Flumazenil Binding Research Articles

GABAA Receptor Benzodiazepine Binding Sites and Motor Impairments in Parkinson's Disease.

Flumazenil is an allosteric modulator of the γ-aminobutyric acid-A receptor (GABAAR) benzodiazepine binding site that could normalize neuronal signaling and improve motor impairments in Parkinson's disease (PD). Little is known about how regional GABAAR availability affects motor symptoms. We investigated the relationship between regional availability of GABAAR benzodiazepine binding sites and motor impairments in PD. Methods: A total of 11 Patients with PD (males; mean age 69.0 ± 4.6 years; Hoehn and Yahr stages 2-3) underwent [11C]flumazenil GABAAR benzodiazepine binding site and [11C]dihydrotetrabenazine vesicular monoamine transporter type-2 (VMAT2) PET imaging and clinical assessment. Stepwise regression analysis was used to predict regional cerebral correlates of the four cardinal UPDRS motor scores using cortical, striatal, thalamic, and cerebellar flumazenil binding estimates. Thalamic GABAAR availability was selectively associated with axial motor scores (R2 = 0.55, F = 11.0, β = -6.4, p = 0.0009). Multi-ligand analysis demonstrated significant axial motor predictor effects by both thalamic GABAAR availability (R2 = 0.47, β = -5.2, F = 7.2, p = 0.028) and striatal VMAT2 binding (R2 = 0.30, β = -3.9, F = 9.1, p = 0.019; total model: R2 = 0.77, F = 11.9, p = 0.0056). Post hoc analysis demonstrated that thalamic [11C]methyl-4-piperidinyl propionate cholinesterase PET and K1 flow delivery findings were not significant confounders. Findings suggest that reduced thalamic GABAAR availability correlates with worsened axial motor impairments in PD, independent of nigrostriatal degeneration. These findings may augur novel non-dopaminergic approaches to treating axial motor impairments in PD.

GABAA receptor availability relates to emotion-induced BOLD responses in the medial prefrontal cortex: simultaneous fMRI/PET with [11C]flumazenil.

The fMRI BOLD response to emotional stimuli highlighting the role of the medial prefrontal cortex (MPFC) has been thoroughly investigated. Recently, the relationship between emotion processing and GABA levels has been studied using MPFC proton magnetic resonance spectroscopy (1H-MRS). However, the role of GABAA receptors in the MPFC during emotion processing remains unexplored. Using [11C]flumazenil PET, we investigated the relationship between the binding potential of GABAA receptors and emotion processing as measured using simultaneous fMRI BOLD. We hypothesized a correlation between the percent signal change in the BOLD signal and the binding potential of GABAA receptors in the MPFC. In a combined simultaneous fMRI and [11C]flumazenil-PET study, we analyzed the data from 15 healthy subjects using visual emotional stimuli. Our task comprised two types of emotional processing: passive viewing and appraisal. Following the administration of a bolus plus infusion protocol, PET and fMRI data were simultaneously acquired in a hybrid 3 T MR-BrainPET. We found a differential correlation of BOLD percent signal change with [11C]flumazenil binding potential in the MPFC. Specifically, [11C]flumazenil binding potential in the ventromedial prefrontal cortex (vMPFC) correlated with passive viewing of emotionally valenced pictures. In contrast, the [11C]flumazenil binding potential and the BOLD signal induced by picture appraisal did show a correlation in the paracingulate gyrus. Our data deliver first evidence for a relationship between MPFC GABAA receptors and emotion processing in the same region. Moreover, we observed that GABAA receptors appear to play different roles in emotion processing in the vMPFC (passive viewing) and paracingulate gyrus (appraisal).

In vivo Illustration of Altered Dopaminergic and GABAergic Systems in Early Parkinson's Disease.

