Adenosine and Seizure Termination: Endogenous Mechanisms

Commentary

Neuronal Adenosine Release, and not Astrocytic ATP Release, Mediates Feedback Inhibition of Excitatory Activity.

Lovatt D, Xu Q, Liu W, Takano T, Smith NA, Schnermann J, Tieu K, Nedergaard M. Proc Natl Acad Sci USA 2012;109:6265–6270

Adenosine is a potent anticonvulsant acting on excitatory synapses through A₁ receptors. Cellular release of ATP, and its subsequent extracellular enzymatic degradation to adenosine, could provide a powerful mechanism for astrocytes to control the activity of neural networks during high-intensity activity. Despite adenosine's importance, the cellular source of adenosine remains unclear. We report here that multiple enzymes degrade extracellular ATP in brain tissue, whereas only Nt5e degrades AMP to adenosine. However, endogenous A₁ receptor activation during cortical seizures in vivo or heterosynaptic depression in situ is independent of Nt5e activity, and activation of astrocytic ATP release via Ca²⁺ photolysis does not trigger synaptic depression. In contrast, selective activation of postsynaptic CA1 neurons leads to release of adenosine and synaptic depression. This study shows that adenosine-mediated synaptic depression is not a consequence of astrocytic ATP release, but is instead an autonomic feedback mechanism that suppresses excitatory transmission during prolonged activity.

Adenosine Release During Seizures Attenuates GABAA Receptor-Mediated Depolarization.

Ilie A, Raimondo JV, Akerman CJ. J Neurosci 2012;32:5321–5332

Seizure-induced release of the neuromodulator adenosine is a potent endogenous anticonvulsant mechanism, which limits the extension of seizures and mediates seizure arrest. For this reason, several adenosine-based therapies for epilepsy are currently under development. However, it is not known how adenosine modulates GABAergic transmission in the context of seizure activity. This may be particularly relevant as strong activation of GABAergic inputs during epileptiform activity can switch GABA_A receptor (GABA_AR) signaling from inhibitory to excitatory, which is a process that plays a significant role in intractable epilepsies. We used gramicidin-perforated patch-clamp recordings to investigate the role of seizure-induced adenosine release in the modulation of postsynaptic GABA_AR signaling in pyramidal neurons of rat hippocampus. Consistent with previous reports, GABA_AR responses during seizure activity transiently switched from hyperpolarizing to depolarizing and excitatory. We found that adenosine released during the seizure significantly attenuated the depolarizing GABA_AR responses and also reduced the extent of the after-discharge phase of the seizure. These effects were mimicked by exogenous adenosine administration and could not be explained by a change in chloride homeostasis mechanisms that set the reversal potential for GABA_ARs, or by a change in the conductance of GABA_ARs. Rather, A₁R-dependent activation of potassium channels increased the cell's membrane conductance and thus had a shunting effect on GABA_AR currents. As depolarizing GABA_AR signaling has been implicated in seizure initiation and progression, the adenosine-induced attenuation of depolarizing GABA_AR signaling may represent an important mechanism by which adenosine can limit seizure activity.

The purine ribonucleoside adenosine is a well-recognized endogenous anticonvulsant of the brain and therapeutic adenosine augmentation is a rational approach for seizure control. Levels of endogenous adenosine are known to rise during seizures and are thought to contribute to seizure arrest and postictal refractoriness (1, 2). Two important questions remain to be answered: First, where does endogenous adenosine come from and, second, how does seizure-induced adenosine-release influence those seizures?

To attenuate neuronal activity, adenosine needs to bind to G-protein coupled pre- and postsynaptic adenosine A₁ receptors (A₁Rs), which promote presynaptic inhibition through blockade of calcium channels and postsynaptic hyperpolarization through activation of potassium channels (3). Concordantly, genetic deficiency of the A₁R or enhanced metabolic clearance of adenosine increase neuronal excitability and promote seizures (4). Synaptic adenosine could originate from neurons or from adjacent astrocytes either directly or through degradation of its metabolic precursor ATP. Several studies have addressed the origin of synaptic adenosine. Synaptically released glutamate was found to increase synaptic adenosine via an N-methyl-d-aspartate mediated mechanism in at least two different brain regions (5, 6), whereas a genetic mouse model of impaired gliotransmission provided robust evidence for the vesicular release of ATP from astrocytes, and it was suggested that subsequent cleavage of astrocyte-derived ATP into adenosine provided the molecular basis for the tonic suppression of synaptic transmission and for activity-dependent, heterosynaptic depression (7).

