Biomedical subjects

S Vicini

Publications and source records attributed to S Vicini.

At least 55 records · Page 3Linked to original sources

PubMed1996

Chronic flumazenil alters GABA(A) receptor subunit mRNA expression, translation product assembly and channel function in neuronal cultures.

Flumazenil competitively blocks the pharmacological effects of both positive and negative allosteric modulators acting at the benzodiazepine binding sites of gamma-aminobutyric acid (GABA(A)) receptors. Using quantitative reverse transcription polymerase chain reaction, label-fracture immunocytochemistry and whole-cell patch-clamp recordings, we determined changes in the contents of selected GABA(A) receptor subunit mRNA(s), in their translation products and in the electrophysiological characteristics of the receptor channels in cultured cerebellar granule cells treated daily with flumazenil (10 microM) for 4 days in vitro. The contents of the alpha1 and alpha6 receptor subunit mRNAs were significantly increased in the flumazenil-treated group as compared with the dimethyl sulfoxide vehicle-treated control group, whereas there were no significant differences in the absolute amounts of the beta2, beta3, gamma2S, gamma2L++ + and delta receptor subunit mRNAs. The gold immunolabeling densities of the alpha1 and delta receptor subunits were significantly increased, whereas those of the alpha6, beta2/beta3 and gamma2 receptor subunits were decreased. Double-immunolabeling experiments using 5- and 10-nm gold particles suggest that after chronic flumazenil treatment, receptor subunit assemblies containing the alpha1/gamma2 and alpha6/delta subunits may be replaced by a receptor assembly containing the alpha1/delta subunits. The GABA potency in eliciting Cl- channel activity decreased significantly, as indicated by the elevated EC50 values, and the positive modulation of GABA action by diazepam also decreased. These results suggest that flumazenil, perhaps by blocking the action of endogenous allosteric modulators of GABA(A) receptors, may trigger a change in the expression and assembly of the subunits of the GABA(A) receptor. This implies that there might be a dynamic state in the regulation of GABA(A) receptor structure.

Animals↗

PubMed1996

Alpha 6 and gamma 2 subunit antisense oligodeoxynucleotides alter gamma-aminobutyric acid receptor pharmacology in cerebellar granule neurons.

To characterize the role of the alpha 6 subunit in gamma-aminobutyric acid (GABA) receptors in cerebellar granule cells, primary cerebellar cultures were treated with antisense oligodeoxynucleotides (ODNs) complementary to and overlapping the initial codon of the alpha 6 subunit cDNA. The specific reduction in the expression of the alpha 6 receptor subunit protein after a 48-hr antisense ODN treatment was assessed with the use of immunoblot assays. Sister cultures were treated in parallel with mismatched (scrambled) ODNs. Inhibition of GABA-gated currents by furosemide, a selective inhibitor of GABAA receptors containing alpha 6 subunits, was attenuated after the alpha 6 antisense treatment. Furosemide was tested in parallel in transfected cells expressing various combinations of the alpha 1 and alpha 6 subunits, which showed that the relative abundance of these subunit mRNAs determines the extent of furosemide-induced inhibition of GABA-gated currents. Compared with control or mismatched ODN-treated cell cultures, treatment of granule neurons with alpha 6 antisense ODNs caused a decrease in GABA-induced maximal current density and increased the half-maximal concentration derived from GABA dose-response curves. Furthermore, the depletion of alpha 6 subunits from cerebellar granule cells enhanced flunitrazepam-induced potentiation of GABA-activated currents. In contrast, gamma 2 antisense ODN treatments of cell cultures increased the receptor sensitivity to GABA and potently decreased the response to flunitrazepam. Our results show that alpha 6 and gamma 2 subunit expression can be blocked with the use of synthetic ODNs and that these subunits are crucial determinants of the pharmacological properties of native GABAA receptors in cerebellar granule cells.

