Pharmacoproteomics pinpoints HSP70 interaction for correction of the most frequent Wilson disease-causing mutant of ATP7B.

Pathogenic mutations in the copper transporter ATP7B have been hypothesized to affect its protein interaction landscape contributing to loss of function and, thereby, to hepatic copper toxicosis in Wilson disease. Although targeting mutant interactomes was proposed as a therapeutic strategy, druggable interactors for rescue of ATP7B mutants remain elusive. Using proteomics, we found that the frequent H1069Q substitution promotes ATP7B interaction with HSP70, thus accelerating endoplasmic reticulum (ER) degradation of the mutant protein and consequent copper accumulation in hepatic cells. This prompted us to use an HSP70 inhibitor as bait in a bioinformatics search for structurally similar Food and Drug Administration-approved drugs. Among the hits, domperidone emerged as an effective corrector that recovered trafficking and function of ATP7B-H1069Q by impairing its exposure to the HSP70 proteostatic network. Our findings suggest that HSP70-mediated degradation can be safely targeted with domperidone to rescue ER-retained ATP7B mutants and, hence, to counter the onset of Wilson disease.

Myocardin and myocardin-related transcription factor-A synergistically mediate actin cytoskeletal-dependent inhibition of liver fibrogenesis.

Activation of hepatic stellate cells (HSCs), characterized by development of a robust actin cytoskeleton and expression of abundant extracellular matrix (ECM) proteins, such as type 1 collagen (COL.1), is a central cellular and molecular event in liver fibrosis. It has been demonstrated that HSCs express both myocardin and myocardin-related transcription factor-A (MRTF-A). However, the biological effects of myocardin and MRTF-A on HSC activation and liver fibrosis, as well as the molecular mechanism under the process, remain unclear. Here, we report that myocardin and MRTF-A's expression and nuclear accumulation are prominently increased during the HSC activation process, accompanied by robust activation of actin cytoskeleton dynamics. Targeting myocardin and MRTF-A binding and function with a novel small molecule, CCG-203971, led to dose-dependent inhibition of HSC actin cytoskeleton dynamics and abrogated multiple functional features of HSC activation (i.e., HSC contraction, migration and proliferation) and decreased COL.1 expression in vitro and liver fibrosis in vivo. Mechanistically, blocking the myocardin and MRTF-A nuclear translocation pathway with CCG-203971 directly inhibited myocardin/MRTF-A-mediated serum response factor (SRF), and Smad2/3 activation in the COL.1α2 promoter and indirectly abrogated actin cytoskeleton-dependent regulation of Smad2/3 and Erk1/2 phosphorylation and their nuclear accumulation. Finally, there was no effect of CCG-203971 on markers of inflammation, suggesting a direct effect of the compound on HSCs and liver fibrosis. These data reveal that myocardin and MRTF-A are two important cotranscriptional factors in HSCs and represent entirely novel therapeutic pathways that might be targeted to treat liver fibrosis.NEW & NOTEWORTHY Myocardin and myocardin-related transcription factor-A (MRTF-A) are upregulated in activated hepatic stellate cells (HSCs) in vitro and in vivo, closely associated with robustly increased actin cytoskeleton remodeling. Targeting myocardin and MRTF-A by CCG-203971 leads to actin cytoskeleton-dependent inhibition of HSC activation, reduced cell contractility, impeded cell migration and proliferation, and decreased COL.1 expression in vitro and in vivo. Dual expression of myocardin and MRTF-A in HSCs may represent novel therapeutic targets in liver fibrosis.

Dissociation of nNOS from PSD-95 promotes functional recovery after cerebral ischaemia in mice through reducing excessive tonic GABA release from reactive astrocytes.