BackgroundChanges in γ-aminobutyric acid (GABA) function are noted in patients with Parkinson's disease (PD) who have some non-motor impairments. However, dopamine-related GABA function and GABA-related cognitive changes are still unclear.MethodsThirteen drug-naive early-stage PD patients underwent a series of PET scans with [11C]flumazenil(FMZ) and [11C]CFT. The [11C]FMZ binding potential (BPND) derived from a Logan plot analysis was compared between PD patients and age-matched controls. The [11C]CFT radioactivity relative to the cerebellar counterpart was estimated as a semiquantitative value [11C]CFT SUVR. Correlations between [11C]FMZ BPND and [11C]CFT SUVR in the same region of interest were also examined.ResultsIn patients in the PD group, [11C]CFT SUVR was significantly lower in the putamen. The levels of [11C]FMZ BPND in the cerebral cortex (frontal lobe dominancy) and the affected-side putamen were also reduced. In addition, [11C]CFT SUVR was negatively correlated with the [11C]FMZ BPND level in the affected-side putamen. In patients in the PD group, the total frontal assessment battery (FAB) score was positively correlated with the [11C]FMZ BPND in the frontal region.ConclusionGABAergic dysfunction coexists with dopaminergic loss not only in the putamen but also over the extrastriatal region in patients with early PD and is related to frontal dysfunction. The negative correlation of [11C]CFT SUVR with [11C]FMZ BPND in the affected putamen suggests that a greater dopaminergic demise would decelerate GABA release (or an increase in tracer binding), resulting in persistent failure of the GABAergic system in PD patients.

Cellular and molecular signatures of in vivo imaging measures of GABAergic neurotransmission in the human brain

Diverse GABAergic interneuron networks orchestrate information processing in the brain. Understanding the principles underlying the organisation of this system in the human brain, and whether these principles are reflected by available non-invasive in vivo neuroimaging methods, is crucial for the study of GABAergic neurotransmission. Here, we use human gene expression data and state-of-the-art imaging transcriptomics to uncover co-expression patterns between genes encoding GABAA receptor subunits and inhibitory interneuron subtype-specific markers, and their association with binding patterns of the gold-standard GABA PET radiotracers [11C]Ro15-4513 and [11C]flumazenil. We found that the inhibitory interneuron marker somatostatin covaries with GABAA receptor-subunit genes GABRA5 and GABRA2, and that their distribution followed [11C]Ro15-4513 binding. In contrast, the inhibitory interneuron marker parvalbumin covaried with GABAA receptor-subunit genes GABRA1, GABRB2 and GABRG2, and their distribution tracked [11C]flumazenil binding. Our findings indicate that existing PET radiotracers may provide complementary information about key components of the GABAergic system.

Effects of Green Lettuce Leaf Extract on Sleep Disturbance Control in Oxidative Stress-Induced Invertebrate and Vertebrate Models.

This study investigated the effect of ethanol-extracted green lettuce leaf (GLE) on sleep behavior in physical stress-induced invertebrate and vertebrate models. In Drosophila melanogaster, the group that experienced vibration stress showed decreased sleep time compared to the no-vibration-stress control group, but the GLE treatment group recovered this lost sleep time. The GLE group also recovered the gene expression of downregulated superoxide dismutase induced by vibration stress conditions. According to electroencephalography analysis of rats, non-rapid eye movement (NREM) sleep significantly decreased with a decrease in sleep time for the group in which immobilization stress was induced. In the GLE group (120 mg/kg), the change in sleep pattern caused by stress was restored, and NREM sleep increased by 68.8%, improving overall sleep quality. In addition, GLE upregulated the expression levels of oxidation-related factors and γ-aminobutyric acid (GABAA) receptor. Quercetin-3-glucuronide (Q3G) was evaluated as a sleep-promoting active substance contained in GLE using the pentobarbital-induced sleep test and showed the effect of prolonged sleep time. Q3G inhibited [3H]-flumazenil binding in a concentration-dependent manner with GLE. Taken together, the results indicate that GLE effectively binds to the GABAA receptor to promote sleep, demonstrating the potential of Q3G as an active substance.

In vivo gamma-aminobutyric acid-A/benzodiazepine receptor availability and genetic liability in asymptomatic individuals with high genetic loading of schizophrenia: A [11C]flumazenil positron emission tomography study.

Whilst reduced signalling and gene expression related to gamma-aminobutyric acid (GABA) play a role in the presumed pathophysiology of schizophrenia, its origin is unclear. Studying asymptomatic individuals with high genetic liability to schizophrenia (AIs) would provide insights. Therefore, this study aimed to investigate the role of genetic liability in GABAergic dysfunction of schizophrenia by exploring in vivo GABA-A/benzodiazepine receptor (GABAR) availability in AIs. A total of 10 AIs with multiple relatives diagnosed as schizophrenia and 11 healthy controls underwent [11C]flumazenil positron emission tomography and neurocognitive function tests. There was no significant difference in [11C]flumazenil availability based on the groups. GABAR availability in caudate nuclei had positive correlations with genetic liability of AIs. GABAR availability in caudate nuclei and verbal memory measures of AIs revealed positive correlations. Only the correlation between right caudate and short-term verbal memory survived multiple-comparison correction (p = 0.030). This study, for the first time, reports correlations between the genetic liability of schizophrenia and GABAR availability. Correlations between [11C]flumazenil binding in caudate of individuals with high genetic liability to schizophrenia suggests that the GABAergic dysfunction may arise from shared genetic factors and also that it may be responsible for cognitive impairment of AIs.