Allosteric Regulation↗

PubMed1995

Melatonin protects primary cultures of cerebellar granule neurons from kainate but not from N-methyl-D-aspartate excitotoxicity.

the antiexcitotoxic efficacy of melatonin, a putative endogenous hydroxyl radical scavenger, was studied in primary cultures of rat cerebellar granule neurons. Excitotoxicity was induced in 7- to 9-day-old cultures by an exposure to glutamate (15 min in the absence of magnesium) or to glutamate receptor agonists, kainate (30 min), and N-methyl-D-aspartate (60 min in the absence of magnesium). Thereafter, cultures were returned to the culture-conditioned medium for 18 h at the end of which time viability was assessed by quantitative staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. Cotreatment with melatonin (500 microM) protected the neurons completely from the toxicity of kainate (up to 1 mM) and shifted the ED50 for glutamate from 55 +/- 2.6 to 97 +/- 3.6 microM. Melatonin cotreatment was ineffective in protecting the neurons from N-methyl-D-aspartate toxicity. When melatonin was added to the cultures only before or after kainate treatment, there was no resultant protection from kainate toxicity. The neuroprotective effect of melatonin does not appear to be related to the direct action of melatonin on ionotropic glutamate receptors. That is, the kainate-stimulated inward currents measured by a patch-clamp technique in voltage clamped neurons and the kainate-stimulated increase in free cytosolic calcium measured at the single-cell level using digital imaging fluorescent microscopy with fura-2 were not affected by melatonin. Moreover, the binding of [3H]glutamate to rat cerebellar membranes was not competed off by melatonin. Further studies are needed to evaluate the pharmacologic relevance of the neuroprotective action of melatonin.

Animals↗

PubMed1995

Characterization of NMDA receptor subunit-specific antibodies: distribution of NR2A and NR2B receptor subunits in rat brain and ontogenic profile in the cerebellum.

Selective antisera for NMDA receptor subunits NR2A and NR2B have been developed. Each antiserum identifies a single band on an immunoblot at approximately 175 kDa that appears to be the appropriate subunit of the NMDA receptor. Using these antisera the relative densities of the subunits in eight areas of adult rat brain have been determined. The NR2A subunit was found to be at its highest level in hippocampus and cerebral cortex, to be at intermediate levels in striatum, olfactory tubercle, mid-brain, olfactory bulb, and cerebellum, and to be at lowest levels in the pons-medulla. The NR2B subunit was found to be expressed at its highest levels in the olfactory tubercle, hippocampus, olfactory bulb, and cerebral cortex. Intermediate levels were expressed in striatum and mid-brain, and low levels were detected in the pons-medulla. No signal for NR2B was found in the cerebellum. These regional distributions were compared with that for [3H]MK-801 binding sites. It was found that although the distribution of the NR2A subunit corresponds well with radioligand binding, the distribution of the NR2B subunit does not. The ontogenic profiles of NR2A and NR2B subunits in the rat cerebellum were also determined. Just following birth [postnatal day (P) 2] NR2A subunits are undetectable, whereas NR2B subunits are expressed at amounts easily measurable. Beginning at about P12 the levels of NR2A rise rapidly to reach adult levels by P22. At the same time (P12), levels of NR2B protein begin to decline rapidly to reach undetectable levels by 22 days after birth.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1995

NMDA-mediated modulation of gamma-aminobutyric acid type A receptor function in cerebellar granule neurons.

GABAA receptors are ligand-gated CI- ion channels with multiple clinically relevant drug-recognition sites. We have previously shown that stimulation of N-methyl-D-aspartic acid (NMDA)-specific glutamate receptors quantitatively alters selected GABAA receptor subunit mRNAs and proteins in primary cultures of rat cerebellar granule neurons. We used whole-cell recordings of GABA-elicited CI- currents and flunitrazepam binding experiments in granule cell cultures maintained in low K+ (12.5 mM), cells maintained in low K+ and treated with a single dose of NMDA (10 microM), and cell cultures maintained in depolarizing concentrations of K+ (25 mM). The EC50 obtained from the dose-response curves for GABA in eliciting a maximal response was comparable in neurons maintained in high K+ or in low K+ and treated with a single dose of NMDA, but that it increased significantly in cells maintained in low K+. The potentiation of GABA-gated CI- currents by flunitrazepam increased significantly, while the negative allosteric modulator methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) was significantly more effective in cultures either maintained in high K+ or treated with NMDA. This was coincident with a twofold increase in the Bmax associated with flunitrazepam binding. To further characterize the receptor assemblies present in the depolarization and NMDA induced paradigms, the Zn(2+)-induced inhibition of GABA-gated CI- currents was reduced as was the inhibition mediated by furosemide. Our data indicate that GABAA receptor assemblies alter their composition in response to excitatory afferent receptor stimulation.

