Pannexin-1-mediated ATP release from area CA3 drives mGlu5-dependent neuronal oscillations

Abstract

The activation of Group I metabotropic glutamate receptors (GI mGluRs) in the hippocampus results in the appearance of persistent bursts of synchronised neuronal activity. Such activity is known to cause the release of the purines ATP and its neuroactive metabolite, adenosine.We have investigated the role of the purines in GI mGluR-induced oscillations in hippocampal areas CA3 and CA1 using pharmacological techniques and microelectrode biosensors for ATP and adenosine. The GI mGluR agonist DHPG induced both persistent oscillations in neuronal activity and the release of adenosine in areas CA1 and CA3. In contrast, the DHPG-induced release of ATP was only observed in area CA3. Whilst adenosine acting at adenosine A 1 receptors suppressed DHPG-induced burst activity, the activation of mGlu5 and P2Y 1 ATP receptors were necessary for the induction of DHPG-induced oscillations. Selective inhibition of pannexin-1 hemichannels with a low concentration of carbenoxolone (10 µM) or probenecid (1 mM) did not affect adenosine release in area CA3, but prevented both ATP release in area CA3 and DHPG-induced bursting. These data reveal key aspects of GI mGluR-dependent neuronal activity that are subject to bidirectional regulation by ATP and adenosine in the initiation and pacing of burst firing, respectively, and which have implications for the role of GI mGluRs in seizure activity and neurodevelopmental disorders.

Combined electrophysiological and biosensor approaches to study purinergic regulation of epileptiform activity in cortical tissue

“…This work has been conducted in wild-type and genetically-modified rodent parasagittal hippocampal and neocortical slices. A thickness of 600 µm is required for hippocampal slices, as opposed to the standard 300 -400 µm, as we find thinner rat slices do not support electrically-evoked seizure activity (Etherington and Frenguelli, 2004) or that induced by the GI mGluR agonist DHPG (Lopatar et al, 2015). We avoid hypoxia in the core of the slice by suspending the slice upon a mesh in the recording chamber and perfusing the submerged tissue on both sides, using gas-impermeable tubing and perfusing aCSF at ~6 ml/min (Etherington and Frenguelli, 2004;Wall and Richardson, 2015).…”

“…Indeed, in a more recent study, we have observed that the application of the GI mGluR agonist DHPG elicits burst firing in area CA1, which is associated with adenosine release from areas CA1 and CA3, but ATP release from only area CA3 (Figure 7) (Lopatar et al, 2015). Importantly, ATP release, but not the release of adenosine, was greatly suppressed by either probenecid or a low concentration of carbenoxolone, suggesting both a spatial and mechanistic dissociation between ATP and adenosine release, and the release of ATP via pannexin-1 hemichannels.…”

“…For example, signals on an ATP biosensor, if produced by ATP, would be predicted to be enhanced if the extracellular breakdown of ATP was inhibited, since more ATP will reach the biosensor. This was the case in the hippocampus where the ecto-ATPase inhibitor, ARL67156 enhanced the ATP signals measured in response to DHPG application (see below and (Lopatar et al, 2015). Conversely ATP signals should be reduced or abolished if the ecto-nucleotidase apyrase is applied.…”

“…Indeed, given the wide array of excitatory and inhibitory receptors for ATP (Burnstock et al, 2011), and the potential for their involvement in epilepsy (Kumaria et al, 2008), we recently investigated the possible release and role of ATP receptors hippocampal slices in different models (Lopatar et al, 2011;Lopatar et al, 2015). In the first model, using brief high-frequency stimulation of the Schaffer pathway in nominally Mg 2+ -free aCSF, we observed that broad spectrum antagonism of ATP P2X (ionotropic) and P2Y (GPCR) receptors had only a minor inhibitory effect on the duration and intensity of electrically-evoked seizures.…”

Modeling microelectrode biosensors: free-flow calibration can substantially underestimate tissue concentrations

“…Biosensors have been used to measure neurotransmitters and neuromodulators including glutamate (Hu et al 1994; Oldenziel et al 2006; Tian et al 2009), acetylcholine (Bruno et al 2006; Zhang et al 2010), adenosine triphosphate (Llaudet et al 2005; Frenguelli et al 2007; Gourine et al 2008; Lalo et al 2014; Lopatář et al 2015; Wells et al 2015), glucose (Lowry et al 1998; Dash et al 2013), adenosine, inosine, and hypoxanthine (Llaudet et al 2003; Klyuch et al 2012; Dale 2013; Van Gompel et al 2014; Wall and Richardson 2015; Frenguelli and Wall 2016). Many microelectrode biosensors developed for brain tissue use oxidative enzymes followed by detection via fixed-potential amperometry.…”