Evans blue reduces macroscopic desensitization of non-NMDA receptor mediated currents and prolongs excitatory postsynaptic currents in cultured rat thalamic neurons

doi:10.1016/0304-3940(92)90160-9

Neuroscience Letters

Volume 146, Issue 1, 26 October 1992, Pages 13-16

https://doi.org/10.1016/0304-3940(92)90160-9 Get rights and content

Abstract

Fast application of l-glutamate, AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) or kainate to cultured rat thalamic neurons revealed properties of non-NMDA ( $N- methyl- d -aspartate$ receptors similar to those described in hippocampal neurons. The kinetics of non-NMDA receptor-mediated currents were altered by the addition of the dye Evans Blue (EB). Macroscopic desensitization was reduced and activation and deactivation kinetics were slowed. Delayed addition of EB, after desensitization of non-NMDA receptors, resulted in reactivation of desensitized receptors. Thus, both ion channel gating and entry into the desensitized state were affected. Evans blue also slowed the activation and the decay of glutamatergic miniature EPSCs (excitatory postsynaptic currents), demonstrating that receptor kinetics determine the time course of the synaptic response.

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Cited by (21)

Effects of paired-pulse and repetitive stimulation on neurons in the rat medial geniculate body
2002, Neuroscience
Many behaviorally relevant sounds, including language, are composed of brief, rapid, repetitive acoustic features. Recent studies suggest that abnormalities in producing and understanding spoken language are correlated with abnormal neural responsiveness to such auditory stimuli at higher auditory levels [Tallal et al., Science 271 (1996) 81–84; Wright et al., Nature 387 (1997) 176–178; Nagarajan et al., Proc. Natl. Acad. Sci. USA 96 (1999) 6483–6488] and with abnormal anatomical features in the auditory thalamus [Galaburda et al., Proc. Natl. Acad. Sci. USA 91 (1994) 8010–8013]. To begin to understand potential mechanisms for normal and abnormal transfer of sensory information to the cortex, we recorded the intracellular responses of medial geniculate body thalamocortical neurons in a rat brain slice preparation. Inferior colliculus or corticothalamic axons were excited by pairs or trains of electrical stimuli. Neurons receiving only excitatory collicular input had tufted dendritic morphology and displayed strong paired-pulse depression of their large, short-latency excitatory postsynaptic potentials. In contrast, geniculate neurons receiving excitatory and inhibitory collicular inputs could have stellate or tufted morphology and displayed much weaker depression or even paired-pulse facilitation of their smaller, longer-latency excitatory postsynaptic potentials. Depression was not blocked by ionotropic glutamate, GABA_A or GABA_B receptor antagonists. Facilitation was unaffected by GABA_A receptor antagonists but was diminished by N-methyl-D-aspartate (NMDA) receptor blockade. Similar stimulation of the corticothalamic input always elicited paired-pulse facilitation. The NMDA-independent facilitation of the second cortical excitatory postsynaptic potential lasted longer and was more pronounced than that seen for the excitatory collicular inputs. Paired-pulse stimulation of isolated collicular inhibitory postsynaptic potentials generated little change in the second GABA_A potential amplitude measured from the resting potential, but the GABA_B amplitude was sensitive to the interstimulus interval. Train stimuli applied to collicular or cortical inputs generated intra-train responses that were often predicted by their paired-pulse behavior. Long-lasting responses following train stimulation of the collicular inputs were uncommon. In contrast, corticothalamic inputs often generated long-lasting depolarizing responses that were dependent on activation of a metabotropic glutamate receptor.
Our results demonstrate that during repetitive afferent firing there are input-specific mechanisms controlling synaptic strength and membrane potential over short and long time scales. Furthermore, they suggest that there may be two classes of excitatory collicular input to medial geniculate neurons and a single class of small-terminal corticothalamic inputs, each of which has distinct features.
Stimulation of large-conductance Ca2+-activated K+ channels by Evans blue in cultured endothelial cells of human umbilical veins
1999, Biochemical and Biophysical Research Communications
The effect of Evans blue (EB) on large-conductance Ca²⁺-activated K⁺(BK_Ca) channels was investigated in cultured endothelial cells of human umbilical veins. In whole-cell configuration, EB (50 μM) reversibly increased the amplitude of K⁺outward currents (I_K). When the patch pipettes were filled with 10 mM EGTA, its stimulatory effect onI_Kwas unaltered. Further application of EB in the presence of suramin, a blocker of P₂-purinergic receptor, or AOPCP, an inhibitor of 5′-nucleotidase, still increasedI_K. However, charybdotoxin (100 nM) suppressed EB-induced increase inI_K. In inside-out configuration, bath application of EB (50 μM) did not change single channel conductance but significantly increased the activity of BK_Cachannels. The EB-induced increase in the activity of BK_Cachannels was independent on internal Ca²⁺. EB (50 μM) shifted the activation curve of BK_Cachannels to less positive membrane potentials by approximately 20 mV. The change in the kinetic behavior of BK_Cachannels caused by EB in these cells is due to an increase in mean open time and a decrease in mean closed time. These results indicate that EB can stimulate the activity of BK_Cachannel in endothelial cells. This effect is unrelated to its blockade of P₂-purinergic receptors or inhibition of 5′-nucleotidase. The direct stimulation of these ionic channels by EB may contribute to its effect on capillary permeability.
Pharmacological differentiation between neuronal and recombinant glutamate receptor channels expressed in Xenopus oocytes