Mechanisms underlying functional recovery after stroke are little known, and effective drug intervention during the delayed stage is desirable. One potential drug target, the protein-protein interaction between neuronal nitric oxide synthase (nNOS) and postsynaptic density protein 95 (PSD-95), is critical to acute ischaemic damage and neurogenesis. We show that nNOS-PSD-95 dissociation induced by microinjection of a recombinant fusion protein, Tat-nNOS-N1-133 , or systemic administration of a small-molecule, ZL006, from day 4 to day 10 after photothrombotic ischaemia in mice reduced excessive tonic inhibition in the peri-infarct cortex and ameliorated motor functional outcome. We also demonstrated improved neuroplasticity including increased dendrite spine density and synaptogenesis after reducing excessive tonic inhibition by nNOS-PSD-95 dissociation. Levels of gamma-aminobutyric acid (GABA) and GABA transporter-3/4 (GAT-3/4) are increased in the reactive astrocytes in the peri-infarct cortex. The GAT-3/4-selective antagonist SNAP-5114 reduced tonic inhibition and promoted function recovery, suggesting that increased tonic inhibition in the peri-infarct cortex was due to GABA release from reversed GAT-3/4 in reactive astrocytes. Treatments with Tat-nNOS-N1-133 or ZL006 after ischaemia inhibited astrocyte activation and GABA production, prevented the reversal of GAT-3/4, and consequently decreased excessive tonic inhibition and ameliorated functional outcome. The underlying molecular mechanisms were associated with epigenetic inhibition of glutamic acid decarboxylase 67 and monoamine oxidase B expression through reduced NO production. The nNOS-PSD-95 interaction is thus a potential target for functional restoration after stroke and ZL006, a small molecule inhibitor of this interaction, is a promising pharmacological lead compound. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Generation and screening of pseudostatic hydrazone libraries derived from 5-substituted nipecotic acid derivatives at the GABA transporter mGAT4.

The γ-aminobutyric acid (GABA) transporter mGAT4 represents a promising drug target for the treatment of epilepsy and other neurological disorders; however, the lack of highly potent and selective inhibitors for mGAT4 still retards its pharmacological elucidation. Herein, the generation and screening of pseudostatic combinatorial hydrazone libraries at the murine GABA transporter mGAT4 for the search of novel GABA uptake inhibitors is described. The hydrazone libraries contained more than 1100 compounds derived from nipecotic acid derivatives substituted at the 5-position instead, as common, at the 1-position of the core structure. Two hits were found and evaluated, which display potencies in the lower micromolar range at mGAT4 and its human equivalent hGAT3. These compounds possess a lipophilic moiety derived from a biphenyl residue attached to the 5-position of the hydrophilic nipecotic acid moiety via a three-atom spacer. Thus, the novel structures with potencies close to that of the bench mark mGAT4 inhibitor (S)-SNAP-5114 add new insights into the structure-activity relationship of mGAT4 inhibitors and could provide a promising starting point for the development of new mGAT4 inhibitors with even higher potencies.

Synthesis and biological evaluation of novel N-substituted nipecotic acid derivatives with a cis-alkene spacer as GABA uptake inhibitors.

To discover new, potent, and selective inhibitors for the murine gamma-aminobutyric acid transporter 4 (mGAT4), the structure-activity relationship (SAR) study of a new cis-alkene analog family based on DDPM-1457 [(S)-2], which previously showed promising inhibitory potency at and subtype selectivity for mGAT4, was conducted. To uncover the importance of the differences between the trans- and the cis-alkene moiety in the spacer, the present publication describes the synthesis of the new compounds via catalytic hydrogenation with Lindlar's catalyst. The biological results collected by the SAR study revealed that analog rac-7j characterized by a four-instead of a three-carbon atom spacer with a cis double bond applying to the majority of the studied compounds displays a surprisingly high potency at mGAT1 (pIC50 = 6.00 ±â€¯0.04) and at the same time a reasonable potency at mGAT4 (pIC50 = 4.82).

Synthesis and biological evaluation of novel N-substituted nipecotic acid derivatives with a trans-alkene spacer as potent GABA uptake inhibitors.

Our study presents the synthesis and structure-activity relationship (SAR) of novel N-substituted nipecotic acid derivatives closely related to DDPM-1457 [(S)-2a], a chemically stable analog of (S)-SNAP-5114 (1), in the pursuit of finding new and potent mGAT4 selective inhibitors. Iminium ion chemistry served as key step for the preparation of the desired, new N-substituted nipecotic acid derivatives containing a variety of different heterocycles attached to the nipecotic acid moiety via a trans-alkene spacer. The target compounds were characterized with regard to their potency at and subtype selectivity for the GABA transporters mGAT1-mGAT4.