Region-specific and dose-specific effects of chronic haloperidol exposure on [3H]-flumazenil and [3H]-Ro15-4513 GABAA receptor binding sites in the rat brain

Postmortem studies suggest that schizophrenia is associated with abnormal expression of specific GABAA receptor (GABAAR) α subunits, including α5GABAAR. Positron emission tomography (PET) measures of GABAAR availability in schizophrenia, however, have not revealed consistent alterations in vivo. Animal studies using the GABAAR agonist [3H]-muscimol provide evidence that antipsychotic drugs influence GABAAR availability, in a region-specific manner, suggesting a potential confounding effect of these drugs. No such data, however, are available for more recently developed subunit-selective GABAAR radioligands. To address this, we combined a rat model of clinically relevant antipsychotic drug exposure with quantitative receptor autoradiography. Haloperidol (0.5 and 2 mg/kg/day) or drug vehicle were administered continuously to adult male Sprague-Dawley rats via osmotic mini-pumps for 28 days. Quantitative receptor autoradiography was then performed postmortem using the GABAAR subunit-selective radioligand [3H]-Ro15-4513 and the non-subunit selective radioligand [3H]-flumazenil. Chronic haloperidol exposure increased [3H]-Ro15-4513 binding in the CA1 sub-field of the rat dorsal hippocampus (p<0.01; q<0.01; d=+1.3), which was not dose-dependent. [3H]-flumazenil binding also increased in most rat brain regions (p<0.05; main effect of treatment), irrespective of the haloperidol dose. These data confirm previous findings that chronic haloperidol exposure influences the specific binding of non-subtype selective GABAAR radioligands and is the first to demonstrate a potential effect of haloperidol on the binding of a α1/5GABAAR-selective radioligand. Although caution should be exerted when extrapolating results from animals to patients, our data support a view that exposure to antipsychotics may be a confounding factor in PET studies of GABAAR in the context of schizophrenia.

T209. EFFECTS OF CHRONIC HALOPERIDOL EXPOSURE ON [3H]RO15-4513 AND [3H]FLUMAZENIL GABA-A RECEPTOR BINDING SITES

BackgroundPost-mortem studies in schizophrenia (SZ) have reported abnormalities in the GABAergic system, more specifically, on αGABA-A receptors (αGABA-A R) (Benes, Kwok, Vincent, & Todtenkopf, 1998; Beneyto, Abbott, Hashimoto, & Lewis, 2011). Consistently, animal models of SZ have also reported changes in these receptor subtypes (Lodge, Behrens, & Grace, 2009; Richetto, Calabrese, Riva, & Meyer, 2014). Preclinical work in the methylazoxymethanol acetate (MAM) animal model of SZ has shown that administration of an α5GABA-A modulating drug rescues the psychosis-like phenotype (hyperdopaminergia) (Gill, Lodge, Cook, Aras, & Grace, 2011). However, this rescue does not occur if the animals are previously administered the first-generation antipsychotic haloperidol (Gill, Cook, Poe, & Grace, 2014). A recent study in patients with first-episode psychosis found increased baseline GABA levels as measured by magnetic resonance spectroscopy (de la Fuente-Sandoval et al., 2017). After 4 weeks of treatment with the antipsychotic risperidone, these GABA levels normalized in the medial prefrontal cortex and decreased at a trend level in the dorsal caudate (de la Fuente-Sandoval et al., 2017). All in all these studies suggest that antipsychotic medication besides directly regulating the dopaminergic system may also be acting on the GABAergic system, but the underlying mechanisms remain unknown. The aim of our study was thus to investigate, for the first time, the effects of clinically-relevant dosages of haloperidol on α5GABA-A R and benzodiazepine-sensitive α1–3;5GABA-A R in healthy rats.MethodsA total of 36 male Sprague-Dawley rats were treated with vehicle, haloperidol 0.5 mg/kg/day or 2 mg/kg/day with an osmotic minipump for 28 days, representing clinically-relevant dosages of antipsychotic administration (Kapur, Vanderspek, Brownlee, & Nobrega, 2003). Quantitative autoradiography was conducted with [3H]Ro15-4513 and [3H]flumazenil, to assess the effects of haloperidol on α5GABA-A R and overall α1–3;5GABA-A R. Optical density measurements from cortical and subcortical regions were obtained from the autoradiographs using the MCID Software and converted into radioactivity values using a robust linear regression on GraphPad Prism. All data were analyzed using a mixed-effects model ANOVA on the same software and considered significant at p<0.05 with FDR correction for multiple comparisons.ResultsAdministration of chronic haloperidol showed a dose-specific increase in [3H]Ro15-4513 in the dorsal CA1 area of the hippocampus, showing an increase in binding in the 0.5 mg/kg/day vs. vehicle (p<0.01; q<0.01) and a decrease in the 2 mg/kg/day vs. the 0.5 group (p<0.05; q<0.01). In the nucleus accumbens, there was a significant reduction in [3H]Ro15-4513 binding in the higher-dose group compared to the 0.5 mg/kg/day group (p<0.001; q<0.001). In contrast, haloperidol exposure relayed a general increase in [3H]flumazenil binding across all the regions of interest explored (p<0.05).DiscussionThis is the first study to investigate, in healthy rats, the effects of clinically-relevant doses of haloperidol on GABA-A R binding in a receptor-subtype specific manner. Our results show that exposure to haloperidol causes changes in α5GABA-A R predominantly in the dorsal CA1 and the nucleus accumbens. An overall change in benzodiazepine-sensitive α1–3;5GABA-A R is also observed, irrespective of region. These results suggest that exposure to antipsychotic medication impacts the GABAergic system and should be considered when testing new pharmacological strategies in humans that have been exposed to antipsychotic medication.

