of many glial cells, including retinal Müller cells, is modified by a host of neurotransmitters, including glutamate. The experiments presented here demonstrate that Müller cells isolated from the tiger salamander retina have metabotropic glutamate receptors that, when activated, lead to the release of calcium ions (Ca2+) from intracellular stores. The Ca2+-sensitive fluorescent dye, Fura-2, and video imaging microscopy were used to monitor changes in cytosolic calcium ion concentration ([Ca2+]i) evoked by glutamate (30-50 microM), (1S,3R)-ACPD (50-200 microM), quisqualate (10-50 microM), and L-AP4 (5-100 microM). Bath application of each of these metabotropic receptor agonists in the absence of extracellular Ca2+ resulted in an increase in [Ca2+]i that often began in the distal end of the cell and occurred later in the endfoot. This wavelike increase in [Ca2+]i is reminiscent of the Ca2+ waves evoked in these cells by other Ca2+ releasing agents such as ryanodine and caffeine. Extracellular application ofATP also evoked increases in [Ca2+] in Müller cells. The presence on Müller cells of receptors for retinal neurotransmitters, such as glutamate and ATP, demonstrates that these glial cells can respond to changes in the retinal extracellular environment and hence neuronal activity. Since Müller cells span almost all layers of the retina, they are likely to be exposed to most retinal neurotransmitters. The Ca2+ waves evoked in Müller cells by neurotransmitters could represent a form of signaling from the outer retinal layers to the inner ones.     

Sensitive non-radioactive PCR detection of clonal T-cell receptor-gamma gene rearrangements in childhood acute lymphoblastic leukaemia

The aim of the present study was to investigate some putative neurotransmitters involved in nociception and pain in parturients during active labour experiencing intense visceral pain. The concentration of the excitatory amino acid aspartate was significantly increased, and there was a tendency for an increase in glutamate, in lumbar cerebrospinal fluid (CSF) of parturients in active vaginal labour compared with control patients without pain subjected to elective caesarean section. The CSF concentration of the nitric oxide breakdown product nitrate was significantly decreased in parturients compared with control patients and healthy volunteers. No significant differences in the concentrations of substance P, substance P-endopeptidase or met-enkephalin were detected between parturients and controls. Our data suggest a paradoxical negative relationship between CSF concentrations of excitatory amino acids and nitric oxide in labour pain. The mechanisms behind this finding is unclear at present.     

Pathophysiology of delirium

Hypotheses about the pathophysiology of delirium are speculative and largely based on animal research. According to the neurotransmitter hypothesis, decreased oxidative metabolism in the brain causes cerebral dysfunction due to abnormalities of various neurotransmitter systems. Reduced cholinergic function, excess release of dopamine, norepinephrine, and glutamate, and both decreased and increased serotonergic and gamma-aminobutyric acid activity may underlie the different symptoms and clinical presentations of delirium. According to the inflammatory hypothesis, increased cerebral secretion of cytokines due to a wide range of physically stressful events plays an important role in the occurrence of delirium. Since cytokines can influence the activity of various neurotransmitter systems, these mechanisms may interact. Also, more fundamental processes like intraneuronal signal transduction, second messenger systems that at the same time use neurotransmitters as first messengers and play an important role in their synthesis and release, may be disturbed. Furthermore, severe illness and physiologic stress may give rise to modification of blood-brain barrier permeability, the sick euthyroid syndrome with abnormalities of thyroid hormone concentrations, and increased activity of the hypothalamic-pituitary-adrenal axis. These circumstances possibly also contribute to changes in neurotransmitter synthesis and release of cytokines in the brain, and consequently to the occurrence of delirium. Elderly patients are more at risk for developing delirium,very likely due to age-related cerebral changes in stress-regulating neurotransmitter and intracellular signal transduction systems. This paper will expand upon these current theories and discuss their applicability to research and clinical work with elderly patients suffering from delirium.     

Effect of glutamate and angiotensin II on whole cell currents and release of nitric oxide in the rat subfornical organ

Blood-borne angiotensin II (AngII) is known to mediate water-intake by its excitatory effect on neurons in the subfornical organ (SFO). Conversely, nitric oxide (NO) has exclusively inhibitory effects on rat SFO-neurons and on SFO-mediated water-intake. Extracellular and patch-clamp recordings from freshly dissociated rat SFO-neurons showed that glutamate activates AngII-sensitive SFO-neurons by opening ligand-gated cation channels. An immunocytochemical study showed that activation of glutamate receptors increased the concentration of the inhibitory second messenger cGMP in the SFO. A model is proposed suggesting that NO protects SFO-neurons from overexcitability by excitatory neurotransmitters.     

Heart rate and mortality]

1. Glutamate is the predominant excitatory neurotransmitter in the brain, but it is also a potent neurotoxin. Following release of glutamate from presynaptic vesicles into the synapse and activation of a variety of ionotropic and metabotropic glutamate receptors, glutamate is removed from the synapse. This is achieved through active uptake of glutamate by transporters located pre- and also post-synaptically or, alternatively, glutamate can diffuse out of the synapse and be taken up by transporters located on the cell surface of glial cells. 2. Complementary DNA encoding a number of glutamate transporters have recently been cloned and form a family of structurally related membrane proteins with a high degree of amino acid sequence conservation. Expression of the cloned glutamate transporters in various cell types has aided in the characterization of the functional properties of the different transporter subtypes. 3. Glutamate transport is coupled to sodium, potassium and pH gradients across the cell membrane creating an electrogenic process. This allows transport to be measured using electrophysiological techniques, which has greatly aided in understanding some of the basic mechanisms of the transport process and has also allowed a detailed understanding of the molecular pharmacology of the different transporter subtypes. 4. In the present review I shall discuss some of the recent advances in understanding the molecular basis for glutamate transporter function and then highlight some of the unanswered questions concerning the physiological roles of these proteins and suggest possible strategies for pharmacological manipulation of transporters for the treatment of neurological disorders.