GAT-1 mediated GABA uptake in rat oligodendrocytes.

Stimulated by the results of a recent paper on the effects of tiagabine, a selective inhibitor of the main GABA transporter GAT-1, on oligodendrogenesis, we verified the possibility that GAT-1 may be expressed in oligodendrocytes using immunocytochemical methods and functional assays. Light microscopic analysis of the subcortical white matter of all animals revealed the presence of numerous GAT-1+ cells of different size (from 3 to 29 µm) and morphology. An electron microscope analysis revealed that, besides fibrous astrocytes and interstitial neurons, GAT-1 immunoreactivity was present in immature and mature oligodendrocytes. Co-localization studies between GAT-1 and markers specific for oligodendrocytes (NG2 and RIP) showed that about 12% of GAT-1 positive cells in the white matter were immature oligodendrocytes, while about 15% were mature oligodendrocytes. In vitro functional assays showed that oligodendrocytes exhibit tiagabine-sensitive Na+ -dependent GABA uptake. Although relationships between GABA and oligodendrocytes have been known for many years, this is the first demonstration that GAT-1 is expressed in oligodendrocytes. The present results on the one hand definitely closes the era of "neuronal" and "glial" GABA transporters, on the other they suggest that oligodendrocytes may contribute to pathophysiology of the several diseases in which GAT-1 have been implicated to date. GLIA 2017;65:514-522.

Neurophysiologically-informed markers of individual variability and pharmacological manipulation of human cortical gamma.

The ability to quantify synaptic function at the level of cortical microcircuits from non-invasive data would be enormously useful in the study of neuronal processing in humans and the pathophysiology that attends many neuropsychiatric disorders. Here, we provide proof of principle that one can estimate inter-and intra-laminar interactions among specific neuronal populations using induced gamma responses in the visual cortex of human subjects - using dynamic causal modelling based upon the canonical microcircuit (CMC; a simplistic model of a cortical column). Using variability in induced (spectral) responses over a large cohort of normal subjects, we find that the predominant determinants of gamma responses rest on recurrent and intrinsic connections between superficial pyramidal cells and inhibitory interneurons. Furthermore, variations in beta responses were mediated by inter-subject differences in the intrinsic connections between deep pyramidal cells and inhibitory interneurons. Interestingly, we also show that increasing the self-inhibition of superficial pyramidal cells suppresses the amplitude of gamma activity, while increasing its peak frequency. This systematic and nonlinear relationship was only disclosed by modelling the causes of induced responses. Crucially, we were able to validate this form of neurophysiological phenotyping by showing a selective effect of the GABA re-uptake inhibitor tiagabine on the rate constants of inhibitory interneurons. Remarkably, we were able to recover the pharmacodynamics of this effect over the course of several hours on a per subject basis. These findings speak to the possibility of measuring population specific synaptic function - and its response to pharmacological intervention - to provide subject-specific biomarkers of mesoscopic neuronal processes using non-invasive data. Finally, our results demonstrate that, using the CMC as a proxy, the synaptic mechanisms that underlie the gain control of neuronal message passing within and between different levels of cortical hierarchies may now be amenable to quantitative study using non-invasive (MEG) procedures.

GABA transporters regulate tonic and synaptic GABAA receptor-mediated currents in the suprachiasmatic nucleus neurons.