T180. REDUCED [3H]RO15-4513 RECEPTOR BINDING IN THE VENTRAL HIPPOCAMPUS IN THE MAM DEVELOPMENTAL DISRUPTION MODEL OF SCHIZOPHRENIA

BackgroundPost-mortem studies in schizophrenia patients have repeatedly found abnormalities in the γ-aminobutyric acid (GABA) inhibitory neurotransmission system in the form of parvalbumin (PV)-expressing GABAergic interneuron reduction and aberrant glutamic acid decarboxylase mRNA expression and receptor distribution. However, these findings cannot be separated from medication effects. Evidence from the methylazoxymethanol acetate (MAM) animal model of schizophrenia has suggested a reduction of hippocampal PV interneurons in MAM-treated rats results in hippocampal dysfunction in the form of increased pyramidal neuron activity, which contributes to dysregulation of the stress response and concurrent disinhibition of dopaminergic neurons in the midbrain. Administration of a positive allosteric modulator of the α5 subunit-containing GABA-A receptor (α5 GABAAR) reversed dopamine neuron hyperactivation in the ventral tegmental area and attenuated schizophrenia-like behaviour in MAM-treated rats, suggesting this receptor subtype’s involvement in the development of psychosis. Conflictingly, an in vivo PET imaging study found no difference in α5 GABAAR receptor binding compared to controls but demonstrated a negative correlation between binding in the hippocampus and negative symptoms of schizophrenia patients. However, this study included previously medicated patients. To understand the involvement of α5 GABAAR in hippocampal dysfunction without a confound of antipsychotic medication, we investigated whether these receptors are differentially expressed in MAM-treated compared to control rats using autoradiography.MethodsMAM-treated (n=21) and saline (vehicle)-treated (n=22) male rats were assessed for schizophrenia- and anxiety-like phenotypes via amphetamine-induced hyperlocomotion (AIH) and the elevated plus maze assays (EPM), respectively. To quantify the density of α5 GABAAR, we used [3H]Ro15-4513, a selective radioligand binding to the benzodiazepine site of this subunit. [3H]Flumazenil, an antagonist of the benzodiazepine-binding site of α1-3,5 GABAAR, was used on adjacent brain slices of the same animals to investigate more general binding to GABAAR. Brain sections were treated with the radioligands and x-ray sensitive films were exposed to the brain sections and radioactivity standards for 4 (flumazenil) or 8 (Ro15-4513) weeks. Developed films were imaged and normalised to background, radioactive binding in images was calculated based on the standard curve using the robust regression method in Graphpad Prism 8. Subsequently, quantified images were sampled for the CA1 of the ventral hippocampus using MCID software. Independent t-tests were used to investigate group differences in time spent in open arms during the EPM, total distance moved during AIH, and receptor binding. The significance threshold was set to p=0.05.ResultsMAM-treatment induced a schizophrenia-like phenotype (AIH: t(39)=‒3.022, p=0.004) and an anxiety-like phenotype (EPM: t(41)=2.810, p=0.008) compared to saline. We found reduced α5 GABAAR binding in the ventral hippocampus CA1 region of MAM-treated rats compared to saline-treated rats (t(41)=2.563, p=0.014), but no differences in flumazenil binding to α1-3,5 GABAAR (t(41)=1.333, p=0.190).DiscussionMAM treatment induced schizophrenia- and anxiety-like phenotypes, accompanied by an α5 GABAAR density decrease in the CA1 region of the ventral hippocampus. Specific reduced binding to α5 was corroborated by the absence of any binding changes in the flumazenil treated sections. These findings reveal part of the molecular mechanisms behind hippocampal dysfunction and implicate the α5 subunit as a potential novel target for treatment of schizophrenia.

