Clustering of Extrasynaptic GABAA Receptors Modulates Tonic Inhibition in Cultured Hippocampal Neurons

Abstract

Tonic inhibition plays a crucial role in regulating neuronal excitability because it sets the threshold for action potential generation and integrates excitatory signals. Tonic currents are known to be largely mediated by extrasynaptic ␥-aminobutyric acid type A (GABA A ) receptors that are persistently activated by submicromolar concentrations of ambient GABA. We recently reported that, in cultured hippocampal neurons, the clustering of synaptic GABA A receptors significantly affects synaptic transmission (Petrini, E. M., Zacchi, P., Barberis, A., Mozrzymas, J. W., and Cherubini, E. (2003) J. Biol. Chem. 278, 16271-16279). In this work, we demonstrated that the clustering of extrasynaptic GABA A receptors modulated tonic inhibition. Depolymerization of the cytoskeleton with nocodazole promoted the disassembly of extrasynaptic clusters of ␦ and ␥ 2 subunitcontaining GABA A receptors. This effect was associated with a reduction in the amplitude of tonic currents and diminished shunting inhibition. Moreover, diffuse GABA A receptors were less sensitive to the GAT-1 inhibitor NO-711 and to flurazepam. Quantitative analysis of GABA-evoked currents after prolonged exposure to submicromolar concentrations of GABA and model simulations suggest that clustering affects the gating properties of extrasynaptic GABA A receptors. In particular, a larger occupancy of the singly and doubly bound desensitized states can account for the modulation of tonic inhibition recorded after nocodazole treatment. Moreover, comparison of tonic currents recorded during spontaneous activity and those elicited by exogenously applied low agonist concentrations allows estimation of the concentration of ambient GABA. In conclusion, receptor clustering appears to be an additional regulating factor for tonic inhibition.Similar to many other neurotransmitter receptors, ␥-aminobutyric acid type A (GABA A ) 1 receptors are localized at synaptic and extrasynaptic levels. Whereas synaptic GABA A receptors are involved in phasic inhibition (1), extrasynaptic ones are responsible for tonic inhibition (2-9). Tonic inhibition is due to persistent inhibitory conductance that contributes to "signal integration" in the brain since it sets the threshold for action potential generation (10, 11) and shunts excitatory synaptic inputs (2,(12)(13)(14)(15). This conductance is maintained by "ambient" GABA, which represents the amount of neurotransmitter present in the extracellular space. Ambient GABA originates from spillover of the neurotransmitter released at neighboring synapses (3, 5, 11), from astrocytes (16, 17), or from non-vesicular release (18,19). Tonic inhibition has been well characterized in the cerebellum, where ␣ 6 subunit-containing receptors act as high affinity sensors for GABA (4,11,20,21). Persistent GABA conductance has also been identified in other brain regions, including the hippocampus (6,8,9,22,23). However, in this structure, the subunit composition of the receptors involved has not been fully elucidated. In the past years, immunocytochemi...

Presynaptic Inhibition of Excitatory Afferents to Hilar Mossy Cells

“…This conclusion is consistent with studies in developing and mature cerebellum that showed a clear link between sIPSCs and tonic currents mediated by ambient GABA (Brickley et al 1996;Carta et al 2004;Kaneda et al 1995;Wall and Usowicz 1997). It is also consistent with cell culture studies of both cerebellar and hippocampal neurons that show TTX-mediated reductions in tonic GABAergic currents (Leao et al 2000;Petrini et al 2004) and with in vitro work in hippocampus directly implicating KA-induced increases in sIPSCs with GABA A -mediated changes in holding current (Frerking et al 1999). Nevertheless, it is clear that action potential-independent mechanisms for regulating ambient GABA have also been implicated in some studies (Rossi et al 2003;Wall and Usowicz 1997).…”

“…However, such measurements must be carefully obtained to prevent contamination by sIPSCs (Prenosil et al 2006;Scimemi et al 2005). Alternatively tonic currents have been characterized using noise analysis (Brickley et al 1996;Mtchedlishvili and Kapur 2006) or by fitting a Gaussian curve to an all-points histogram of sampled data (Glykys and Mody 2006a;Glykys et al 2006;Petrini et al 2004;Wall and Usowicz 1997). Unfortunately, these techniques generally fail to simultaneously describe changes in both tonic and phasic currents.…”

GABAAReceptor γ2 Subunit Mutations Linked to Human Epileptic Syndromes Differentially Affect Phasic and Tonic Inhibition

“…Beta subunits could escape such ER trapping because mutation of R43 of the ␥2 subunit is thought to modify interactions with ␤ but not ␣ subunits (Hales et al, 2005). Alternatively, mutant R43Q ␥2 may compete for ␣5-associated proteins, such as radixin (Loebrich et al, 2006), and so prevent aggregation of extrasynaptic receptors thus decreasing tonic inhibition (Petrini et al, 2004). Tonic GABA currents of hippocampal pyramidal cells are probably mediated by both ␣5␤3␥2 and ␣4␤2/3␦ assemblies, in proportions that may depend on agonist concentration (Caraiscos et al, 2004;Scimemi et al, 2005;Glykys and Mody, 2006).…”

Inhibitory synapse deficits caused by familial α1 GABAA receptor mutations in epilepsy

“…Clusters of GABA A Rs could be observed on neuronal processes. These were either co-localised with synaptotagmin and thus considered to be synaptic, or were otherwise considered to be extrasynaptic (39,40). Diffuse labelling was also present, likely reflecting the existence of a mobile extrasynaptic GABA A R population (41).…”