GABA is a principal neurotransmitter in the hypothalamic suprachiasmatic nucleus (SCN) that contributes to intercellular communication between individual circadian oscillators within the SCN network and the stability and precision of the circadian rhythms. GABA transporters (GAT) regulate the extracellular GABA concentration and modulate GABAA receptor (GABAAR)-mediated currents. GABA transport inhibitors were applied to study how GABAAR-mediated currents depend on the expression and function of GAT. Nipecotic acid inhibits GABA transport and induced an inward tonic current in concentration-dependent manner during whole cell patch-clamp recordings from SCN neurons. Application of either the selective GABA transporter 1 (GAT1) inhibitors NNC-711 or SKF-89976A, or the GABA transporter 3 (GAT3) inhibitor SNAP-5114, produced only small changes of the baseline current. Coapplication of GAT1 and GAT3 inhibitors induced a significant GABAAR-mediated tonic current that was blocked by gabazine. GAT inhibitors decreased the amplitude and decay time constant and increased the rise time of spontaneous GABAAR-mediated postsynaptic currents. However, inhibition of GAT did not alter the expression of either GAT1 or GAT3 in the hypothalamus. Thus GAT1 and GAT3 functionally complement each other to regulate the extracellular GABA concentration and GABAAR-mediated synaptic and tonic currents in the SCN. Coapplication of SKF-89976A and SNAP-5114 (50 µM each) significantly reduced the circadian period of Per1 expression in the SCN by 1.4 h. Our studies demonstrate that GAT are important regulators of GABAAR-mediated currents and the circadian clock in the SCN.NEW & NOTEWORTHY In the suprachiasmatic nucleus (SCN), the GABA transporters GAT1 and GAT3 are expressed in astrocytes. Inhibition of these GABA transporters increased a tonic GABA current and reduced the circadian period of Per1 expression in SCN neurons. GAT1 and GAT3 showed functional cooperativity: inhibition of one GAT increased the activity but not the expression of the other. Our data demonstrate that GABA transporters are important regulators of GABAA receptor-mediated currents and the circadian clock.

Rho Kinase Regulates Aortic Vascular Smooth Muscle Cell Stiffness Via Actin/SRF/Myocardin in Hypertension.

BACKGROUND/AIMS: Our previous studies demonstrated that intrinsic aortic smooth muscle cell (VSMC) stiffening plays a pivotal role in aortic stiffening in aging and hypertension. However, the underlying molecular mechanisms remain largely unknown. We here hypothesized that Rho kinase (ROCK) acts as a novel mediator that regulates intrinsic VSMC mechanical properties through the serum response factor (SRF) /myocardin pathway and consequently regulates aortic stiffness and blood pressure in hypertension. METHODS: Four-month old male spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were studied. Aortic stiffness was measured by echography. Intrinsic mechanical properties of VSMCs were measured by atomic force microscopy (AFM) in vitro. RESULTS: Compared to WKY rats, SHR showed a significant increase in aortic stiffness and blood pressure, which is accompanied by a remarkable cell stiffening and ROCK activation in thoracic aortic (TA) VSMCs. Theses alterations in SHR were abolished by Y-27632, a specific inhibitor of ROCK. Additionally, boosted filamentous/globular actin ratio was detected in TA VSMCs from SHR versus WKY rats, resulting in an up-regulation of SRF and myocardin expression and its downstream stiffness-associated genes including α-smooth muscle actin, SM22, smoothelin and myosin heavy chain 11. Reciprocally, these alterations in SHR TA VSMCs were also suppressed by Y-27632. Furthermore, a specific inhibitor of SRF/myocardin, CCG-100602, showed a similar effect to Y-27632 in SHR in both TA VSMCs stiffness in vitro and aorta wall stiffness in vivo. CONCLUSION: ROCK is a novel mediator modulating aortic VSMC stiffness through SRF/myocardin signaling which offers a therapeutic target to reduce aortic stiffening in hypertension.

Preclinical Comparison of Mechanistically Different Antiseizure, Antinociceptive, and/or Antidepressant Drugs in a Battery of Rodent Models of Nociceptive and Neuropathic Pain.

