The anterior retrosplenial cortex is required for short-term object in place recognition memory retrieval: Role of ionotropic glutamate receptors in male and female Long-Evans rats.

The anterior retrosplenial cortex (aRSC) integrates multimodal sensory information into cohesive associative recognition memories. Little is known about how information is integrated during different learning phases (i.e., encoding and retrieval). Additionally, sex differences are observed in performance of some visuospatial memory tasks; however, inconsistent findings warrant more research. We conducted three experiments using the 1-h delay object-in-place (1-h OiP) test to assess recognition memory retrieval in male and female Long-Evans rats. (i) We found both sexes performed equally in three repeated 1-h OiP test sessions. (ii) We showed infusions of a mixture of muscimol/baclofen (GABAA/B receptor agonists) into the aRSC ~15-min prior to the test phase disrupted 1-h OiP in both sexes. (iii) We assessed the role of aRSC ionotropic glutamate receptors in 1-h OiP retrieval using another squad of cannulated rats and confirmed that infusions of either the competitive AMPA/Kainate receptor antagonist CNQX (3 mM) or competitive NMDA receptor antagonist AP-5 (30 mM) (volumes = 0.50 uL/side) significantly impaired 1-h OiP retrieval in both sexes compared to controls. Taken together, findings challenge reported sex differences and clearly establish a role for aRSC ionotropic glutamate receptors in short-term visuospatial recognition memory retrieval. Thus, modulating neural activity in the aRSC may alleviate some memory processing impairments in related disorders.

Subunit-selective iGluR antagonists can potentiate heteromeric receptor responses by blocking desensitization.

Ionotropic glutamate receptors (iGluRs) are key molecules for synaptic signaling in the central nervous system, which makes them promising drug targets. Intensive efforts are being devoted to the development of subunit-selective ligands, which should enable more precise pharmacologic interventions while limiting the effects on overall neuronal circuit function. However, many AMPA and kainate receptor complexes in vivo are heteromers composed of different subunits. Despite their importance, little is known about how subunit-selective ligands affect the gating of heteromeric iGluRs, namely their activation and desensitization properties. Using fast ligand application experiments, we studied the effects of competitive antagonists that block glutamate from binding at part of the four subunits. We found that UBP-310, a kainate receptor antagonist with high selectivity for GluK1 subunits, reduces the desensitization of GluK1/GluK2 heteromers and fully abolishes the desensitization of GluK1/GluK5 heteromers. This effect is mirrored by subunit-selective agonists and heteromeric receptors that contain binding-impaired subunits, as we show for both kainate and GluA2 AMPA receptors. These findings are consistent with a model in which incomplete agonist occupancy at the four receptor subunits can provide activation without inducing desensitization. However, we did not detect significant steady-state currents during UBP-310 dissociation from GluK1 homotetramers, indicating that antagonist dissociation proceeds in a nonuniform and cooperativity-driven manner, which disfavors nondesensitizing occupancy states. Besides providing mechanistic insights, these results have direct implications for the use of subunit-selective antagonists in neuroscience research and envisioned therapeutic interventions.

Polyamine-mediated channel block of ionotropic glutamate receptors and its regulation by auxiliary proteins.

Most excitatory neurotransmission in the mammalian brain is mediated by a family of plasma membrane-bound signaling proteins called ionotropic glutamate receptors (iGluRs). iGluRs assemble at central synapses as tetramers, forming a central ion-channel pore whose primary function is to rapidly transport Na+ and Ca2+ in response to binding the neurotransmitter l-glutamic acid. The pore of iGluRs is also accessible to bulkier cytoplasmic cations, such as the polyamines spermine, spermidine, and putrescine, which are drawn into the permeation pathway, but get stuck and block the movement of other ions. The degree of this polyamine-mediated channel block is highly regulated by processes that control the free cytoplasmic polyamine concentration, the membrane potential, or the iGluR subunit composition. Recently, an additional regulation by auxiliary proteins, most notably transmembrane AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor regulatory proteins (TARPs), cornichons, and neuropilin and tolloid-like proteins (NETOs), has been identified. Here, I review what we have learned of polyamine block of iGluRs and its regulation by auxiliary subunits. TARPs, cornichons, and NETOs attenuate the channel block by enabling polyamines to exit the pore. As a result, polyamine permeation occurs at more negative and physiologically relevant membrane potentials. The structural basis for enhanced polyamine transport remains unresolved, although alterations in both channel architecture and charge-screening mechanisms have been proposed. That auxiliary subunits can attenuate the polyamine block reveals an unappreciated impact of polyamine permeation in shaping the signaling properties of neuronal AMPA- and kainate-type iGluRs. Moreover, enhanced polyamine transport through iGluRs may have a role in regulating cellular polyamine levels.

