Biomedical subjects

H Lüddens

Publications and source records attributed to H Lüddens.

At least 19 recordsLinked to original sources

PubMed2002

Anxiolytic-like effects of acute and chronic GABA transporter inhibition in rats.

Acute GABA transporter inhibition can induce anxiolytic-like behaviors. The present analysis addressed whether chronic treatment (23 days via drinking water) with a GABA transporter inhibitor affects rat behavior similar to acute treatment and interferes with additional benzodiazepine-receptor agonistic treatment. Seventy-one rats divided into seven groups were acutely treated with either vehicle, diazepam (2 mg/kg), zolpidem (0.05 mg/kg), tiagabine (19 mg/kg) or chronically with tiagabine with or without acute diazepam or zolpidem. Animals were behaviorally characterized in an elevated plus-maze. None of the treatments induced changes in the activity of the animals. Acute and chronic treatment with tiagabine induced anxiolytic-like effects, similar to acute doses of diazepam. Acute diazepam did not enhance chronic tiagabine effects, whereas acute zolpidem attenuated the anxiolytic-like effects of chronic tiagabine. It is concluded that anxiolytic effects of acute GABA-uptake inhibition by tiagabine persist under chronic treatment and are sensitive to concomitant use of benzodiazepine receptor ligands.

Animals↗

PubMed2002

Pharmacological heterogeneity of gamma-aminobutyric acid receptors during development suggests distinct classes of rat cerebellar granule cells in situ.

The gamma-aminobutyric acid receptor (GABA(A)R) represents a ligand-gated Cl(-)-channel assembling as heteropentamere from 19 known subunits. Cerebellar granule cells contain a unique subset, namely the alpha1-, alpha6-, beta2-, gamma2- and delta-subunits. We studied their GABAergic pharmacology in situ using whole-cell patch-clamp recordings in brain slices and a modified Y-tube application system. The distribution of the EC50s for GABA in young (P8-P14) and medium aged animals (P15-P28) could be fitted with the sum of two Gaussian distributions with means of 60 and 185 microM and 27 and 214 microM, respectively. In older animals (P29-P48) the observed homogeneous range of sensitivities fitted a single Gaussian distribution (11 microM). In young animals (< or =P14) GABA-responses were largely insensitive towards 300 microM of the alpha6-specific inhibitor furosemide (82% of control response). The sensitivity increased in older animals at the EC5-20 of GABA (31% of control responses), supporting an increased expression of alpha6-subunits as molecular basis for the observed developmental changes. Approximately 50% of cells in the age range P15-P48 were potentiated by 1 microM diazepam and by 3 microM methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), suggesting the concurrent presence of alpha1- and alpha6-containing receptors, whereas the remaining of cells were neither potentiated by diazepam nor did they show the alpha6-typical DMCM potentiation, though they were potentiated by loreclezole. These properties indicate unknown pharmacological characteristics of cerebellar receptor-subunit combinations in approximately 50% of granule cells in situ.

Aging↗

PubMed2002

Altered receptor subtypes in the forebrain of GABA(A) receptor delta subunit-deficient mice: recruitment of gamma 2 subunits.

A GABA(A) receptor delta subunit-deficient mouse line was created by homologous recombination in embryonic stem cells to investigate the role of the subunit in the brain GABA(A) receptors. High-affinity [(3)H]muscimol binding to GABA sites as studied by ligand autoradiography was reduced in various brain regions of delta(-/-) animals. [(3)H]Ro 15-4513 binding to benzodiazepine sites was increased in delta(-/-) animals, partly due to an increment of diazepam-insensitive receptors, indicating an augmented forebrain assembly of gamma 2 subunits with alpha 4 subunits. In the western blots of forebrain membranes of delta(-/-) animals, the level of gamma 2 subunit was increased and that of alpha 4 decreased, while the level of alpha1 subunits remained unchanged. In the delta(-/-) forebrains, the remaining alpha 4 subunits were associated more often with gamma 2 subunits, since there was an increase in the alpha 4 subunit level immunoprecipitated by the gamma 2 subunit antibody. The pharmacological properties of t-butylbicyclophosphoro[(35)S]thionate binding to the integral ion-channel sites were slightly altered in the forebrain and cerebellum, consistent with elevated levels of alpha 4 gamma 2 and alpha 6 gamma 2 subunit-containing receptors, respectively.The altered pharmacology of forebrain GABA(A) receptors and the decrease of the alpha 4 subunit level in delta subunit-deficient mice suggest that the delta subunit preferentially assembles with the alpha 4 subunit. The delta subunit seems to interfere with the co-assembly of alpha 4 and gamma 2 subunits and, therefore, in its absence, the gamma 2 subunit is recruited into a larger population of alpha 4 subunit-containing functional receptors. These results support the idea of subunit competition during the assembly of native GABA(A) receptors.