Animals↗

PubMed1995

gamma-Aminobutyric acid gating of Cl- channels in recombinant GABAA receptors.

We studied gamma-aminobutyric acid (GABA) gated whole-cell Cl- currents after rapid transmitter application (< 10 ms) to cells expressing structurally different recombinant GABAA receptors. A variety of recombinant receptors were transiently transfected in a human cell line with three cDNAs encoding for different molecular forms of alpha-, beta- and gamma-subunits of the GABAA receptors. The maximal current amplitude elicited by GABA on recombinant GABAA receptors was greater when the alpha-subunits were combined with beta 2 gamma 2-subunits than with beta 1 gamma 2-subunits, with the exception of receptors including the alpha 6-subunit. The maximal current amplitude elicited by GABA was greater when the beta-subunits were combined with gamma 2 alpha 1-subunits than with gamma 1 alpha 1 subunits, with the exception of receptors including the beta 3-subunit. Furthermore, receptors comprising the beta 3-subunit had a greater sensitivity to GABA than those including the beta 1-or beta 2-subunits. When the gamma-subunits were substituted in receptors including alpha- and beta-subunits, the greatest current amplitude elicited by GABA was obtained with the alpha 1 beta 2 gamma 2 combination, whereas the GABA potency was greater with alpha 1 beta 2 gamma 1 and alpha 1 beta 1 gamma 3 than with the alpha 1 beta 2 gamma 2 combination. In receptors including alpha 6 beta 2-subunits the rank order of GABA maximal current amplitude was gamma 1 > gamma 2 > delta subunits.

Chloride Channels↗

PubMed1995

Time- and voltage-dependent block of delayed rectifier potassium channels by docosahexaenoic acid.

Docosahexaenoic acid (22:6n3) acts at an extracellular site to produce a voltage- and time-dependent block of the delayed rectifier current (IK) similar to that classically described for intracellularly applied quaternary ammonia compounds. In dissociated cells from the pineal gland, some long-chain polyunsaturated fatty acids reduced both late sustained (IK) (for 22:6n3, IC50 = 2.5 +/- 0.3 microM) and early transient (IA) (IC50 = 2.0 +/- 0.1 microM) components of potassium current when applied extracellularly, whereas the monounsaturate oleic acid had minimal efficacy. From comparisons of other related fatty acids, it was determined that there is a structural requirement for polyunsaturation to block IK. In contrast, chain-elongated 22-carbon polyunsaturates acted similarly to their precursor 20-carbon fatty acids (arachidonic acid and eicosapentanoic acid). Block of IK by 22:6n3 was accompanied by a dose-dependent acceleration of the current decay in both whole-cell and outside-out membrane patches, and 22:6n3 increased the macroscopic inactivation rate of IA. The combined "eicosanoid" inhibitor eicosatetraenoic acid, when included in the patch pipette, did not antagonize the action of 22:6n3. Instead, eicosatetraenoic acid produced a direct block of IK when applied extracellularly at high concentrations (25 microM). Analyses of voltage- and time-dependent block by 22:6n3 support the hypothesis that certain fatty acids directly interact with and preferentially block the open state of some potassium channels. We also describe an interaction between fatty acid block and zinc; 22:6n3 failed to block either IA or IK in the presence of zinc or cadmium, whereas extracellular calcium did not affect the response. These studies suggest a possible biological function for 22:6n3 in the nervous system, which may underlie its essential role during neural development.

Animals↗

PubMed1994

Changes in gamma-aminobutyrate type A receptor subunit mRNAs, translation product expression, and receptor function during neuronal maturation in vitro.

