4> 0.05 rotenone-succinate SBI-0206965 + MCS vs rotenone-succinate alone; = 5) (Fig. release, implicating mitochondrial H2O2 in release modulation. In contrast, inhibitors of MAO or Nox had LEPR no effect on dopamine release, suggesting a limited role for these metabolic enzymes in rapid H2O2 production in the SBI-0206965 striatum. These data provide the first demonstration that respiring mitochondria are the primary source of H2O2 generation for dynamic neuronal signaling. Introduction Beginning with Ramn y Cajal’s discovery of gaps between neurons (Ramn y Cajal, 1909), neurotransmission has been considered to be hard-wired, with point-to-point synaptic connections providing interneuronal communication. However, nonsynaptic communication by diffusion-based volume transmission (Fuxe and Agnati, 1991; Vizi, 2000) is also increasingly appreciated as playing a critical role. For example, dopamine, a key motor-system transmitter in the striatum, acts by volume transmission to activate predominantly extrasynaptic receptors after synaptic release (Sesack et al., 1994; Yung et al., 1995; Cragg and Rice, 2004; Rice and Cragg, 2008). In this context, an emerging diffusible messenger is the reactive oxygen species (ROS), hydrogen peroxide (H2O2) (Atkins and Sweatt, 1999; Avshalumov et al., 2003, 2007; Avshalumov and Rice, 2003; Kamsler and Segal, 2004). Importantly, H2O2 mediates the regulation of striatal dopamine release by the classical synaptic transmitter, glutamate (Avshalumov et al., 2003, 2008), in the absence of glutamate synapses or ionotropic receptors on dopaminergic axons (Smith and Bolam, 1990; Bernard et al., 1997; Bernard and Bolam, 1998; Chen et al., 1998). Evidence for H2O2 involvement in modulation of striatal dopamine release by glutamate comes from several avenues. Blockade of glutamatergic AMPA receptors (AMPARs) causes an increase in locally evoked dopamine release, which is prevented by the H2O2-metabolizing enzymes glutathione (GSH) peroxidase or catalase (Avshalumov et al., 2003). Conversely, locally evoked dopamine release is suppressed when H2O2 levels are amplified by inhibition of GSH peroxidase; this suppression is lost when AMPARs are blocked, demonstrating that modulatory H2O2 generation is glutamate dependent (Avshalumov et al., 2003). The mechanism of release inhibition by H2O2 is the activation of ATP-sensitive K+ (KATP) channels (Avshalumov and Rice, 2003; Avshalumov et al., 2008), and key cellular sources of modulatory H2O2 are striatal medium spiny neurons (MSNs) (Avshalumov et al., 2008). The subcellular source of H2O2 generation has been elusive, however. Three potential sources might contribute. The first is mitochondrial respiration, which produces superoxide anion (O2?) by the one-electron reduction of molecular oxygen (O2), with subsequent conversion of O2? to H2O2 by superoxide dismutase or spontaneous dismutation (Boveris et al., 1973; Adam-Vizi, 2005). The second is monoamine oxidase (MAO), which catalyzes deamination of dopamine through a two-electron reduction of O2 to H2O2 (Maker et al., 1981) and is expressed abundantly in striatum (Azzaro et al., 1985). The third is NADPH oxidase (Nox), a family of enzymes that catalyze the one-electron reduction of O2 to form O2? and consequently H2O2 (Lambeth, 2004; Infanger et al., 2006; Rhee, 2006; Bedard and Krause, 2007). Nox has been implicated in a variety of signaling pathways and is also found in striatum (Infanger et al., 2006; Kishida and Klann, 2007). Here, we examined contributions from these subcellular sources to rapid H2O2-dependent signaling. Synaptic release of dopamine was elicited by pulse-train stimulation in guinea-pig striatal slices; manipulation of mitochondrial H2O2 generation was monitored in MSNs using fluorescence imaging. The data show that mitochondrial respiration is the primary subcellular source of modulatory H2O2 and reveal an exquisite interplay among neuronal activity, mitochondrial respiration, and transmitter release, bridged by a unique signaling molecule, H2O2. Materials and Methods Brain slice preparation. All animal handling procedures were in accordance with National Institutes of SBI-0206965 Health guidelines and were approved by the New York University School of Medicine Animal Care and Use Committee. Young adult guinea pigs (male, Hartley, 150C250 g) were deeply anesthetized with 50 mg/kg (i.p.) pentobarbital and decapitated. For voltammetric recording, coronal brain slices (400 m) containing striatum were prepared as described previously (Chen and Rice, 2001; Avshalumov et al., 2003). In some experiments, ROS generation was monitored in striatal MSNs. Brain slices for these studies were prepared from animals that were perfused intracardially with ice-cold modified artificial CSF (ACSF) (Bao et al., 2005; Avshalumov et al., 2008). SBI-0206965 Slices were maintained in a holding chamber for at least 1 h at room temperature before experimentation in HEPES-buffered ACSF containing (in mm): 120 NaCl, 5 KCl, 20 NaHCO3, 6.7 HEPES acid, 3.3 HEPES salt, 2 CaCl2, 2 MgSO4,.