Affinity Labels↗

PubMed2001

Magnesium potentiation of the function of native and recombinant GABA(A) receptors.

Mg2+ decreased basal and GABA-inhibited t-butylbicyclophosphoro[35S]thionate binding to GABAA receptor ion channels in rat brain sections up to 1 mM, but increased the binding at 10 mM. The Mg2+-effect was detectable in the presence of a specific GABA site competitive antagonist. Two-electrode voltage clamp recordings of recombinant alpha1beta2gamma2S, alpha1beta2, alpha2beta2gamma2S and alpha2beta2 GABAA receptors revealed a potentiation by 0.1-1 mM Mg2+ of EC20 GABA-evoked ion currents. At 10 mM, Mg2+ decreased the currents. In the absence of GABA, Mg2+ did not evoke any currents. The results show that physiologically relevant Mg2+ concentrations affect the GABA responses on GABAA receptors in native and the main recombinant receptor subtypes, suggesting putative Mg2+ binding sites on the receptor complex.

Action Potentials↗

PubMed2001

Altered atypical coupling of gamma-aminobutyrate type A receptor agonist and convulsant binding sites in subunit-deficient mouse lines.

We searched for subunit correlations for GABA(A) receptor-associated atypically GABA-insensitive [35S]TBPS binding. The homomeric beta3 subunit receptors could be excluded, as GABA-insensitive [35S]TBPS binding was present in beta3-/- mice. Localization of GABA-insensitive [35S]TBPS binding correlated best with those of delta, alpha4 and alpha6 subunit mRNAs. The amounts of GABA-insensitive [35S]TBPS binding components were increased in delta-/- mice, but dramatically reduced in alpha6-/- mice, suggesting a role for alpha6 but excluding delta subunits.

Animals↗

PubMed2001

Characterization of gamma-aminobutyrate type A receptors with atypical coupling between agonist and convulsant binding sites in discrete brain regions.

Gamma-ainobutyric acid type A (GABA(A)) receptor ionophore ligand t-[35S]butylbicyclophosphorothionate ([35S]TBPS) was used in an autoradiographic assay on brain cryostat sections to visualize and characterize atypical GABA-insensitive [35S]TBPS binding previously described in certain recombinant GABA(A) receptors and the cerebellar granule cell layer. Picrotoxinin-sensitive but 1-mM GABA-insensitive [35S]TBPS binding was present in the rat cerebellar granule cell layer, many thalamic nuclei, subiculum and the internal rim of the cerebral cortex, amounting in these regions up to 6% of the basal binding determined in the absence of exogenous GABA. Similar binding properties were detected also in human and chicken brain sections. Like the GABA-sensitive [35S]TBPS binding, GABA-insensitive binding was profoundly decreased by pentobarbital, pregnanolone, loreclezole and Mg2+. The binding was reversible and apparently dependent on Cl- ions. Localization of the GABA-insensitive [35S]TBPS binding was not identical to that of high-affinity [3H]muscimol binding and diazepam-insensitive [3H]Ro 15-4513 binding, two previously established receptor subtype-dependent binding heterogeneities in the rat brain. The present study reveals a component of the GABA-ionophore enriched in the thalamus and cerebellar granule cells, possibly representing poorly desensitized or desensitizing receptors.