The amounts of mRNAs encoding alpha 1, alpha 6, beta 2, beta 3, gamma 2, and delta subunits of gamma-aminobutyrate type A (GABAA) receptors and the gold immunolabeling density of their translation products were monitored during the growth of neonatal rat granule cells in primary culture. We investigated possible correlations (i) between temporal changes in mRNA content and expression density of their respective translation products and (ii) between the quantitative changes of receptor subunit expression, the GABA EC50 for Cl- channel activation, and diazepam efficacy in modulating GABA action on the Cl- channels. At 3 days in vitro, the amount of GABAA receptor subunit mRNAs and the expression of their respective translation products were very low. During the next 2 weeks both parameters for every subunit studied increased asynchronously; moreover, at 14 days in vitro the sum of gamma 2 and delta subunit expression was smaller than the expression of the alpha 1 or alpha 6 or beta 2/beta 3 subunits. This suggests that during in vitro maturation each subunit may be regulated independently and invites speculation as to possible changes in specific GABAA receptor subtype abundance during development in vitro. The maximal current intensity elicited by GABA failed to increase from 5 to 14 days in vitro, though the amount of mRNA encoding various subunits and the expression density of their respective translation products increased. Thus, qualitative changes in the GABAA receptor subtypes expressed and/or abnormalities in the subunit assembly very likely account for the uniformity of the maximal current intensity elicited by GABA during in vitro development. Also, during maturation of neuronal cultures from 5 to 20 days in vitro the extent of the positive modulation of GABA action by diazepam decreased dramatically. This finding might be related to an increase in the abundance of GABAA receptors including the alpha 6 subunit and/or to the expression, during granule cell maturation in vitro, of GABAA receptors devoid of gamma 2 subunits.

Animals↗

PubMed1994

Functional diversity of GABA-activated Cl- currents in Purkinje versus granule neurons in rat cerebellar slices.

In rat cerebellar slices, we compared whole-cell recordings of spontaneous inhibitory postsynaptic currents (sIPSCs) with Cl- currents resulting from pulses of GABA (1 mM, < 2 ms) to outside-out patches from Purkinje and granule neurons. sIPSCs in Purkinje cells decayed with a single fast exponential, as previously reported, whereas in granule cells sIPSC decay was best described by the sum of a fast and a slow exponential curve, with a variable contribution of the slow component to the peak current. GABA pulses to nucleated patches from granule cells elicited Cl- currents with decays similar to sIPSC decays, whereas in patches from Purkinje neurons GABA pulses produced Cl- currents decaying largely with a fast component, but often followed by a slower exponential. GABA concentration steps produced rapidly desensitizing currents in patches from both cerebellar neurons. In distinct cerebellar neurons, specific functional properties of GABAA receptors may relate to the presence of distinct receptor subtypes.

Animals↗

PubMed1994

Analysis by polymerase chain reaction of alpha 1 and alpha 6 GABAA receptor subunit mRNAs in individual cerebellar neurons after whole-cell recordings.

With the use of the single-cell polymerase chain reaction (PCR), the GABAA receptor subunit mRNA content was analyzed in granule and Purkinje neurons from rat cerebellar slices. We used an experimental protocol to assess simultaneously the presence of two subunits in each cell while electrophysiological recordings were performed with the whole-cell patch-clamp technique. Based on a computer alignment of the nucleotide sequence corresponding to alpha 1 and alpha 6 GABAA receptor subunits, homologous regions were identified that allowed coamplification of both mRNAs using a single primer combination. The presence of selective restriction sites within the targeted templates allowed us to identify which receptor subunit mRNAs were coamplified by performing restriction enzyme-mediated cleavage of the amplification products. In all Purkinje neurons assayed, alpha 1 subunit mRNA but not alpha 6 mRNA was detected. In contrast, among individual granule neurons we found a heterogeneous distribution of the mRNA for the alpha 1 and alpha 6 GABAA receptor subunits. A comparison of the results of the PCR amplification and the analysis of GABA-mediated inhibitory synaptic currents does not allow us to identify kinetic characteristics of synaptic currents that clearly correlate with the presence or the absence of alpha 6 subunit mRNA.

Base Sequence↗

PubMed1994

Mechanism of early anoxia-induced suppression of the GABAA-mediated inhibitory postsynaptic current.