The somewhat controversial source of synaptic adenosine was recently revisited in the study from Lovatt and colleagues. While several extracellular enzymes can degrade ATP into ADP and AMP, it was shown for the first time that only one enzyme—5'-ectonucleotidase (CD73 or Nt5e)—can degrade AMP into adenosine. Adenosine formation was almost completely abrogated in brain slices from CD73 knockout mice or when the enzyme was inhibited pharmacologically. Further, the authors have demonstrated that disruption or inhibition of CD73 did not alter latency to seizure onset or heterosynaptic depression induced by high-frequency stimulation, whereas deletion or blockade of the A₁R eliminated those functions. These findings led the authors to conclude that adenosine is not generated in the extracellular space from ATP but rather released directly. This conclusion was further corroborated by findings that selective activation of ATP release from astrocytes through photolysis of caged Ca²⁺ did not inhibit excitatory transmission. Thus, it seems that endogenous ATP released from astrocytes during highfrequency stimulation, or after uncaging of Ca²⁺, might not sufficiently raise adenosine at the right location to have a major impact on neuronal excitability. Using an elegant approach to selectively increase firing of a single excitatory neuron, Lovatt and colleagues finally demonstrated the direct neuronal release of adenosine. Importantly, adenosine release and presynaptic inhibition were not dependent on CD73 but were sensitive to transport blockade using an approach that competitively antagonized both types of equilibrative neucluoside transporters (ENTs) by administration of inosine through the patch pipette. These findings are conceptually intriguing since they suggest an inhibitory feedback mechanism by which overexcitation of neurons can be prevented by the release of sufficiently large quantities of adenosine in a highly localized manner. While astrocytes modulate neuronal activity through the release of ATP globally (7), they also play a key role in the metabolic clearance of adenosine (8). Adenosine homeostasis in the brain largely depends on astrocyte-based metabolic clearance of adenosine through adenosine kinase (ADK), and even minor changes in astrocyte ADK can produce major changes in adenosine and seizure susceptibility (8). Interestingly, ADK in the adult brain is mostly astrocyte-specific, whereas pyramidal neurons of the adult hippocampus lack ADK (8). The concentration gradient-driven release of adenosine from neurons through ENTs is likely possible only because neurons lack ADK. Thus, neuronal adenosine as a major source of synaptic adenosine might be a mechanism to permit highly localized actions of adenosine, whereas astrocytes—forming a major sink for metabolic clearance of adenosine—allow a directed flow of synaptic adenosine through ENTs into astrocytes, which might be a crucial mechanism to prevent spillover and to maintain the high local specificity afforded by neuronal adenosine release.

The paper from Ilie and colleagues addressed the question how seizure-derived adenosine, that is, adenosine derived most likely directly from “seizing neurons,” could contribute to the termination of seizures. In the adult brain, GABA_A receptor (GABA_AR) mediated Cl⁻ currents flow into the postsynaptic neuron, leading to hyperpolarization and inhibition of neurotransmission. However, due to excessive intracellular Cl⁻ accumulation, the neuronal Cl⁻ gradient can reverse during the course of a seizure and cause a transient switch in GABAergic signaling from inhibitory to depolarizing and excitatory (9, 10). Thus, GABAergic drugs become less effective and, in particular, during prolonged seizures, may aggravate seizures. Using an organotypic hippocampal seizure model in which single seizures were induced by transient washout of Mg²⁺, and gramicidin-perforated patch-clamp and current-clamp recordings from CA3 pyramidal neurons under conditions that preserved the intracellular Cl⁻ concentration, the authors first demonstrated that pharmacological blockade of adenosine receptors prolonged both the ictal period of a seizure as well as its afterdischarge, indicating that endogenous adenosine limited the extent of the seizure. Using puffs of muscimol to trigger postsynaptic GABA_AR responses during a seizure and current-clamp recordings, they next demonstrated that the activation of adenosine receptors by endogenous adenosine attenuated the GABA_AR mediated depolarization during epileptiform activity. The underlying mechanism for the attenuation of postsynaptic GABA_AR was found to depend on the increase of membrane conductance via the activation of K⁺-channels, which are downstream of the A₁R. Importantly, a cocktail of K⁺-channel blockers designed to block all major downstream targets of A₁Rs was found to abolish the effects of adenosine on GABA_ARs. Control experiments ruled out the alteration of Cl⁻ homeostasis or any effects on GABA_AR conductance.

Together, these publications suggest an attractive endogenous mechanism for the termination of seizure activity: Excessive neuronal activation, as occurs during a seizure, will lead directly to neuronal release of high enough amounts of adenosine that act in situ within the excited synapses. Since depolarizing GABA_AR-mediated signaling has been implicated in seizure initiation and progression, the adenosineinduced attenuation of depolarizing GABA_AR signaling may represent an important new mechanism by which adenosine can limit seizure activity. It becomes evident that pathophysiological conditions that limit the adenosine-based feedback inhibition, that is, excessive metabolic clearance of adenosine through astrocytes, will impede endogenous mechanisms of seizure termination and may ultimately permit the transition of a seizure into status epilepticus (SE). Whereas depolarizing GABA_AR-mediated signaling might be a plausible explanation for diazepam-resistance in SE, the augmentation of adenosine signaling might be a promising strategy to terminate drug-refractory SE. Further understanding of endogenous adenosine-based self-regulatory mechanisms, such as those discussed above, will be essential for the development of novel therapeutics that capitalize on endogenous regulatory feedback mechanisms.