Affinity Labels↗

PubMed2001

[18F]Fluoroethylflumazenil: a novel tracer for PET imaging of human benzodiazepine receptors.

5-(2'-[18F]Fluoroethyl)flumazenil ([18F]FEF) is a fluorine-18 labelled positron emission tomography (PET) tracer for central benzodiazepine receptors. Compared with the established [11C]flumazenil, it has the advantage of the longer half-life of the fluorine-18 label. After optimisation of its synthesis and determination of its in vitro receptor affinities, we performed first PET studies in humans. PET studies in seven healthy human volunteers were performed on a Siemens ECAT EXACT whole-body scanner after injection of 100-280 MBq [L8F]FEF. In two subjects, a second PET scan was conducted after pretreatment with unlabelled flumazenil (1 mg or 2.5 mg i.v., 3 min before tracer injection). A third subject was studied both with [18F]FEF and with [11C]flumazenil. Brain radioactivity was measured for 60-90 min p.i. and analysed with a region of interest-oriented approach and on a voxelwise basis with spectral analysis. Plasma radioactivity was determined from arterial blood samples and metabolites were determined by high-performance liquid chromatography. In human brain, maximum radioactivity accumulation was observed 4 +/- 2 min p.i., with a fast clearance kinetics resulting in 50% and 20% of maximal activities at about 10 and 30 min, respectively. [18F]FEF uptake followed the known central benzodiazepine receptor distribution in the human brain (occipital cortex >temporal cortex >cerebellum >thalamus >pons). Pretreatment with unlabelled flumazenil resulted in reduced tracer uptake in all brain areas except for receptor-free reference regions like the pons. Parametric images of distribution volume and binding potential generated on a voxelwise basis revealed two- to three-fold lower in vivo receptor binding of [18F]FEF compared with [11C]flumazenil, while relative uptake of [18F]FEF was higher in the cerebellum, most likely owing to its relatively higher affinity for benzodiazepine receptors containing the alpha6 subunit. Metabolism of [18F]FEF was very rapid. Polar metabolites represented about 50%-60% of total plasma radioactivity at 5 min and 80%-90% at 20 min p.i. Although [11C]flumazenil has some advantages over [18F]FEF (higher affinity, slower metabolism, slower kinetics), our results indicate that [18F]FEF is a suitable PET ligand for quantitative assessment of central benzodiazepine receptors, which can be used independently of an on-site cyclotron.

Brain↗

PubMed2001

Behavioral analysis indicates benzodiazepine-tolerance mediated by the benzodiazepine binding-site at the GABA(A)-receptor.

1. GABA(A)-receptor induced changes in locomotion and anxiety-like behaviors were studied in rats using an open-field and an elevated plus-maze. Acute and chronic doses of the benzodiazepine diazepam without and in combination with the GABA uptake inhibitor SKF-89976A were investigated. 2. Fifty-six male rats of the strain PVG/OlaHsd (PVG; 180-200 g body wt) were used to assess the influence of the benzodiazepine binding-site to the development of tolerance. Rats were divided into six groups: The first receiving saline (0.9%), the second and third diazepam (10.0 mg/kg) daily for 23 days with or without an acute challenge of 2.0 mg/kg diazepam. The fourth group received diazepam (10.0 mg/kg) daily and acutely SKF-89976A (15.0 mg/kg) plus diazepam and the fifth and sixth group received acute treatment with diazepam (2.0 mg/kg) or SKF-89976A (15.0 mg/kg). 3. Under chronic treatment with diazepam the animals became tolerant to acute doses of diazepam in activity and anxiety-related behaviors. Acute treatment with SKF-89976A increased exploration. Parameters expressing anxiolytic-like behaviors were increased, too, but not all of them significantly. In diazepam tolerant animals SKF-89976A produced anxiolytic-like behaviors 4. We conclude that the BZ- and not the GABA-binding site at the GABA(A)-receptor is involved in the development of BZ-tolerance.

Animals↗

PubMed2001

The inhibitory neural circuitry as target of antiepileptic drugs.