Metabotropic and ionotropic glutamate receptors mediate the modulation of acetylcholine release at the frog neuromuscular junction.

There is some evidence that glutamate (Glu) acts as a signaling molecule at vertebrate neuromuscular junctions where acetylcholine (ACh) serves as a neurotransmitter. In this study, performed on the cutaneous pectoris muscle of the frog Rana ridibunda, Glu receptor mechanisms that modulate ACh release processes were analyzed. Electrophysiological experiments showed that Glu reduces both spontaneous and evoked quantal secretion of ACh and synchronizes its release in response to electrical stimulation. Quisqualate, an agonist of ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptors and metabotropic Group I mGlu receptors, also exerted Glu-like inhibitory effects on the secretion of ACh but had no effect on the kinetics of quantal release. Quisqualate's inhibitory effect did not occur when a blocker of Group I mGlu receptors (LY 367385) or an inhibitor of phospholipase C (U73122) was present. An increase in the degree of synchrony of ACh quantal release, such as that produced by Glu, was obtained after application of N-methyl-D-aspartic acid (NMDA). The presence of Group I mGlu and NMDA receptors in the neuromuscular synapse was confirmed by immunocytochemistry. Thus, the data suggest that both metabotropic Group I mGlu receptors and ionotropic NMDA receptors are present at the neuromuscular synapse of amphibians, and that the activation of these receptors initiates different mechanisms for the regulation of ACh release from motor nerve terminals. © 2016 Wiley Periodicals, Inc.

Neuronal activity is required for the development of specific cortical interneuron subtypes.

Electrical activity has been shown to regulate development in a variety of species and in various structures, including the retina, spinal cord and cortex. Within the mammalian cortex specifically, the development of dendrites and commissural axons in pyramidal cells is activity-dependent. However, little is known about the developmental role of activity in the other major cortical population of neurons, the GABA-producing interneurons. These neurons are morphologically and functionally heterogeneous and efforts over the past decade have focused on determining the mechanisms that contribute to this diversity. It was recently discovered that 30% of all cortical interneurons arise from a relatively novel source within the ventral telencephalon, the caudal ganglionic eminence (CGE). Owing to their late birth date, these interneurons populate the cortex only after the majority of other interneurons and pyramidal cells are already in place and have started to functionally integrate. Here we demonstrate in mice that for CGE-derived reelin (Re)-positive and calretinin (Cr)-positive (but not vasoactive intestinal peptide (VIP)-positive) interneurons, activity is essential before postnatal day 3 for correct migration, and that after postnatal day 3, glutamate-mediated activity controls the development of their axons and dendrites. Furthermore, we show that the engulfment and cell motility 1 gene (Elmo1), a target of the transcription factor distal-less homeobox 1 (Dlx1), is selectively expressed in Re(+) and Cr(+) interneurons and is both necessary and sufficient for activity-dependent interneuron migration. Our findings reveal a selective requirement for activity in shaping the cortical integration of specific neuronal subtypes.

Disrupted Ionic Homeostasis in Ischemic Stroke and New Therapeutic Targets.

BACKGROUND: Stroke is a leading cause of long-term disability. All neuroprotectants targeting excitotoxicity have failed to become stroke medications. In order to explore and identify new therapeutic targets for stroke, we here reviewed present studies of ionic transporters and channels that are involved in ischemic brain damage. METHOD: We surveyed recent literature from animal experiments and clinical reports in the databases of PubMed and Elsevier ScienceDirect to analyze ionic mechanisms underlying ischemic cell damage and suggest promising ideas for stroke therapy. RESULTS: Dysfunction of ionic transporters and disrupted ionic homeostasis are most early changes that underlie ischemic brain injury, thus receiving sustained attention in translational stroke research. The Na+/K+-ATPase, Na+/Ca2+ Exchanger, ionotropic glutamate receptor, acid-sensing ion channels (ASICs), sulfonylurea receptor isoform 1 (SUR1)-regulated NCCa-ATP channels, and transient receptor potential (TRP) channels are critically involved in ischemia-induced cellular degenerating processes such as cytotoxic edema, excitotoxicity, necrosis, apoptosis, and autophagic cell death. Some ionic transporters/channels also act as signalosomes to regulate cell death signaling. For acute stroke treatment, glutamate-mediated excitotoxicity must be interfered within 2 hours after stroke. The SUR1-regulated NCCa-ATP channels, Na+/K+-ATPase, ASICs, and TRP channels have a much longer therapeutic window, providing new therapeutic targets for developing feasible pharmacological treatments toward acute ischemic stroke. CONCLUSION: The next generation of stroke therapy can apply a polypharmacology strategy for which drugs are designed to target multiple ion transporters/channels or their interaction with neurotoxic signaling pathways. But a successful translation of neuroprotectants relies on in-depth analyses of cell death mechanisms and suitable animal models resembling human stroke.