A Multi-Ligand Imaging Study Exploring GABAergic Receptor Expression and Inflammation in Multiple Sclerosis.

The γ-aminobutyric acid (GABA) is the main inhibitory neurotransmitter and essential for normal brain function. The GABAergic system has been shown to have immunomodulatory effects and respond adaptively to excitatory toxicity. The association of the GABAergic system and inflammation in patients with multiple sclerosis (MS) remains unknown. In this pilot study, the in vivo relationship between GABAA binding and the innate immune response is explored using positron emission tomography (PET) with [11C] flumazenil (FMZ) and [11C]-PK11195 PET (PK-PET), a measure of activated microglia/macrophages. Sixteen MS patients had dynamic FMZ-PET and PK-PET imaging. Ten age-matched healthy controls (HC) had a single FMZ-PET. GABAA receptor binding was calculated using Logan reference model with the pons as reference. Distribution of volume ratio (VTr) for PK-PET was calculated using image-derived input function. A hierarchical linear model was fitted to assess the linear association between PK-PET and FMZ-PET among six cortical regions of interest. GABAA receptor binding was higher throughout the cortex in MS patients (5.72 ± 0.91) as compared with HC (4.70 ± 0.41) (p = 0.002). A significant correlation was found between FMZ binding and PK-PET within the cortex (r = 0.61, p < 0.001) and among the occipital (r = 0.61, p = 0.012), parietal (r = 0.49, p = 0.041), and cingulate (r = 0.32, p = 0.006) regions. A higher GABAA receptor density in MS subjects compared with HC was observed and correlated with innate immune activity. Our observations demonstrate that immune-driven GABAergic abnormalities may be present in MS.

Probing the molecular basis for affinity/potency- and efficacy-based subtype-selectivity exhibited by benzodiazepine-site modulators at GABAA receptors

The extracellular α(+)/γ2(−) interface in the α1,2,3,5βγ2 GABAA receptor harbours the allosteric binding site targeted by benzodiazepines and newer generations of subtype-selective modulators. We have probed the molecular determinants for the affinity/potency-based α1-preference exhibited by the hypnotic zolpidem (Ambien®, Stilnox®) and the efficacy-based α3-over-α1 selectivity displayed by the analgesic NS11394. Binding affinities and functional properties of the modulators were characterized at wild-type, concatenated, mutant and chimeric α1,3β2γ2S receptors expressed in tsA201 cells and Xenopus oocytes by [3H]flumazenil binding and two-electrode voltage clamp electrophysiology. Substitution of Gly201 in α1 with the corresponding Glu in α3 completely eliminated the α1-over-α3 preference exhibited by zolpidem. In contrast, the reverse α3-E225G mutation did not yield corresponding increases in the binding affinity or modulatory potency of zolpidem at α3β2γ2S, and two additional molecular elements in the extracellular domain of the α-subunit were found also to contribute to its α1-preference. Interestingly, the α1-Gly201/α3-Glu225 residue was also a key determinant of the efficacy-based α3-over-α1 selectivity exhibited by NS11394, and a pronounced correlation existed between the side-chain bulkiness of this residue and the modulatory efficacy of NS11394 at the receptor. The subtype-selectivity determinants identified for zolpidem and NS11394 were found also to apply in different degrees to the α1-preferring modulator indiplon and the α3-over-α1 selective modulator L-838,417, respectively. In conclusion, the molecular origins of subtype-selectivity exhibited by benzodiazepine-site modulators at the α1,2,3,5βγ2 GABAA receptor seem more complex than previously appreciated, and the importance of the α1-Gly201/α3-Glu225 residue for both potency- and efficacy-based subtype-selective modulation through this site is likely to be rooted in different molecular mechanisms.

