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Treatment with the neurotransmitters glutamate, GABA or BzATP significantly increased superoxide production when compared with control un-treated cells. Glutamate significantly elevated superoxide production when used at 1 µM, which is within the physiological concentration range of 1-4 µM found at non-synaptic sites (Baker et al. 2002; Nyitrai et al.

2006). However, microdialysis has shown that glutamate is present at lower concentrations in the CNS, with levels of 25 nM reported in resting hippocampal slices (Herman & Jahr 2007).

It has however been shown that physiological stimulation of glutamate receptors occurs within the micromolar range, thereby indicating that the glutamate concentration used here may be physiological. Furthermore, the EC₅₀ for the NMDA receptor is reported to be 2.3 µM (Patneau & Mayer 1990), and the EC50 for the group I mGluRs is between 3-13 µM, for mGluR3 is 4-5 µM and for the group III mGluRs is between 0.02 and 1000 µM (Conn &

Pin, 1997), thereby suggesting that micromolar and therefore physiological levels of glutamate may promote the stimulation of microglial glutamate receptors. According to Herman & Jahr (2007) the concentration of glutamate used here may not lie within the basal range, and may be more representative of either physiological stimulation, or excitotoxic injury seen during ischaeimia and in AD, in which degenerating neurons and activated astrocytes release glutamate into the extracellular space (Vesce et al. 2007). Here, treatment

193 of BV2 microglia with lower levels of glutamate (100 nM) did not significantly elevate NBT reduction, suggesting activation of the microglial NADPH oxidase following exposure to glutamate may only occur under physiological stimulatory conditions or during pathology, rather than in response to basal glutamate levels.

GABA is present at extrasynaptic sites at a concentration of 2.9 µM (Lerma et al. 1986), whilst here, exposure of microglia to 100 µM GABA induced a more significant increase in NBT reduction, and subsequent superoxide production when compared with exposure to lower concentrations. Treatment of BV2 microglia with 1 µM GABA significantly elevated NBT reduction; however, as treatment with the higher concentration of 100 µM induced a more significant increase in NBT reduction, this was used in later experiments. These data do however suggest that microglia respond to physiological concentrations of GABA as well as higher concentrations, which may mimic the increase in GABA tone seen during AD pathology (DiFiglia 1990). GABA is released from neurons during AD (Marczynski 1998), and these elevated GABA levels exacerbate neuronal vulnerability (Erdö et al. 1991). It could be suggested that the GABA released in neurodegenerative processes could act on microglia to enhance reactivity and induce ROS production.

Whilst treatment of BV2 and primary microglia with GABA (100 µM) siginifcantly elevated superoxide production that could be inhibited by apocynin as determined by the NBT assay and by dHEth fluorescence microscopy, flow cytometry analysis showed that GABA induced dHEth fluoresenence could not be significantly attenuated upon co-treatment with apocynin, suggesting the involvement of another oxidase system or activation of other NADPH oxidase isoforms, such as Nox4, which are insensitive to apocynin (Harrigan et al. 2008).

Furthermore, HPLC analysis showed that GABA treatment significantly increased Eth production, indicative of H₂O₂ generation (Zielonka et al. 2008), which was significantly reduced by apocynin, demonstrating that GABA induces ROS, rather than superoxide

194 production through activation of the NADPH oxidase. The NADPH oxidase activity assay showed that enzymatic activity was increased upon treatment with GABA, which could be attenuated by co-treatment with apocynin, supporting the findings that GABA induced ROS production was a consequence of NADPH oxidase activity, however this finding does not rule out the possibility that GABA may induce the activation of additional NADPH oxidase isoforms.

Superoxide production from the NADPH oxidase isoforms Nox1 and Nox4, shown to be expressed in microglia (Harrigan et al. 2008; Chéret et al. 2008), is more rapidly dismutased to H₂O₂ than superoxide produced from Nox2 (Dikalov et al. 2008; Lassègue & Griendling 2010), which suggests that GABA induces microglial Nox1 or Nox4 activation. The findings could however also suggest that exposure of BV2 microglia to GABA may enhance the enzymatic activity or expression of SOD, which converts superoxide to H₂O₂ (Vaziri et al.

2004). This has been demonstrated in an in vivo model of rat renal failure, in which administration of GABA to the kidney increased SOD expression, which protected against oxidative damage through the enhanced production of H2O2 rather than superoxide (Sasaki et al. 2006). There are no reports of this in the CNS; however it could be an interesting point for further investigation.

Treatment of BV2 and primary microglia with 250 µM BzATP significantly increased superoxide production in line with published findings (Parvathenani et al. 2003; Skaper et al.

2006). BzATP was used in place of ATP, which was readily hydrolysed in the culture media, and had no effect on microglial reactivity or the production of superoxide in vitro (Skaper et al. 2006), however, ATP is released from dying neurons as an activating signal for microglia, and therefore exerts an effect in vivo (North & Verkhratsky 2006). Whilst BzATP induced superoxide production in primary and BV2 microglia, it did not significantly elevate NADPH oxidase activity. These findings therefore suggest that treatment of BV2 microglia with

195 BzATP may induce superoxide production through NADPH oxidase activation, and also by another superoxide generating system.

Superoxide production following treatment of BV2 microglia with BzATP was observed by HPLC analysis, showing a significant increase in 2-OH-E⁺, which was attenuated by apocynin. However, Eth production was also elevated, which could not be attenuated by co-treatment with apocynin, suggesting a low level of activity of another H2O2 generating system, or enhanced activation of the mitochondrial respiratory chain, leading to increased release of H2O2 (Rigoulet et al. 2011). There are reports that activation of microglia with Aβ promotes ATP release which activates the NADPH oxidase in an autocrine manner, elevating ROS production rather than superoxide specifically (Moon et al. 2008), which supports the elevated Eth seen here by HPLC. Furthermore, activation of the microglial P2X7 receptor with BzATP promotes TNFα production (Suzuki et al. 2004) which elevates ROS generation from the mitochondria (Goossens et al. 1999). It could therefore be suggested that whilst BzATP induces superoxide production through the NADPH oxidase, the production of TNFα as a consequence of increased microglial reactivity following activation of the P2X7 receptor could increase H2O2 production through mitochondrial pathways (Morgan & Liu 2010). In addition, TNFα release from microglia following activation of the P2X7 receptor (Suzuki et al. 2004) can feed-back onto microglial TNF receptors (TNFR‟s) in an autocrine manner (Kuno et al. 2005), resulting in elevated ROS production through enhanced expression and activity of the NADPH oxidase (Mir et al. 2009) and also mitochondrial pathways (Suzuki et al. 2004). The contribution of TNFα induced ROS, produced as a consequence of P2X7 receptor activation of microglia could explain why here, increased superoxide production is observed in the assays detecting ROS production, but that the NADPH oxidase activity assay shows a non-significant increase in enzymatic activity. The NADPH oxidase does indeed

196 play a role in BzATP induced production of superoxide in microglia, however the levels may be enhanced by TNFα induced activation of the mitochondrial respiratory pathway.

3.3.2 Modulation of neurotransmitter receptors induces superoxide production in