1997, Neuropharmacology
To determine the molecular components of neuronal glutamate receptors, it is important to identify pharmacological tools that allow differentiation between different glutamate receptor types. Here, we utilized the naphthalene derivative Evans Blue (EB) and a collection of other subtype-specific compounds (polyamine toxins, concanavalin A, cyclothiazide) to compare the pharmacological profile of neuronal and recombinant glutamate receptors GluR1-GluR6 expressed in Xenopus oocytes. Submicromolar concentrations of EB selectively reduced the activity of homomeric glutamate receptors GluR1, GluR2(Q) and GluR4. Applied at concentrations above 100 μM, EB potentiated kainate responses of receptors GluR1, GluR3 and GluR4, while receptors GluR2(Q) and GluR6(Q) were completely blocked. Similar experiments were performed on identified neurones in brain slices and after injection of rat brain RNA in Xenopus oocytes. Neuronal kainate responses were (i) potentiated by 100 μM cyclothiazide, (ii) slightly blocked after preincubation in 10 μM concanavalin A, and (iii) not significantly affected by either low (< 1 μM) or high (>100 μM) concentrations of EB. Their pharmacological properties were markedly different from those of recombinant glutamate receptor channels GluR1-GluR6 investigated in heterologous expression systems.
The vital dye Evans blue mimics limbic seizures induced by kainate or pilocarpine
1997, Brain Research
Evans blue dye, given i.c.v. in rats in a dose of 208 nmol, causes electrical and behavioural seizures which resemble those induced by the glutamate analogue, kainate, or by electrical kindling of the amygdala. Chicago sky blue, 201 nmol i.c.v., produces similar seizures. The principal elements of the seizures are wet-rat-shakes, facial and forelimb clonus, rearing and spike-and-waves in the EEG. A non-NMDA receptor antagonist, GYKI 52466 and a benzodiazepine, diazepam, significantly delay the onset to the occurrence of the first forelimb clonus. The cholinergic antagonist, scopolamine, significantly reduces the delay to onset of first facial clonus. The competitive NMDA receptor antagonist, d-CPPene, the non-specific dopamine antagonist, haloperidol, and the purinergic agonist, 2-chloroadenosine, have no effect on the measured parameters. During the induction of seizures by Evans blue, the average extracellular glutamate concentration in hippocampus or cortex does not increase statistically significantly in comparison to pre-seizure values. Histological examination of limbic areas indicates that the moderate to severe Evans blue-induced cell damage is similar to that seen after limbic seizures induced by pilocarpine and in the hippocampus is partially preventable by d-CPPene but not by diazepam or GYKI 52466. It is proposed that Evans blue-induced seizures may be useful as a new model for studying the mechanisms of intractable epilepsy of the complex partial seizure type. © 1997 Elsevier Science B.V. All rights reserved.
Trophic and outgrowth-promoting effects of K+-induced depolarization on developing thalamic cells in organotypic culture
1996, Neuroscience
The aim of this study was to investigate how different levels of K⁺-induced depolarization affect the survival and growth of isolated, cultured thalamic explants from mice aged embryonic day 13 to postnatal day 2. K⁺ was added to explants in serum-free culture medium. After culture for three days, explants were sectioned and Nissl-stained or photographed under phase contrast for quantification of neurite outgrowth. Viable and pyknotic cells were counted in sectioned material. The results revealed that, with no added K⁺, both viability and neurite outgrowth decreased as the age of the thalamic explant increased: most cells survived in embryonic day 13 explants, most died in postnatal day 2 explants. Adding K⁺ had an age- and dose-dependent effect on viability and neurite outgrowth. The greatest viability-promoting effect of adding K⁺ was at embryonic day 19: adding 5 mM K⁺ rescued the majority of these cells, although there was no effect on neurite outgrowth at this age (i.e. enhanced viability did not necessarily produce increased outgrowth). This same dose of K⁺ had its greatest effect on neurite outgrowth at embryonic day 17. No dose of added K⁺ had a stimulatory effect on viability and neurite outgrowth after embryonic day 19. The highest dose of K⁺ used here (50 mM) inhibited thalamic cell survival.
We suggest that the survival and growth of the prenatal thalamus can occur without external influences. This intrinsic control may use an autocrine mechanism that becomes increasingly reliant on neural activity for its maintenance as it ages. After birth, when thalamic cells may switch their dependence to cortex-derived growth factors, this intrinsic control may become ineffective.
New developments in the molecular pharmacology of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate and kainate receptors
1996, Pharmacology and Therapeutics
Separation of $non -N- methyl - d - aspartate$ subtypes of glutamate receptors, known as α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate receptors, is traced through conventional pharmacology to molecular biology. The physiology and pharmacology of recombinant receptor subtypes (GluR1-7 andK A1-2) are described. Competitive antagonists, e.g., the quinoxalinedione, 2,3-dihydroxy-6-nitro-7-sulphamoyl-benz(F)quinoxaline, and the decahydroisoquinoline, 3S,4aR,6R,8aR-6-[2-(1(2)H-tetrazol'5-yl)ethyl] decahydroisoquinoline-3-carboxylate, have a broad antagonist spectrum, except that the latter is inactive on GluR6. The 2,3-benzodiazepines noncompetitively antagonise the AMPA receptor GluR1-4. Desensitisation of AMPA (GluR1-4) and kainate (GluR5-7 and KA1-2) receptors is blocked by cyclothiazide and concanavalin A, respectively. Polyamine toxins block AMPA receptors not containing GluR2 and unedited kainate receptors (GluR5-6). These data correlate well with results on native neurons characterised by techniques such as in situ hybridisation.