Impairments and defects in the inhibitory neurotransmission in the CNS can contribute to various seizure disorders, i.e., gamma-aminobutyric acid (GABA) and glycine as the main inhibitory neurotransmitters in the brain play a crucial role in some forms of epilepsy. Recent advances in deciphering the molecular basis of the GABAergic and glycinergic systems has been achieved by means of cloning techniques and gene targeting strategies in animals, contributing to the understanding of drug action. As well, several anticonvulsive substances emerged which target key molecules of the inhibitory systems. Employment of recombinant expression systems, including, but not restricted to the inhibitory circuitry, will further facilitate drug screening and rational approaches to design novel specific antiepileptic drugs, which act highly efficiently to prevent or reduce generation and spread of seizures.

Animals↗

PubMed2000

Assembly of functional alpha6beta3gamma2delta GABA(A) receptors in vitro.

Transgenic mice deficient in the alpha6 subunit of the GABA(A) receptor show reduced levels of the delta subunit protein and an altered GABA(A) receptor pharmacology, suggesting selective assembly mechanisms. Delta reduced the binding of [3H]Ro15-4513 or t-butylbicyclophosphoro[35S]thionate and, to a lesser extent, [3H]muscimol to recombinant alpha1beta1gamma2(delta), alpha4beta1gamma2(delta) and alpha6beta1gamma2(delta) receptors, paralleled by diminished GABA-evoked maximal currents in electrophysiological recordings for the latter one. The delta subunit gave rise to a lower EC50 for GABA and a slowed desensitization indicating its assembly in alpha6beta2delta, alpha6beta1gamma2delta and alpha6beta2gamma2delta receptors. The data show that the delta subunits assemble in various functional GABA(A) receptor subtypes in vitro to reduce GABA-evoked maximal currents and ligand binding, but increase the potency for GABA.

Affinity Labels↗

PubMed2000

Coupling between agonist and chloride ionophore sites of the GABA(A) receptor: agonist/antagonist efficacy of 4-PIOL.

Eight gamma-aminobutyric acid (GABA) mimetics were tested on their ability to differentiate native GABA(A) receptor subtypes present in various rat brain regions. In rat brain cryostat sections, little regional variations by the agonistic actions of muscimol, thiomuscimol, 4,5,6,7-tetrahydroisoazolo(5,4-c)pyridin-3-ol, piperidine-4-sulphonic acid, taurine and beta-alanine on [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to GABA(A) receptor channels were found. They were very similar to those found for GABA itself and indicated no direct correlation with single subunit distributions for any of these compounds. Only the low-efficacy GABA mimetic 5-(4-piperidyl)isoxazol-3-ol (4-PIOL) acted like a weak partial agonist or antagonist depending on the brain area. As the cerebellar granule cell layer was relatively insensitive to both modes of action, we tested 4-PIOL in recombinant alpha1beta2gamma2 (widespread major subtype) and alpha6beta2gamma2 (cerebellar granule cell restricted) receptors where it had different effects on GABA-modulated [35S]TBPS binding and on electrophysiological responses. 4-PIOL may thus serve as a potential lead for receptor subtype selective compounds.

Animals↗

PubMed2000

Quantification of GABA(A) receptor subunit mRNAs by non-radioisotopic competitive RT-PCR utilizing plate-based EIA methodology.

We developed a non-radioisotopic quantitative competitive RT-PCR method for the measurement of gamma-aminobutyric acid (GABA) type A receptor subunit mRNA levels. The specificity of the method was optimized by the use of four subunit-specific oligonucleotides in the sequential steps: reverse transcription, polymerase chain reaction (PCR), and detection. The biotinylated PCR products were bound on streptavidin-coated microtiter plates allowing detection of the products using dinitrophenyl (DNP)-labeled probes and anti-DNP alkaline phosphatase conjugate. The method was set up for the six major cerebellar GABA(A) receptor subunits: alpha1; alpha6; beta2; beta3; gamma2 and delta. The method is quantitative and rapid. With a large dynamic range from 10 fg to 1 ng of subunit mRNA, the accuracy was 12 and 19% (intra- and interassay coefficients of variation, respectively), which might be improved by using a smaller range of standards. The use of a double logarithmic standard curve [log (standard to competitor signal) vs. log (standard mRNA originally present)] requires only one reaction from each sample, allowing the analysis of a large number of samples in one experiment.

