Understanding your inhibitions. A functional metabolomic approach to the GABAergic system

Introduction The GABAergic system is complex; GABA may act at multiple subtypes of ionotropic and metabotropic GABA receptors, at GABA transporters and it may be metabolized in distinct metabolic pools of GABA. Discriminating this plethora of activities is problematic. Here, we describe a novel approach where the system is perturbed by specific GABA receptor ligands with known (or partially known) properties, the response of the system as a whole is evaluated and inferences made based on an objective overview of multiple effects of such perturbations. Methods We used a Guinea pig cortical tissue slice model, incubating the slices with [3-C]pyruvate (control) and one of at least two concentrations of a range of ligands active at GABAergic sites. After one hour, metabolic activity was stopped and the slices extracted for analysis by {C-decoupled}H and {H-decoupled}C NMR spectroscopy (600 MHz, Bruker AVANCE system). A metabolic fingerprint unique for each ligand was generated consisting of 14 variables comprising both net flux and pool size information (e.g. Fig. 1, 1.0 μM Baclofen). These fingerprints were then subjected to principal components analysis and analysed for clustering based on similarity in metabolic outcomes, using SIMCA P+ (V 11.5). Results and Discussion The data were determined to cluster into five separate groups which corresponded not to receptor pharmacology but clustered around profiles generated by concentrations of exogenous GABA (Fig. 2). Black squares are patterns typifying mainstream GABAergic synaptic activity, responding to ~10 μM GABA, green stars α5-containing receptors (4.0 μM GABA), red circles extrasynaptic high affinity receptors (0.1 – 1.0 μM GABA), blue diamonds perisynaptic activity responding to synaptic spillover (40 μM GABA) and yellow diamonds correspond to an as yet