Isolation of a sleep-promoting compound from Polygonatum sibiricum rhizome.

The aim of this study was to identify sleep-promoting substance from Polygonatum sibiricum rhizome extract (PSE) with the regulation of sleep architecture. PSE showed a decrease in sleep latency time and an increase in the sleeping time. In the electroencephalography analysis of rats, PSE (150mg/kg) showed an increase of non-rapid eye movement by 38% and a decrease of rapid eye movement by 31% compared to the control. This sleep-promoting activity was found to be involved in the GABAA-BDZ receptor. The chemical structure of the pure compound was determined by the 1H and 13C nuclear magnetic resonance spectroscopy and gas chromatography mass spectrometry analysis; active compound was glyceryl-1-monolinoleate. The commercial standard glyceryl-1-monolinoleate showed a similar inhibitory concentration on [3H]-flumazenil binding to GABAA-BDZ receptors with final active fraction of PSE. The results indicate that glyceryl-1-monolinoleate is a major active compound responsible for the PSE-derived sleep promotion.

Engineered Flumazenil Recognition Site Provides Mechanistic Insight Governing Benzodiazepine Modulation in GABAA Receptors.

The anxiolytic, anticonvulsant, muscle-relaxant, and sedative-hypnotic effects of benzodiazepine site ligands are mainly elicited by allosteric modulation of GABAA receptors via their extracellular αx+/γ2- ( x = 1, 2, 3, 5) interfaces. In addition, a low affinity binding site at the homologous α+/β- interfaces was reported for some benzodiazepine site ligands. Classical benzodiazepines and pyrazoloquinolinones have been used as molecular probes to develop structure-activity relationship models for benzodiazepine site activity. Considering all possible α+/β- and α+/γ- interfaces, such ligands potentially interact with as many as 36 interfaces, giving rise to undesired side effects. Understanding the binding modes at their binding sites will enable rational strategies to design ligands with desired selectivity profiles. Here, we compared benzodiazepine site ligand interactions in the high affinity α1+/γ2- site with the homologous α1+/β3- site using a successive mutational approach. We incorporated key amino acids known to contribute to high affinity benzodiazepine binding of the γ2- subunit into the β3- subunit, resulting in a quadruple mutant β3(4mut) with high affinity flumazenil (Ro 15-1788) binding properties. Intriguingly, some benzodiazepine site ligands displayed positive allosteric modulation in the tested recombinant α1β3(4mut) constructs while diazepam remained inactive. Consequently, we performed in silico molecular docking in the wildtype receptor and the quadruple mutant. The results led to the conclusion that different benzodiazepine site ligands seem to use distinct binding modes, rather than a common binding mode. These findings provide structural hypotheses for the future optimization of both benzodiazepine site ligands, and ligands that interact with the homologous α+/β- sites.

Pharmacodynamics of Interaction between Propoxazepam and a GABA-Benzodiazepine Receptor-Ionofor Complex

Effects of a propyloxy derivative of 1.4-benzodiazepine, propoxazepam, on GABA-benzodiazepine receptor complexes (GABA RCs) were examined in vitro and in vivo. The parameters of propoxazepam binding to synaptosomes from the rat brain were estimated in vitro. The Ki constant for inhibition of [3H] flumazenil binding by this agent was 3.5 ± 0.3 nM, on average. Considering the value of the GABA shift (1.9), propoxazepam can be considered as GABA-RC full agonist. On the model of picrotoxin-induced seizures in vivo, the propoxazepam average effective dose was estimated as 4.1 ± ± 0.21 mmol/kg. It was found that the parameters of myoclonic components (latent period of the onset of myoclonic seizures and their number), as well as death time of the tested animals, characterize adequately an anticonvulsant action of propoxazepam of picrotoxin-induced seizures in mice. Competitive interaction with picrotoxin is the possible mechanism of these effects at the level of GABAARCs. Significant deviations from a competitive model of monomolecular and cooperative binding of the agent at the receptor level have been found.

Correction to: Parametric Imaging of [11C]Flumazenil Binding in the Rat Brain.

The name of the second author was incorrectly cited and is corrected now, as displayed here.

Loss of inhibition in sensorimotor networks in focal hand dystonia.