Animals↗

PubMed2000

Behavioral effects of GABA(A) receptor stimulation and GABA-transporter inhibition.

The present analysis addressed behavioral changes after treatment with 4.5 mg/kg or 18.5 mg/kg of the GABA-uptake inhibitor tiagabine combined with either the benzodiazepine diazepam (1.5 mg/kg) or the imidazopyridine zolpidem (0.05 mg/kg), the latter two acting differentially on GABA(A) receptor subtypes. The study included 97 male PVG/OIaHsd rats. A standard open field, an enriched open field, and an elevated plus-maze was used to study rat behavior. Treatment with the low dose of tiagabine alone induced no specific behavioral effects, whereas the high dose had an anxiolytic-like potential. Furthermore, diazepam but not zolpidem displayed anxiolytic-like effects. Combination of each benzodiazepine receptor agonist with tiagabine at the low dose decreased explorative activity. Diazepam plus the high dose of tiagabine increased the activity in the open-field test. Zolpidem together with 18.5 mg/kg tiagabine had an angiogenic-like effect compared to pure tiagabine treatment. These results provide evidence for a pharmacodynamic interaction between the GABA-uptake inhibitor tiagabine and diazepam or zolpidem. The interaction might be relevant in the clinic when combining the anticonvulsant tiagabine and a benzodiazepine receptor agonist.

Animals↗

PubMed2000

Neonatal 6-hydroxydopamine treatment affects GABA(A) receptor subunit expression during postnatal development of the rat cerebellum.

Neurotoxic elimination of noradrenergic terminals by 6-hydroxydopamine (6-OHDA) leads to alteration of the granule cell layer formation. We have studied the developmental expression of GABA(A) receptor subunits in rat cerebellum after neonatal administration of 6-OHDA during the first postnatal month of life. 6-OHDA was injected subcutaneously. The expression of GABA(A) receptor subunits was studied by in situ hybridization and immunohistochemistry. The alterations were observed in the neocerebellum - the part of the cerebellum which starts development postnatally. The migration of granule cells was delayed, and the total area of the granule cell layer in the neocerebellum from 6-OHDA-treated rats was reduced to 22.6+/-5% of the corresponding area from control rats. In situ hybridization with subunit-specific antisense oligonucleotide probes was performed for alpha1, alpha2, alpha3, alpha5, alpha6, beta1, beta2, gamma1 and gamma2 subunits of the GABA(A) receptor. In neocerebellum, 6-OHDA treatment caused a significant reduction in the alpha1, alpha6 and gamma2 subunit mRNA levels. The expression of the other subunits was not changed. It has been shown that in the postnatal cerebellum alpha1 and alpha6 subunits can be detected in granule cells only when the cells had migrated to their final destination. Our findings indicate that a noradrenergic influence may be necessary for the normal maturation and migration of cerebellar granule cells.

Adrenergic Agents↗

PubMed2000

Neonatal 6-hydroxydopamine treatment affects GABA(A) receptor subunit expression in the frontal cortex but not the hippocampus of rats during postnatal development.