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¹: Present address: Ruhr-Universität Bochum, Department of Molecular Neurobiochemistry, Universitätsstr. 150, D-4630 Bochum, FRG.

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Neuroscience Letters

Abstract

References (24)

Rapid activation and desensitization by glutamate of excitatory, cation-selective channels in locust muscle

Neurosci. Lett.

Rapid activation, desensitization, and resensitization of synaptic channels of crayfish muscle after glutamate pulses

Biophys. J.

Membrane and action potential responses evoked by excitatory amino acids acting at $N- methyl- d -aspartate$ receptors and $non -N- methyl- d -aspartate$ receptors in the rat thalamus in vivo

Neuroscience

Liquid filament switch for ultra-fast exchanges of solutions at excised patches of synaptic membrane of crayfish muscle

Neurosci. Lett.

Glutamate receptor channels in rat DRG neurons: activation by kainate and quisqualate and blockade of desensitization by Con A

Neuron

Excitatory amino acid receptors in hippocampal neurons: kainate fails to desensitize them

Neurosci. Lett.

The physiology of excitatory amino acids in the vertebrate central nervous system

Prog. Neurobiol.

Kinetic analysis of interactions between kainate and AMPA: evidence for activation of a single receptor in mouse hippocampal neurons

Neuron

Glutamate receptor desensitization and its role in synaptic transmission

Neuron

Blockade of desensitization augments quisqualate excitotoxicity in hippocampal neurons

Neuron

Some properties of ovarian connective tissue in relation to parenchymatous changes

J. Endocrinol.

Site and mechanism of activation of proton-induced sodium current in chick dorsal root ganglion neurones

J. Physiol.

Cited by (21)

Effects of paired-pulse and repetitive stimulation on neurons in the rat medial geniculate body

Stimulation of large-conductance Ca<sup>2+</sup>-activated K<sup>+</sup> channels by Evans blue in cultured endothelial cells of human umbilical veins

Pharmacological differentiation between neuronal and recombinant glutamate receptor channels expressed in Xenopus oocytes

The vital dye Evans blue mimics limbic seizures induced by kainate or pilocarpine

Trophic and outgrowth-promoting effects of K<sup>+</sup>-induced depolarization on developing thalamic cells in organotypic culture

New developments in the molecular pharmacology of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate and kainate receptors