The series of experiments herein evaluated prototype drugs representing different mechanisms of antiseizure, antinociceptive or antidepressant action in a battery of preclinical pain models in adult male CF#1 mice (formalin, writhing, and tail flick) and Sprague Dawley rats partial sciatic nerve ligation (PSNL). In the formalin assay, phenytoin (PHT, 6 mg/kg), sodium valproate (VPA, 300 mg/kg), amitriptyline (AMI, 7.5 and 15 mg/kg), gabapentin (GBP, 30 and 70 mg/kg), tiagabine (TGB, 5 and 15 mg/kg), and acetominophen (APAP, 250 and 500 mg/kg) reduced both phases of the formalin response to ≤ 25% of vehicle-treated mice. In the acetic acid induced writhing assay, VPA (300 mg/kg), ethosuximide (ETX, 300 mg/kg), morphine (MOR, 5 & 10 mg/kg), GBP (10, 30, and 60 mg/kg), TGB (15 mg/kg), levetiracetam (LEV, 300 mg/kg), felbamate (FBM, 80 mg/kg) and APAP (250 mg/kg) reduced writhing to ≤ 25% of vehicle-treated mice. In the tail flick test, MOR (1.25-5 mg/kg), AMI (15 mg/kg) and TGB (5 mg/kg) demonstrated significant antinociceptive effects. Finally, carbamazepine (CBZ, 20 and 50 mg/kg), VPA, MOR (2 and 4 mg/kg), AMI (12 mg/kg), TPM (100 mg/kg), lamotrigine (LTG, 40 mg/kg), GBP (60 mg/kg), TGB (15 mg/kg), FBM (35 mg/kg), and APAP (250 mg/kg) were effective in the PSNL model. Thus, TGB was the only prototype compound with significant analgesic effects in each of the four models, while AMI, GBP, APAP, and MOR each improved three of the four pain phenotypes. This study highlights the importance evaluating novel targets in a variety of pain phenotypes.

Significant reductions in human visual gamma frequency by the gaba reuptake inhibitor tiagabine revealed by robust peak frequency estimation.

The frequency of visual gamma oscillations is determined by both the neuronal excitation-inhibition balance and the time constants of GABAergic processes. The gamma peak frequency has been linked to sensory processing, cognitive function, cortical structure, and may have a genetic contribution. To disentangle the intricate relationship among these factors, accurate and reliable estimates of peak frequency are required. Here, a bootstrapping approach that provides estimates of peak frequency reliability, thereby increasing the robustness of the inferences made on this parameter was developed. The method using both simulated data and real data from two previous pharmacological MEG studies of visual gamma with alcohol and tiagabine was validated. In particular, the study by Muthukumaraswamy et al. [] (Neuropsychopharmacology 38(6):1105-1112), in which GABAergic enhancement by tiagabine had previously demonstrated a null effect on visual gamma oscillations, contrasting with strong evidence from both animal models and very recent human studies was re-evaluated. After improved peak frequency estimation and additional exclusion of unreliably measured data, it was found that the GABA reuptake inhibitor tiagabine did produce, as predicted, a marked decrease in visual gamma oscillation frequency. This result demonstrates the potential impact of objective approaches to data quality control, and provides additional translational evidence for the mechanisms of GABAergic transmission generating gamma oscillations in humans. Hum Brain Mapp 37:3882-3896, 2016. © 2016 Wiley Periodicals, Inc.

Inhibition of myocardin-related transcription factor/serum response factor signaling decreases lung fibrosis and promotes mesenchymal cell apoptosis.

Myofibroblasts are crucial to the pathogenesis of tissue fibrosis. Their formation of stress fibers results in the release of myocardin-related transcription factor (MRTF), a transcriptional coactivator of serum response factor (SRF). MRTF-A (Mkl1)-deficient mice are protected from lung fibrosis. We hypothesized that the SRF/MRTF pathway inhibitor CCG-203971 would modulate myofibroblast function in vitro and limit lung fibrosis in vivo. Normal and idiopathic pulmonary fibrosis lung fibroblasts were treated with/without CCG-203971 (N-[4-chlorophenyl]-1-[3-(2-furanyl)benzoyl]-3-piperidine carboxamide) and/or Fas-activating antibody in the presence/absence of transforming growth factor (TGF)-ß1, and apoptosis was assessed. In vivo studies examined the effect of therapeutically administered CCG-203971 on lung fibrosis in two distinct murine models of fibrosis induced by bleomycin or targeted type II alveolar epithelial injury. In vitro, CCG-203971 prevented nuclear localization of MRTF-A; increased the apoptotic susceptibility of normal and idiopathic pulmonary fibrosis fibroblasts; blocked TGF-ß1-induced myofibroblast differentiation; and inhibited TGF-ß1-induced expression of fibronectin, X-linked inhibitor of apoptosis, and plasminogen activator inhibitor-1. TGF-ß1 did not protect fibroblasts or myofibroblasts from apoptosis in the presence of CCG-203971. In vivo, CCG-203971 significantly reduced lung collagen content in both murine models while decreasing alveolar plasminogen activator inhibitor-1 and promoting myofibroblast apoptosis. These data support a central role of the SRF/MRTF pathway in the pathobiology of lung fibrosis and suggest that its inhibition can help resolve lung fibrosis by promoting fibroblast apoptosis.