Hypocretin (orexin) regulates glutamate input to fast-spiking interneurons in layer V of the Fr2 region of the murine prefrontal cortex.

We studied the effect of hypocretin 1 (orexin A) in the frontal area 2 (Fr2) of the murine neocortex, implicated in the motivation-dependent goal-directed tasks. In layer V, hypocretin stimulated the spontaneous excitatory postsynaptic currents (EPSCs) on fast-spiking (FS) interneurons. The effect was accompanied by increased frequency of miniature EPSCs, indicating that hypocretin can target the glutamatergic terminals. Moreover, hypocretin stimulated the spontaneous inhibitory postsynaptic currents (IPSCs) on pyramidal neurons, with no effect on miniature IPSCs. This action was prevented by blocking 1) the ionotropic glutamatergic receptors; 2) the hypocretin receptor type 1 (HCRTR-1), with SB-334867. Finally, hypocretin increased the firing frequency in FS cells, and the effect was blocked when the ionotropic glutamate transmission was inhibited. Immunolocalization confirmed that HCRTR-1 is highly expressed in Fr2, particularly in layer V-VI. Conspicuous labeling was observed in pyramidal neuron somata and in VGLUT1+ glutamatergic terminals, but not in VGLUT2+ fibers (mainly thalamocortical afferents). The expression of HCRTR-1 in GABAergic structures was scarce. We conclude that 1) hypocretin regulates glutamate release in Fr2; 2) the effect presents a presynaptic component; 3) the peptide control of FS cells is indirect, and probably mediated by the regulation of glutamatergic input onto these cells.

Effects of NMDA and non-NMDA ionotropic glutamate receptors in the medial preoptic area on body temperature in awake rats.

Glutamate when microinjected at the medial preoptic area (mPOA) influences brain temperature (Tbr) and body temperature (Tb) in rats. Glutamate and its various receptors are present at the mPOA. The aim of this study was to identify the contribution of each of the ionotropic glutamatergic receptors at the mPOA on changes in Tbr and Tb in freely moving rats. Adult male Wistar rats (n=40) were implanted with bilateral guide cannula with indwelling styli above the mPOA. A telemetric transmitter was implanted at the peritoneum to record Tb and locomotor activity (LMA). A precalibrated thermocouple wire implanted near the hypothalamus was used to assess Tbr. Specific agonist for each ionotropic glutamate receptor was microinjected into the mPOA and its effects on temperature and LMA were measured in the rats. The rats were also microinjected with the respective ionotropic receptor antagonists, 15min prior to the microinjection of each agonist. Amongst amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-d-aspartate (NMDA) and kainic acid, AMPA increased Tb and LMA when injected at the mPOA. Specific antagonists for AMPA receptors was able to attenuate this increase (p<0.005). Pharmacological blockade of NMDA was able to lower Tbr only. Microinjection of kainic acid and its antagonist had no effect on the variables. The finding of the study suggests that activation of the AMPA receptors at the mPOA, leads to the rise in body temperature.

Amide of Lambertian Acid Suppresses Hyperactivation of Inotropic Glutamate Receptors, but not Synaptic Potentiation in Hippocampal Sections.

Amide of lambertian acid suppresses hyperactivation of inotropic glutamate receptors in hippocampal sections induced by a decrease in the level of magnesium ions (a selective blocker of glutamate NMDA receptors). Treatment of the sections with amide of lambertian acid in standard physiological saline does not prevent development of NMDA-dependent synaptic potentiation. Lambertian acid isolated from needles and turpentine of Siberian pine (Pinus sibirica R. Mayr), and its derivatives may become a source of substances with glutamatergic mechanism of action for treatment of cognitive and neurodegenerative disorders.