1. We investigated the mechanism of hypoxia-induced depression of gamma-aminobutyric acid-A (GABAA)-mediated inhibitory postsynaptic currents (IPSCs) in CA1 neurons of hippocampal slices from 21- to 28-day-old rats. Cells were examined by whole-cell patch-clamp recording and hypoxia was induced by switching perfusion of the slice from oxygenated artificial cerebral spinal fluid (ACSF) to ACSF saturated with 95% N2-5% CO2. 2. Synaptic responses evoked by stimulation of the Schaffer collateral-commissural projection at a fixed holding potential (VH = -60 mV) during anoxia revealed that the IPSC appeared more sensitive than the excitatory postsynaptic current to anoxia-induced depression. All subsequent studies examined the GABAA-mediated IPSC synaptic responses in isolation by direct stimulation of GABA interneurons in the stratum radiatum in the presence of extracellular 3-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid (CPP) (20 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (50 microM) to block glutamatergic currents and intracellular QX-314 (lidocaine N-ethyl bromide, 1 mM) to block GABAB-mediated currents. When studied in this manner (VH = -60 mV) the GABAA-mediated IPSC appeared to change from an outward to inward current after exposure to anoxia. 3. The current-voltage relationship of GABAA-mediated IPSCs revealed that these changes resulted from a positive shift in the IPSC reversal potential without a significant change in the conductance. Thus under patch clamp apparent IPSC inhibition may result from a decrease in the extracellular concentration of chloride ions. Similar findings were observed with micropipettes that contained high intracellular chloride concentrations. 4. Miniature spontaneous IPSCs were examined in the presence of tetrodotoxin (1 microM) with micropipettes containing high intracellular chloride concentrations. The miniature IPSCs (mIPSCs) appeared as spontaneous transient inward currents. Consistent with an anoxia-induced decrease in extracellular chloride, the mean amplitude of the mIPSCs increased after the onset of anoxia. A significant decrease in rise and decay time was also noted during anoxia. The frequency of the mIPSCs also increased by approximately 300%. 5. The resting input resistance of the cells was examined by measuring the current resulting from a 20-mV hyperpolarizing pulse. A significant reduction in resistance was observed 2 min after the onset of anoxia. This still occurred, although to a lesser degree, in the presence of glutamatergic blockers (20 microM CPP plus 50 microM CNQX). In the presence of both GABAergic (picrotoxin, 100 microM) and glutamatergic blockers no significant reduction in resting input resistance was apparent after 2 min of anoxia.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

PubMed1993

Single-cell analysis of Hodgkin and Reed-Sternberg cells: molecular heterogeneity of gene expression and p53 mutations.

We have used a single-cell based polymerase chain reaction (PCR) amplification technique to examine the gene expression pattern in single Hodgkin's and Reed-Sternberg (H&RS) cells from seven patients with Hodgkin's disease. Single cells were isolated from lymph nodes obtained at diagnosis (5 of 7 patients) or in first or second relapse (2 of 7 patients). Gene expression was examined by hybridization to a panel of 22 cDNA probes. Forty-nine H&RS cells (and 23 CD3+ or CD20+ lymphocytes as controls) from four patients with nodular sclerosing Hodgkin's disease (HD) and one patient each with lymphocyte predominant and mixed-cellularity HD were successfully analyzed by PCR. This analysis provides evidence that single H&RS cells can coexpress genes characteristic of several hematopoietic lineages (monocytes and lymphocytes). Genes characteristic of activated lymphoid cells are expressed in most H&RS cells. Heterogeneity of expression for certain genes between different cases was found and may eventually define molecular subgroups of HD. These findings indicate that H&RS cells of HD resemble activated hematopoietic cells. Phenotypically similar cells from different cases exhibit characteristic molecular differences. In one patient, 5 of 7 single RS cells showed identical p53 cDNA mutations at codon 246 on specific reverse transcriptase [RT]-PCR and sequencing of exons 5 through 8. The novel experimental approach may provide a valuable tool for understanding the molecular events in newly diagnosed Hodgkin's disease and progression of the disease.

Adolescent↗

PubMed1993

Triazolam is more efficacious than diazepam in a broad spectrum of recombinant GABAA receptors.

Benzodiazepine-induced modifications of GABA (gamma-aminobutyric acid) activated Cl- currents were studied in native GABAA receptors expressed in neonatal rat brain cortical neurons in primary cultures and in recombinant GABAA receptors expressed in transformed human embryonic kidney cells (293) after a transient transfection with cDNAs encoding for different molecular forms of alpha, beta, and gamma subunits of GABAA receptors. The efficacy of triazolam in cortical neurons was higher than that of diazepam. In transfected cells, triazolam showed a greater efficacy as a positive modulator of GABA-elicited Cl- currents in alpha 1 beta 1 gamma 1, alpha 1 beta 1 gamma 2, alpha 1 beta 1 gamma 3, alpha 6 beta 1 gamma 2 and alpha 1 beta 3 gamma 2 receptors than diazepam, except in alpha 3 beta 1 gamma 2 receptors where diazepam was more efficacious. When triazolam and diazepam were applied together to GABAA receptors assembled by transfecting cDNAs encoding for alpha 1 beta 1 gamma 1 subunits, the action of triazolam was curtailed in a manner related to the dose of diazepam. In recombinant receptors assembled with alpha 1 beta 1 gamma 1 receptors, maximally active doses of triazolam were more efficacious than those of clonazepam, alpidem, zolpidem, diazepam or bretazenil.

