The extracellular space of the brain contains -aminobutyric acid (GABA) that activates extrasynaptic GABAA receptors mediating tonic inhibition. 1986; Lerma 1986; Ding 1998; Kuntz 2004). Extra- and peri-synaptic GABAA receptors (GABAARs) are in a NES favored position to be activated by the low levels of ambient GABA, due to their high GABA affinity in contrast to the lower affinity of synaptic GABAARs (Saxena & MacDonald, 1994; Mody, 2001; Brown 2002; Farrant & Nusser, 2005). The high-affinity extrasynaptic GABAARs consist of specific subunit combinations differentially expressed in various brain regions. These include the subunit-containing GABAARs of dentate gyrus and cerebellar granule cells, cortical and thalamic neurons (Nusser 1998; Sur 19992000; Nusser & Mody, 2002; Stell 2003; Sun 2004; Jia 2005; Cope 2005; Ruxolitinib enzyme inhibitor Drasbek & Jensen, 2006), and the 5 subunit-containing GABAARs in CA1 and CA3 pyramidal cells (PCs) (Sperk 1997; Caraiscos 2004; Glykys & Mody, 2006). The current mediated by these extrasynaptic receptors has been termed tonic inhibition (Brickley 1996; Farrant & Nusser, 2005), which is usually highly sensitive to the extracellular GABA concentration ([GABA]). It is enhanced when ambient [GABA] is usually increased by blocking GABA transporters, by adding GABA to the aCSF to mimic that normally present Ruxolitinib enzyme inhibitor in the extracellular space, or by preventing GABA degradation (Nusser & Mody, 2002; Stell & Mody, 2002; Wu 2003; Glykys Ruxolitinib enzyme inhibitor & Mody, 2006). Numerous sources have been proposed for the normal amounts of GABA found in the extracellular space. These include: astrocytic release (Liu 2000; Kozlov 2006), reversal of GABA transporter (Gaspary 1998; Ruxolitinib enzyme inhibitor Richerson & Wu, 2003), non-vesicular release as well as action potential-mediated release (Attwell 1993; Brickley 1996; Zoli 1999; Rossi 2003; Bright 2007). The cerebellar granule cells (CGCs) show a tonic current early in development that depends on action potential firing (Kaneda 1995; Brickley 1996). However, this source of extracellular GABA decreases during development and in adult CGCs tonic currents are mediated by action potential-independent mechanisms (Wall & Usowicz, 1997; Rossi 2003). Yet, the inhibitory inputs onto CGCs are a part of a unique synaptic structure, the glial ensheathed glomerulus. Thus, transmitter release, diffusion and overspill may be unique to this highly specialized structure (Brickley 1996; Wall & Usowicz, 1997; Rossi & Hamann, 1998; Mitchell & Silver, 2000). In the rest of the brain, where less specialized GABA synapses are the norm, it is unclear whether GABA released by action potential-dependent mechanisms can actually increase the level of tonic inhibitory current and be correlated with phasic inhibitory current. It has been recently shown in glomerular synapses of the dorsal lateral geniculate nucleus (dLGN) thalamic relay neurons that tonic inhibition does depend around the global level of inhibititory activity and vesicular release Ruxolitinib enzyme inhibitor (Bright 2007). In most cases a proper correlation between tonic and phasic currents cannot be established due to the low frequency of spontaneous inhibitory postsynaptic (phasic) currents (sIPSCs) recorded at room heat and because of the methods utilized for the analysis of phasic and tonic inhibitory currents. We have developed a method to simultaneously measure both mean currents (double knockout mice on C57/Bl6 background were obtained from our breeding colonies maintained by the UCLA Division of Laboratory Animal Medicine. Mice were anaesthetized with halothane and decapitated according to a protocol approved by the UCLA Chancellor’s Animal Research Committee. The brain was removed and placed in ice-cold artificial cerebrospinal fluid (aCSF) made up of (mm): NaCl (126), KCl (2.5), CaCl2 (2), MgCl2 (2), NaH2PO4 (1.25), NaHCO3 (26) and d-glucose (10) with pH 7.3C7.4 when bubbled.