[The kynurenic acid hypothesis - a new look at etiopathogenesis and treatment of schizophrenia]. / Koncepcja kynureninowa ­ nowe spojrzenie na etiopatogeneze i leczenie schizofrenii.

Kynurenic acid (KYNA) is a neuroactive metabolite of tryptophan formed in the brain and in the periphery, known to block ionotropic glutamate receptors and α7 nicotinic receptors, and to act as a ligand of G protein-coupled GPR35 receptors and human aryl hydrocarbon (AHR) receptors. KYNA seems to modulate a number of mechanisms involved in the pathogenesis of schizophrenia including dopaminergic transmission in mesolimbic and mesocortical areas or glutamatemediated neurotransmission. The kynurenine hypothesis of schizophrenia links the occurrence of positive and negative symptoms of schizophrenia and cognitive impairments characteristic for the disease with the disturbances of kynurenine pathway function. Available data suggest that antipsychotic drugs may restore balance among kynurenine pathway metabolites, and that co-administration of glycine with antipsychotics may reduce extrapyramidal symptoms in patients suffering from schizophrenia. Central level of KYNA may increase in the course of inflammation, which is consistent with the inflammatory hypothesis of schizophrenia. Alterations of immune response and disturbed functioning of kynurenine pathway may lead to disproportion between neuroprotective and neurotoxic mechanisms in the brain. Currently, intense research efforts are focused on the role of kynurenine pathway metabolites in pathogenesis of schizophrenia, their association with the response to antipsychotic treatment, and search for novel medications modulating the function of kynurenine pathway.

An iGlu receptor antagonist and its simultaneous use with an anticancer drug for cancer therapy.

Glutamate receptor antagonists have been known to play a crucial role in the treatment of many neuronal diseases. Recently, these antagonists have also shown therapeutic effects in the treatment of cancer. In this study, an ionotropic glutamate (iGlu) receptor antagonist, 4-hydroxyphenylacetyl spermine (L1), was used concurrently with a common anticancer drug, doxorubicin (Dox), for simultaneous cancer therapy. Mesoporous silica nanoparticles (MSNPs) were employed as the delivery vehicle for both L1 and Dox by conjugating the iGlu receptor antagonist on the surface and encapsulating Dox within the mesopores. Dox was then trapped within the mesopores by functionalizing a redox-cleavable capping group on the MSNP surface, and it could be released upon exposure to the reductive glutathione. In vitro studies on B16F10 and NIH3T3 cell lines revealed that the iGlu receptor antagonist L1 exhibited therapeutic as well as targeting effects. In addition, the simultaneous use of therapeutic L1 and Dox proved to be synergistic in the treatment of cancer. The present work demonstrated the feasibility of employing a delivery system to deliver both neuroprotective drug and anticancer drug for efficient anticancer treatment.

Structure-activity relationships of IKM-159: Diverted synthesis and biological evaluation of a series of C5-oxy analogs.

Ligands for neuronal receptors are important for understanding the biological functions as well as for treatment of neuronal diseases associated with. Here, we report diverted synthesis and biological evaluation of four C-ring analogs of IKM-159, a subtype-selective inhibitor for (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA)-type ionotropic glutamate receptor. Starting from iodinated 7-oxanorbornene 7, those analogs 3-6 were successfully synthesized in 7.0-33% yields over 8-11 steps via a common intermediate 13. Intracerebroventricular injection of those analogs on mice showed that introduction of oxo group on the C-ring (analogs 4, 5) or cleavage of the C-ring (analog 6) caused significant loss of the activity, while the ether analog 3 still retain the suppressed motor activity, indicating the importance of the C-ring in the neuronal activity of IKM-159.

Dopamine and glutamate interaction mediates reinstatement of drug-seeking behavior by stimulation of the ventral subiculum.