Animals↗

PubMed1993

Glutamate-activated currents in outside-out patches from spiny versus aspiny hilar neurons of rat hippocampal slices.

The desensitization rate of non-NMDA glutamate receptors was investigated in outside-out membrane patches obtained from morphologically identified spiny "mossy cells" (SMCs) and aspiny hilar interneurons (AHIs) in young rat hippocampal slices. The fast application of a 1 mM step of L-glutamate for 50-100 msec in the presence of TTX and dizolcipine (MK-801) onto patches excised from these neurons produced large glutamate-activated currents (GACs) that decayed with a single or double exponential time course despite the continued presence of agonist. These desensitization rates of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-sensitive receptors differed markedly between patches obtained from the two cell types. The fast time constant of desensitization in AHIs (n = 34) averaged 3.3 +/- 0.93 msec (mean +/- SD), while that of SMCs (n = 57) averaged 6.8 +/- 2.0 msec. Current-voltage relationships of the GACs did not differ between SMCs and AHIs, with comparable reversal potentials and no evidence of inward rectification. We also failed to observe significant Ca2+ permeability in either cell type. However, brief (< 1 msec) pulses of 1 mM glutamate produced rapidly decaying GACs with distinct kinetics in the two neuronal classes. Furthermore, analysis of the single glutamate-activated channel currents in outside-out patches from hilar neurons revealed a larger predominant single-channel current in AHIs versus SMCs. Lastly, we observed a greater sensitivity to cyclothiazide in SMCs versus AHIs, with half-maximal removal of desensitization being 90 mM and 200 mM, respectively. Taken together, these differences in GACs between SMCs and AHIs might indicate a functional correlate to the substantial heterogeneity in the molecular structure of glutamate receptor subunits or might be related to posttranslational modifications of these subunits, perhaps provided by the unique microenvironment in the spines covering SMCs.

Action Potentials↗

PubMed1993

Modulation of AMPA/kainate receptors by analogues of diazoxide and cyclothiazide in thin slices of rat hippocampus.

Among the non-NMDA (non-N-methyl-D-aspartic acid) glutamate receptors, the AMPA (alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid) selective receptors are characterized by a fast occurring desensitization. We and others have searched for specific modifiers of the rapid desensitization of AMPA responses in hippocampal slices using the patch-clamp technique. Aniracetam (1-(4-methoxybenzoyl)-2-pyrrolidinone) and diazoxide (7-chloro-3-methyl-2H-1,2,4-benzo-thiadiazine 1,1-dioxide) (1 mM) increased glutamate-activated currents recorded from voltage-clamped CA1 pyramidal neurons in presence of 5 microM MK-801 (dizocilpine; 10,11-dihydro-5-methyl-5H-dibenzo[a,d]cyclohepten-5,10-imine) by 2.5 fold. Cyclothiazide (3-bicyclo[2.2.1]hept-5-en-2-yl-6-chloro-3,4-dihydro-2H-1,2,4-benzoth ia diazine-7-sulfonamide 1,1-dioxide) (100 microM), a chemical congener of diazoxide, completely removed the desensitization of the AMPA response measured with fast application in excised outside-out patches. At this concentration cyclothiazide produced an 18 fold enhancement of the glutamate current. Eighteen diazoxide analogues (2H-1,2,4-benzothiadizines: IDRA 2-19) were then tested but none of them was as effective as diazoxide. Three analogues of cyclothiazide (3,4-dihydro-2H-1,2,4-benzothiadiazines: IDRA 20-22) were also tested and none of them were as potent as the parent compound. However, IDRA 21 produced a response 3 times larger than diazoxide. Moreover, while cyclothiazide and diazoxide potentiated kainate responses for all the doses that decreased AMPA receptor desensitization, IDRA 21, similarly to aniracetam, inhibited AMPA receptor desensitization preferentially. These results suggest that similarly to NMDA receptors the structure of AMPA receptors may include a center that regulates desensitization.