Synthesis of 4-substituted nipecotic acid derivatives and their evaluation as potential GABA uptake inhibitors.

In this study, we disclose the design and synthesis of novel 4-susbtituted nipecotic acid derivatives as inhibitors of the GABA transporter mGAT1. Based on molecular modeling studies the compounds are assumed to adopt a binding pose similar to that of the potent mGAT1 inhibitor nipecotic acid. As substitution in 4-position should not cause an energetically unfavorable orientation of nipecotic acid as it is the case for N-substituted derivatives this is expected to lead to highly potent binders. For the synthesis of novel 4-substituted nipecotic acid derivatives a linear synthetic strategy was employed. As a key step, palladium catalyzed cross coupling reactions were used to attach the required biaryl moieties to the ω-position of the alkenyl- or alkynyl spacers of varying length in the 4-position of the nipecotic acid scaffold. The resulting amino acids were characterized with respect to their binding affinities and inhibitory potencies at mGAT1. Though the biological activities found were generally insignificant to poor, two compounds, one of which possesses a reasonable binding affinity for mGAT1, rac-57, the other a notable inhibitory potency at mGAT4, rac-84, both displaying a slight subtype selectivity for the individual transporters, could be identified.

Postnatal maturation of GABAergic modulation of sensory inputs onto lateral amygdala principal neurons.

KEY POINTS: Throughout life, fear learning is indispensable for survival and neural plasticity in the lateral amygdala underlies this learning and storage of fear memories. During development, properties of fear learning continue to change into adulthood, but currently little is known about changes in amygdala circuits that enable these behavioural transitions. In recordings from neurons in lateral amygdala brain slices from infant up to adult mice, we show that spontaneous and evoked excitatory and inhibitory synaptic transmissions mature into adolescence. At this time, increased inhibitory activity and signalling has the ability to restrict the function of excitation by presynaptic modulation, and may thus enable precise stimulus associations to limit fear generalization from adolescence onward. Our results provide a basis for addressing plasticity mechanisms that underlie altered fear behaviour in young animals. ABSTRACT: Convergent evidence suggests that plasticity in the lateral amygdala (LA) participates in acquisition and storage of fear memory. Sensory inputs from thalamic and cortical areas activate principal neurons and local GABAergic interneurons, which provide feed-forward inhibition that tightly controls LA activity and plasticity via pre- and postsynaptic GABAA and GABAB receptors. GABAergic control is also critical during fear expression, generalization and extinction in adult animals. During rodent development, properties of fear and extinction learning continue to change into early adulthood. Currently, few studies have assessed physiological changes in amygdala circuits that may enable these behavioural transitions. To obtain first insights, we investigated changes in spontaneous and sensory input-evoked inhibition onto LA principal neurons and then focused on GABAB receptor-mediated modulation of excitatory sensory inputs in infant, juvenile, adolescent and young adult mice. We found that spontaneous and sensory-evoked inhibition increased during development. Physiological changes were accompanied by changes in dendritic morphology. While GABAB heteroreceptors were functionally expressed on sensory afferents already early in development, they could only be physiologically recruited by sensory-evoked GABA release to mediate heterosynaptic inhibition from adolescence onward. Furthermore, we found GABAB -mediated tonic inhibition of sensory inputs by ambient GABA that also emerged in adolescence. The observed increase in GABAergic drive may be a substrate for providing modulatory GABA. Our data suggest that GABAB -mediated tonic and evoked presynaptic inhibition can suppress sensory input-driven excitation in the LA to enable precise stimulus associations and limit generalization of conditioned fear from adolescence onward.