BACKGROUND: Drug addiction is a chronic brain disease characterized by recurrent episodes of relapse to drug-seeking/-taking behaviors. The ventral subiculum, the primary output of the hippocampus, plays a critical role in mediating drug-seeking behavior. METHODS: A d-amphetamine intravenous self-administration rat model was employed along with focal electrical stimulation of the ventral subiculum (20 Hz/200 pulses) to examine its role in reinstatement of drug-seeking behavior. Dopamine efflux in the nucleus accumbens was measured by in vivo microdialysis and subsequent HPLC-ED analyses. Pharmacological antagonism of dopamine and ionotropic glutamate receptors locally within the nucleus accumbens was employed to assess the role of glutamate and dopamine in reinstatement of drug-seeking behavior induced by stimulation of the ventral subiculum. RESULTS: Here, we demonstrate that reinstatement of drug-seeking behavior following extinction of d-amphetamine self-administration by rats was induced by electrical stimulation in the ventral subiculum but not the cortex. This reinstatement was accompanied by a significant increase in dopamine efflux in the nucleus accumbens and was disrupted by microinfusion of a dopamine D1 or D2 antagonist into the nucleus accumbens. Inhibition of N-methyl-D-aspartate or non- N-methyl-D-aspartate receptors had no effect on the reinstatement induced by ventral subiculum stimulation, whereas co-infusion of D1 and N-methyl-D-aspartate antagonists at formerly ineffective doses prevented drug-seeking behavior. CONCLUSIONS: These data support the hypothesis that dopamine/glutamate interactions within the ventral striatum related to memory processes are involved in relapse to addictive behavior.

Brainstem mechanisms controlling cardiovascular reflexes in channel catfish.

Microinjections of kynurenic acid and kainic acid into the general visceral nucleus (nGV), homologous to the mammalian nucleus tractus solitarius of the medulla, in anesthestized, spontaneously breathing catfish were used to identify central areas and mechanisms controlling resting normoxic heart rate and blood pressure and the cardiovascular responses to hypoxia. Kynurenic acid, an antagonist of ionotropic glutamate receptors, significantly reduced resting normoxic heart rate but did not block the bradycardia associated with aquatic hypoxia. Kainic acid (an excitotoxic glutamatergic receptor agonist) also significantly reduced normoxic heart rate, but blocked the hypoxia-induced bradycardia. Neither kynurenic acid nor kainic acid microinjections affected blood pressure in normoxia or hypoxia. The results of this study indicate that glutamatergic receptors in the nGV are involved in the maintenance of resting heart rate and the destruction of these neurons with kainic acid abolishes the bradycardia associated with aquatic hypoxia.

[How do antiepileptic drugs work?]. / Hvordan virker antiepileptika?

BACKGROUND: There are currently around 25 antiepileptic drugs in use in Norway, of which 15 have entered the market in the last 20 years. All have somewhat different effect- and adverse effect profiles and mechanisms of action. Here we present a brief overview of current knowledge regarding the basic mechanisms of action of these drugs. METHOD: The review is based on a discretionary selection of relevant articles found through a literature search in PubMed and our own clinical and research experience. RESULTS: There are, roughly speaking, four main mechanisms; 1) modulation of ion channels (sodium and calcium channel blockers, potassium channel openers), 2) potentiation of GABAergic inhibition, 3) reduction of glutamatergic excitation and 4) modulation of presynaptic neurotransmitter release. Some of the drugs have several mechanisms of action, and for some of them it is unclear which mechanism is clinically most important. To some extent, the drugs' mechanisms of action predict their effect against different types of epilepsy and seizures. For instance, sodium channel blockers work best against focal seizures, while calcium channel blockers work best against absences, a type of generalised seizure. INTERPRETATION: Optimal treatment of patients with epilepsy requires not only thorough knowledge of seizure- and epilepsy classification, but also insight into the mechanisms of action of antiepileptic drugs.

Effects of intrathecal kynurenate on arterial pressure during chronic osmotic stress in conscious rats.

Increased plasma osmolality elevates mean arterial pressure (MAP) through activation of the sympathetic nervous system, but the neurotransmitters released in the spinal cord to regulate MAP during osmotic stress remain unresolved. Glutamatergic neurons of the rostral ventrolateral medulla project to sympathetic preganglionic neurons in the spinal cord and are likely activated during conditions of osmotic stress; however, this has not been examined in conscious rats. This study investigated whether increased MAP during chronic osmotic stress depends on activation of spinal glutamate receptors. Rats were chronically instrumented with an indwelling intrathecal (i.t.) catheter for antagonist delivery to the spinal cord and a radiotelemetry transmitter for continuous monitoring of MAP and heart rate. Osmotic stress induced by 48 h of water deprivation (WD) increased MAP by ~15 mmHg. Intrathecal kynurenic acid, a nonspecific antagonist of ionotropic glutamate receptors, decreased MAP significantly more after 48 h of WD compared with the water-replete state. Water-deprived rats also showed a greater fall in MAP in response to i.t. 2-amino-5-phosphonovalerate. Finally, i.t. kynurenic acid also decreased MAP more in an osmotically driven model of neurogenic hypertension, the DOCA-salt rat, compared with normotensive controls. Our results suggest that spinally released glutamate mediates increased MAP during 48-h WD and DOCA-salt hypertension.