ObjectiveTo investigate GABA-ergic receptor density and associated brain functional and grey matter changes in focal hand dystonia (FHD).Methods18 patients with FHD of the right hand and 18 age and gender matched healthy volunteers (HV) participated in this study. We measured the density of GABA-A receptors using [11C] Flumazenil and perfusion using [15O] H2O. Anatomical images were also used to measure grey matter volume with voxel-based morphometry (VBM).ResultsIn FHD patients compared to HV, the vermis VI of the right cerebellum and the left sensorimotor cortex had a decrease of Flumazenil binding potential (FMZ-BP), whereas the striatum and the lateral cerebellum did not show significant change. Bilateral inferior prefrontal cortex had increased FMZ-BP and an increase of perfusion, which correlated negatively with disease duration. Only the left sensorimotor cortex showed a decrease of grey matter volume.InterpretationImpairments of GABAergic neurotransmission in the cerebellum and the sensorimotor cortical areas could explain different aspects of loss of inhibitory control in FHD, the former being involved in maladaptive plasticity, the latter in surround inhibition. Reorganization of the inferior prefrontal cortices, part of the associative network, might be compensatory for the loss of inhibitory control in sensorimotor circuits. These findings suggest that cerebellar and cerebral GABAergic abnormalities could play a role in the functional imbalance of striato-cerebello-cortical loops in dystonia.

Parametric Imaging of [11C]Flumazenil Binding in the Rat Brain

PurposeThis study evaluates the performance of several parametric methods for assessing [11C]flumazenil binding distribution in the rat brain.ProceduresDynamic (60 min) positron emission tomography data with metabolite-corrected plasma input function were retrospectively analyzed (male Wistar rats, n = 10). Distribution volume (VT) images were generated from basis function method (BFM), Logan graphical analysis (Logan), and spectral analysis (SA). Using the pons as pseudo-reference tissue, binding potential (BPND and DVR–1) images were obtained from receptor parametric imaging algorithms (RPM and SRTM2) and reference Logan (RLogan). Standardized uptake value images (SUV and SUVR) were also computed for different intervals post-injection. Next, regional averages were extracted from the parametric images, using pre-defined volumes of interest, which were also applied to the regional time-activity curves from the dynamic data. Parametric data were compared to their regional counterparts and to two-tissue compartment model (2TCM)-based values (previously defined as the model of choice for rats). Parameter agreement was assessed by linear regression analysis and Bland-Altman plots.ResultsAll parametric methods strongly correlated to their regional counterparts (R2 > 0.97) and to the 2TCM values (R2 ≥ 0.95). SA and RLogan underestimated VT and BPND (slope of 0.93 and 0.86, respectively), while SUVR-1 overestimated BPND (slope higher than 1.07 for all intervals). While BFM and SRTM2 had the smallest bias to 2TCM values (0.05 for both), ratio Bland-Altman plots showed Logan and RLogan displayed relative errors which were comparable between different regions, in contrast with the other methods. Although SUV consistently underestimated VT, the bias in this method was also constant across regions.ConclusionsAll parametric methods performed well for the analysis of [11C]flumazenil distribution and binding in the rat brain. However, Logan and RLogan slightly outperformed the other methods in terms of precision, providing robust parameter estimation and constant bias. Yet, other methods can be of interest, because they can provide tissue perfusion (i.e., K1 with BFM and SA), relative flow (i.e., R1 with RPM and SRTM2), and model order (SA) images.

Contribution of neuroinflammation to changes in [11C]flumazenil binding in the rat brain: Evaluation of the inflamed pons as reference tissue