The influence of neonatal administration of 6-hydroxydopamine (6-OHDA) on the maturation of GABA(A) receptors in the frontal cortex and hippocampus was studied using 5- to 40-day-old rats. In situ hybridization with antisense oligonucleotide probes was performed for alpha(1), alpha(2), alpha(5), beta(2), beta(3) and gamma(2) subunit mRNAs of the GABA(A) receptor. We demonstrated that neonatal treatment with 6-OHDA temporarily delays the postnatal transcription of the alpha(1) and gamma(2) subunits in the rat prefrontal cortex, as assessed by in situ hybridization histochemistry. The effect was selective for these subunits (the alpha(2), alpha(5), beta(2), and beta(3) subunit mRNAs remained unchanged) and for this region (the mRNA levels in the hippocampus were not changed). The reduction in mRNA levels at early postnatal stages (postnatal day 5, PD5, and PD10) also affected the subunit protein levels, as shown by immunohistochemistry for the alpha(1) subunit, and the formation of GABA(A) receptor-associated picrotoxinin-insensitive TBPS binding sites, as shown by autoradiography. Our findings indicate that without a noradrenergic influence, the maturation of GABAergic interneurons in the frontal cortex is transiently delayed (from PD5 to PD40). However, it is possible that this transient reduction of the expression of certain GABA subunits - caused by depletion of noradrenergic innervation - cannot cause a lasting alteration to the GABAergic function in the prefrontal cortex.

Animals↗

PubMed1999

Furosemide action on cerebellar GABA(A) receptors in alcohol-sensitive ANT rats.

Furosemide increases the basal tert-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding and reverses the inhibition of the binding by gamma-aminobutyric acid (GABA) in the cerebellar GABA(A) receptors containing the alpha6 and beta2/beta3 subunits. These effects are less pronounced in the alcohol-sensitive (ANT) than in the alcohol-insensitive (AT) rat line. The difference between the rat lines in the increase of basal [35S]TBPS binding was removed after a longer preincubation with ethylendiaminetetraacetic acid (EDTA) containing buffer, but long preincubation did not reduce the GABA content of the incubation fluid or remove the difference in GABA antagonism by furosemide. The GABA sensitivity of the [35S]TBPS binding did not differ between the rat lines. There was no nucleotide sequence difference in the beta2 or beta3 subunits between the rat lines and similar beta2/3 subunit-dependent agonistic actions by methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) in the rat lines were detected. The data suggest that there are still unknown structural alterations in the cerebellar GABA(A) receptors between the AT and ANT rat lines, possibly associated with differential alcohol sensitivity.

Alcoholism↗

PubMed1998

The main determinant of furosemide inhibition on GABA(A) receptors is located close to the first transmembrane domain.

Inhibitory GABA(A) receptors are regulated by numerous allosteric modulators, the most receptor-subtype specific of which is furosemide. It recognises receptors of the subunit composition alpha6beta2/3gamma2, restricted to cerebellar granule cells. To locate furosemide's site of action we constructed chimeras of the furosemide-sensitive alpha6 and the furosemide-insensitive alpha1 subunit, and expressed and studied them together with the beta3 and gamma2 subunits in Xenopus oocytes by the two-electrode voltage clamp technique. The inhibition of GABA-induced currents by furosemide mainly depended on a short domain proximal to the first transmembrane region of the alpha6 subunit.

Allosteric Regulation↗

PubMed1998

Structure-activity relationship of furosemide-derived compounds as antagonists of cerebellum-specific GABA(A) receptors.

The Na+-K+-2Cl- cotransporter blocker furosemide inhibits gamma-aminobutyric acid (GABA)-gated chloride currents and reverses GABA-mediated inhibition of [35S]-t-butylbicyclophosphorothionate ([35S]TBPS) binding of the cerebellar alpha6 subunit-containing GABA(A) receptors much more potently than the cerebrocortical non-alpha6 subunit-containing receptors. Of the 44 compounds studied, all precursors or derivatives of diuretics, one compound [hydrazinosulfonyl-furosemide (PF 1885)] reversed 5-microM GABA-induced inhibition of [35S]TBPS binding to cerebellar and cerebrocortical receptors. Three other compounds, all of which are structurally closely related to furosemide, were selective antagonists for the cerebellar receptors comparable to the lead compound. Still, the diuretic and GABAergic structure-activity relationships differ, since we found potent diuretic structures lacking GABA antagonistic activity. Further development of the GABAergic potency of furosemide derivatives can now focus on the modification of the carboxyl group, replaceable by tetrazole but not by sulfonic or phosphinic acids and the furanyl moiety which could be substituted by thienyl and benzyl groups.

Animals↗