Action Potentials↗

PubMed1993

Does neurosteroid modulatory efficacy depend on GABAA receptor subunit composition?

The modulation of GABA activity by 3 alpha-OH-DHP (allopregnanolone, 3 alpha-hydroxy-5 alpha-pregnan-20-one) and PS (pregnenolone sulfate) has been studied in native GABAA receptors of rat cortical neurons in primary cultures and in structurally different recombinant GABAA receptors of rat cortical neurons in primary cultures and in structurally different recombinant GABAA receptors expressed in the 293 human embryonic kidney cell line (HEK 293). In cortical neurons 3 alpha-OH-DHP positively modulates GABA elicited Cl- currents while PS at 10 microM negatively modulates (50% decrease) this GABA response, but at 10 nM PS positively modulates the GABA current (40% increase). Both neurosteroids are equally active on various types of recombinant GABAA receptors, except for alpha 6 beta 1 gamma 2 receptors which are less sensitive to the positive allosteric modulation by 3 alpha-OH-DHP. In contrast the presence of the gamma 1 subunit doubles the efficacy of 3 alpha-OH-DHP. The negative modulation of PS is similar in recombinant GABAA receptors including various molecular forms of alpha or gamma units. A direct activation of Cl- current by 3 alpha-OH-DHP was observed in native and recombinant receptors but its efficacy on the various molecular forms of GABAA receptor tested was always smaller than that of identical concentrations (10 microM) of GABA.

Allosteric Regulation↗

PubMed1992

Activity-dependent decrease in NMDA receptor responses during development of the visual cortex.

Plasticity of the developing visual system has been regarded as the best model for changes of neuronal connections under the influence of the environment. N-methyl-D-aspartate (NMDA) receptors are crucial for experience-dependent synaptic modifications that occur in the developing visual cortex. NMDA-mediated excitatory postsynaptic currents (EPSCs) in layer IV neurons of the visual cortex lasted longer in young rats than in adult rats, and the duration of the EPSCs became progressively shorter, in parallel with the developmental reduction in synaptic plasticity. This decrease in NMDA receptor-mediated EPSC duration is delayed when the animals are reared in the dark, a condition that prolongs developmental plasticity, and is prevented by treatment with tetrodotoxin, a procedure that inhibits neural activity. Application of L-glutamate to outside-out patches excised from layer IV neurons of young, but not of adult, rats activated prolonged bursts of NMDA channel openings. A modification of the NMDA receptor gating properties may therefore account for the age-dependent decline of visual cortical plasticity.

Action Potentials↗

PubMed1992

Molecular mechanisms of the partial allosteric modulatory effects of bretazenil at gamma-aminobutyric acid type A receptor.

In central nervous system gamma-aminobutyric acid (GABA) inhibits neuronal activity by acting on GABA type A (GABAA) receptors. These heterooligomeric integral membrane proteins include a GABA-gated Cl- channel and various allosteric modulatory sites where endogenous modulators and anxiolytic drugs act to regulate GABA action. In vivo, various anxiolytic drugs exhibit a wide range of variability in their modulatory efficacy and potency of GABA action. For instance, bretazenil modulatory efficacy is much lower than that of diazepam. Such low efficacy could be due either to a preferential modulation of specific GABAA receptor subtypes or to a low modulatory efficacy at every GABAA receptor subtype. To address these questions we studied drug-induced modifications of GABA-activated Cl- currents in native GABAA receptors of cortical neurons in primary cultures and in recombinant GABAA receptors transiently expressed in transformed human embryonic kidney cells (293) after transfection with cDNAs encoding different molecular forms of alpha, beta, and gamma subunits of GABAA receptors. In cortical neurons the efficacy of bretazenil was lower than that of diazepam, whereas the potency of the two drugs was similar. In cells transfected with gamma 2 subunits and various molecular forms of alpha and beta subunits bretazenil efficacy was always lower than that of diazepam. However, in cells transfected with gamma 1 or gamma 3 subunits and various forms of alpha and beta subunits the efficacy of both diazepam and bretazenil was lower and always of similar magnitude. When bretazenil and diazepam were applied together to GABAA receptors including a gamma 2 subunit, the action of diazepam was curtailed in a manner related to the dose of bretazenil.

Allosteric Regulation↗