Introduction[11C]Flumazenil is a well–known PET tracer for GABAA receptors and is mainly used as an imaging biomarker for neuronal loss. Recently, GABAA receptors on immune cells have been investigated as target for modulation of inflammation. Since neuronal loss is often accompanied by neuroinflammation, PET imaging with [11C]flumazenil is potentially affected by infiltrating immune cells. This may also compromise the validity of using the pons as reference tissue in quantitative pharmacokinetic analysis. This study aims to evaluate whether inflammatory processes in the brain can influence [11C]flumazenil uptake and affect the outcome of pharmacokinetic modeling when the pons is used as reference tissue. MethodsThe herpes simplex encephalitis (HSE) rat model is known to cause neuroinflammation in the brainstem. Dynamic [11C]flumazenil PET scans of 60-min, accompanied by arterial blood sampling and metabolite analysis, were acquired at day 6–7days post-infection of male Wistar rats (HSE, n=5 and control, n=6). Additionally, the GABAA receptor was saturated by injection of unlabeled flumazenil prior to the tracer injection in 4 rats per group. PET data were analyzed by pharmacokinetic modeling. ResultsNo statistically significant differences were found in the volume of distribution (VT) or non-displaceable binding potential (BPND) between control and HSE rats in any of the brain regions. Pre-saturation with unlabeled flumazenil resulted in a statistically significant reduction in [11C]flumazenil VT in all brain regions. The BPND obtained from SRTM exhibited a good correlation to DVR – 1 values from the two-tissue compartment model, coupled with some level of underestimation. ConclusionReliable quantification of [11C]flumazenil binding in rats can be obtained by pharmacokinetic analysis using the pons as a pseudo-reference tissue even in the presence of strong acute neuroinflammation.

Pharmacokinetic modeling of [11C]flumazenil kinetics in the rat brain

BackgroundPreferred models for the pharmacokinetic analysis of [11C]flumazenil human studies have been previously established. However, direct translation of these models and settings to animal studies might be sub-optimal. Therefore, this study evaluates pharmacokinetic models for the quantification of [11C]flumazenil binding in the rat brain.Dynamic (60 min) [11C]flumazenil brain PET scans were performed in two groups of male Wistar rats (tracer dose (TD), n = 10 and pre-saturated (PS), n = 2). Time-activity curves from five regions were analyzed, including the pons (pseudo-reference region). Distribution volume (VT) was calculated using one- and two-tissue compartment models (1TCM and 2TCM) and spectral analysis (SA). Binding potential (BPND) was determined from full and simplified reference tissue models with one or two compartments for the reference tissue (FRTM, SRTM, and SRTM-2C). Model preference was determined by Akaike information criterion (AIC), while parameter agreement was assessed by linear regression, repeated measurements ANOVA and Bland-Altman plots.Results1TCM and 2TCM fits of regions with high specific binding showed similar AIC, a preference for the 1TCM, and good VT agreement (0.1% difference). In contrast, the 2TCM was markedly preferred and necessary for fitting low specific-binding regions, where a worse VT agreement (17.6% difference) and significant VT differences between the models (p < 0.005) were seen. The PS group displayed results similar to those of low specific-binding regions. All reference models (FRTM, SRTM, and SRTM-2C) resulted in at least 13% underestimation of BPND.ConclusionsAlthough the 1TCM was sufficient for the quantification of high specific-binding regions, the 2TCM was found to be the most adequate for the quantification of [11C]flumazenil in the rat brain based on (1) higher fit quality, (2) lower AIC values, and (3) ability to provide reliable fits for all regions. Reference models resulted in negatively biased BPND and were affected by specific binding in the pons of the rat.

GABAA receptor occupancy by subtype selective GABAA\u03b12,3 modulators: PET studies in humans

RationaleSedation, dependence, and abuse liability limit the use of non-selective γ-aminobutyric acid (GABAA) receptor positive modulators for the treatment of anxiety. AZD7325 and AZD6280 are novel, subtype-selective GABAAα2,3 receptor positive modulators with limited sedative effects.ObjectivesThe current study aimed to confirm target engagement at GABAA receptors by AZD7325 and AZD6280 in humans and to determine the relationship between exposure, GABAA receptor occupancy, and tolerability.MethodTwo PET studies, using high-resolution research tomography (HRRT) and the radioligand [11C]flumazenil, were performed in 12 subjects at baseline and after administration of single oral doses of AZD7325 (0.2 to 30 mg) and AZD6280 (5 to 40 mg). PET images were analyzed using a simplified reference tissue model, and regional binding potentials (BPND) were obtained. The relationship between plasma concentration of AZD7325 or AZD6280 and GABAA receptor occupancy was described by hyperbolic function, and Ki,plasma (plasma concentration required for 50% receptor occupancy) was estimated. Assessments of safety and tolerability included recording of adverse events, vital signs, electrocardiogram, and laboratory tests.ResultsThe [11C]flumazenil binding was reduced in a dose-dependent, saturable manner by both agents. Maximum receptor occupancy could be reached for both compounds without causing sedation or cognitive impairment. The Ki,plasma estimates for AZD7325 and AZD6280 were 15 and 440 nmol/l, respectively.ConclusionHigh GABAA receptor occupancy by AZD7325 and AZD6280 could be reached without clear sedative effects.