Effects of γ-Aminobutyric acid transporter 1 inhibition by tiagabine on brain glutamate and γ-Aminobutyric acid metabolism in the anesthetized rat In vivo.

γ-Aminobutyric acid (GABA) clearance from the extracellular space after release from neurons involves reuptake into terminals and astrocytes through GABA transporters (GATs). The relative flows through these two pathways for GABA released from neurons remains unclear. This study determines the effect of tiagabine, a selective inhibitor of neuronal GAT-1, on the rates of glutamate (Glu) and GABA metabolism and GABA resynthesis via the GABA-glutamine (Gln) cycle. Halothane-anesthetized rats were administered tiagabine (30 mg/kg, i.p.) and 45 min later received an intravenous infusion of either [1,6-(13)C2]glucose (in vivo) or [2-(13)C]acetate (ex vivo). Nontreated rats served as controls. Metabolites and (13)C enrichments were measured with (1)H-[(13)C]-nuclear magnetic resonance spectroscopy and referenced to their corresponding endpoint values measured in extracts from in situ frozen brain. Metabolic flux estimates of GABAergic and glutamatergic neurons were determined by fitting a metabolic model to the (13)C turnover data measured in vivo during [1,6-(13)C2]glucose infusion. Tiagabine-treated rats were indistinguishable (P > 0.05) from controls in tissue amino acid levels and in (13)C enrichments from [2-(13)C]acetate. Tiagabine reduced average rates of glucose oxidation and neurotransmitter cycling in both glutamatergic neurons (↓18%, CMR(glc(ox)Glu): control, 0.27 ± 0.05 vs. tiagabine, 0.22 ± 0.04 µmol/g/min; ↓11%, V(cyc(Glu-Gln)): control 0.23 ± 0.05 vs. tiagabine 0.21 ± 0.04 µmol/g/min and GABAergic neurons (↓18-25%, CMR(glc(ox)GABA): control 0.09 ± 0.02 vs. tiagabine 0.07 ± 0.03 µmol/g/min; V(cyc(GABA-Gln)): control 0.08 ± 0.02 vs. tiagabine 0.07 ± 0.03 µmol/g/min), but the changes in glutamatergic and GABAergic fluxes were not significant (P > 0.10). The results suggest that any reduction in GABA metabolism by tiagabine might be an indirect response to reduced glutamatergic drive rather than direct compensatory effects.

Influence of agonist induced internalization on [3H]Ro15-4513 binding-an application to imaging fluctuations in endogenous GABA with positron emission tomography.

Low affinity α1/α2 containing GABAA receptors are significantly less able to bind [(11) C]/[(3) H]Ro15-4513 following translocation to the endosomal environment. The alterations in [(11) C]Ro15-4513 binding observed in vivo following perturbations in endogenous GABA are likely driven by both alterations in receptor binding parameters following agonist induced internalisation and the GABA Shift.

Silencing the α2 subunit of γ-aminobutyric acid type A receptors in rat dorsal root ganglia reveals its major role in antinociception posttraumatic nerve injury.

BACKGROUND: Neuropathic pain (NPP) is likely the result of repetitive high-frequency bursts of peripheral afferent activity leading to long-lasting changes in synaptic plasticity in the spinal dorsal horn. Drugs that promote γ-aminobutyric acid (GABA) activity in the dorsal horn provide partial relief of neuropathic symptoms. The authors examined how in vivo silencing of the GABA receptor type A (GABAA) α2 gene in dorsal root ganglia (DRG) controls NPP. METHODS: After crush injury to the right sciatic nerve of female rats, the α2 GABAA antisense and mismatch oligodeoxynucleotides or NO-711 (a GABA uptake inhibitor) were applied to the L5 DRG. In vivo behavioral assessment of nociception was conducted before the injury and ensuing 10 days (n = 4 to 10). In vitro quantification of α2 GABAA protein and electrophysiological studies of GABAA currents were performed on acutely dissociated L5 DRG neurons at relevant time points (n = 6 to 14). RESULTS: NPP postcrush injury of a sciatic nerve in adult female rats coincides with significant down-regulation of the α2 subunit expression in the ipsilateral DRG (approximately 30%). Selective down-regulation of α2 expression in DRGs significantly worsens mechanical (2.55 ± 0.75 to 5.16 ± 1.16) and thermal (7.97 ± 0.96 to 5.51 ± 0.75) hypersensitivity in crush-injured animals and causes development of significant mechanical (2.33 ± 0.40 to 5.00 ± 0.33) and thermal (10.80 ± 0.29 to 7.34 ± 0.81) hypersensitivity in sham animals (data shown as mean ± SD). Conversely, up-regulation of endogenous GABA via blockade of its uptake in DRG alleviates NPP. CONCLUSION: The GABAA receptor in the DRG plays an important role in pathophysiology of NPP caused by sciatic nerve injury and represents promising target for novel pain therapies.