Regulation of taurine release in the hippocampus of developing and adult mice.

Taurine release in mouse hippocampal slices is regulated by several neurotransmitter receptor systems. The ionotropic glutamate receptors and the adenosine receptor A(1) are the most effective. The effect of N-methyl-D-aspartate receptors is mediated via activation of the pathway involving nitric oxide and 3',5'-cyclic guanosine monophosphate. The activation of excitatory amino acid receptors causes at the same time an increase in taurine release. The activation of adenosine A(1) receptors also potentiates taurine release. The taurine released may counteract any excitotoxic effects of glutamate, particularly in the developing hippocampus.

Radiosynthesis of carbon-11 and fluorine-18 labelled radiotracers to image the ionotropic and metabotropic glutamate receptors.

l-Glutamate is the major neurotransmitter in the central nervous system and activates both ionotropic and metabotropic receptors. Here the radiosynthesis of radiotracers developed for both types of receptors are reviewed with a highlight on the radiopharmaceuticals used or evaluated in humans. At first, radiotracers were developed for ionotropic N-methyl-d-aspartate receptors without any success to obtain radiopharmaceuticals useable for clinical or even preclinical positron emission tomography (PET) imaging purposes. Some compounds were radiolabelled and evaluated for α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors without any successful results. The recent development of radiotracers for metabotropic glutamate receptors was more efficient because radiopharmaceuticals are currently evaluated or used in clinical trials to study the mGluR1, mGluR2 or mGluR5 receptors by PET. Although the majority of the radiotracers were classically labelled with carbon-11 by O- or N-[(11) C]-methylation or with fluorine-18 nucleophilic substitution of aromatic nitro or halogeno precursors using krypofix 2.2.2/potassium [(18) F]fluoride complex, some radiosyntheses were performed with recent radiolabelling reactions like the use of iodionium salt for [(18) F]-labelling.

Electrical synaptic input to ganglion cells underlies differences in the output and absolute sensitivity of parallel retinal circuits.

Parallel circuits throughout the CNS exhibit distinct sensitivities and responses to sensory stimuli. Ambiguities in the source and properties of signals elicited by physiological stimuli, however, frequently obscure the mechanisms underlying these distinctions. We found that differences in the degree to which activity in two classes of Off retinal ganglion cell (RGC) encode information about light stimuli near detection threshold were not due to obvious differences in the cells' intrinsic properties or the chemical synaptic input the cells received; indeed, differences in the cells' light responses were largely insensitive to block of fast ionotropic glutamate receptors. Instead, the distinct responses of the two types of RGCs likely reflect differences in light-evoked electrical synaptic input. These results highlight a surprising strategy by which the retina differentially processes and routes visual information and provide new insight into the circuits that underlie responses to stimuli near detection threshold.

Determination of transmembrane water fluxes in neurons elicited by glutamate ionotropic receptors and by the cotransporters KCC2 and NKCC1: a digital holographic microscopy study.

Digital holographic microscopy (DHM) is a noninvasive optical imaging technique that provides quantitative phase images of living cells. In a recent study, we showed that the quantitative monitoring of the phase signal by DHM was a simple label-free method to study the effects of glutamate on neuronal optical responses (Pavillon et al., 2010). Here, we refine these observations and show that glutamate produces the following three distinct optical responses in mouse primary cortical neurons in culture, predominantly mediated by NMDA receptors: biphasic, reversible decrease (RD) and irreversible decrease (ID) responses. The shape and amplitude of the optical signal were not associated with a particular cellular phenotype but reflected the physiopathological status of neurons linked to the degree of NMDA activity. Thus, the biphasic, RD, and ID responses indicated, respectively, a low-level, a high-level, and an "excitotoxic" level of NMDA activation. Moreover, furosemide and bumetanide, two inhibitors of sodium-coupled and/or potassium-coupled chloride movement strongly modified the phase shift, suggesting an involvement of two neuronal cotransporters, NKCC1 (Na-K-Cl) and KCC2 (K-Cl) in the genesis of the optical signal. This observation is of particular interest since it shows that DHM is the first imaging technique able to monitor dynamically and in situ the activity of these cotransporters during physiological and/or pathological neuronal conditions.