Impaired GABAergic inhibition in the prefrontal cortex of early postnatal phencyclidine (PCP)-treated rats.

A compromised γ-aminobutyric acid (GABA)ergic system is hypothesized to be part of the underlying pathophysiology of schizophrenia. N-methyl-D-aspartate (NMDA) receptor hypofunction during neurodevelopment is proposed to disrupt maturation of interneurons causing an impaired GABAergic transmission in adulthood. The present study examines prefrontal GABAergic transmission in adult rats administered with the NMDA receptor channel blocker, phencyclidine (PCP), for 3 days during the second postnatal week. Whole-cell patch-clamp recordings from pyramidal cells in PCP-treated rats showed a 22% reduction in the frequency of miniature inhibitory postsynaptic currents in layer II/III, but not in layer V pyramidal neurons of the prefrontal cortex. Furthermore, early postnatal PCP treatment caused insensitivity toward effects of the GABA transporter 1 (GAT-1) inhibitor, 1,2,5,6-tetrahydro-1-[2-[[(diphenyl-methylene)amino]oxy]ethyl]-3-pyridinecarboxylic acid, and also diminished currents passed by δ-subunit-containing GABAA receptors in layer II/III pyramidal neurons. The observed impairments in GABAergic function are compatible with the alteration of GABAergic markers as well as cognitive dysfunction observed in early postnatal PCP-treated rats and support the hypothesis that PCP administration during neurodevelopment affects the functionality of interneurons in later life.

Differential GABAB-receptor-mediated effects in perisomatic- and dendrite-targeting parvalbumin interneurons.

Inhibitory parvalbumin-containing interneurons (PVIs) control neuronal discharge and support the generation of theta- and gamma-frequency oscillations in cortical networks. Fast GABAergic input onto PVIs is crucial for their synchronization and oscillatory entrainment, but the role of metabotropic GABA(B) receptors (GABA(B)Rs) in mediating slow presynaptic and postsynaptic inhibition remains unknown. In this study, we have combined high-resolution immunoelectron microscopy, whole-cell patch-clamp recording, and computational modeling to investigate the subcellular distribution and effects of GABA(B)Rs and their postsynaptic effector Kir3 channels in rat hippocampal PVIs. Pre-embedding immunogold labeling revealed that the receptors and channels localize at high levels to the extrasynaptic membrane of parvalbumin-immunoreactive dendrites. Immunoreactivity for GABA(B)Rs was also present at lower levels on PVI axon terminals. Whole-cell recordings further showed that synaptically released GABA in response to extracellular stimulation evokes large GABA(B)R-mediated slow IPSCs in perisomatic-targeting (PT) PVIs, but only small or no currents in dendrite-targeting (DT) PVIs. In contrast, paired recordings demonstrated that GABA(B)R activation results in presynaptic inhibition at the output synapses of both PT and DT PVIs, but more strongly in the latter. Finally, computational analysis indicated that GABA(B) IPSCs can phasically modulate the discharge of PT interneurons at theta frequencies. In summary, our results show that GABA(B)Rs differentially mediate slow presynaptic and postsynaptic inhibition in PVIs and can contribute to the dynamic modulation of their activity during oscillations. Furthermore, these data provide evidence for a compartment-specific molecular divergence of hippocampal PVI subtypes, suggesting that activation of GABA(B)Rs may shift the balance between perisomatic and dendritic inhibition.