ANÁLISIS DE RESULTADOS

Because KA was not directly affecting microglia, we searched for an alternative mediator between seizures and microglia. The extracellular adenosine triphosphate (ATP) was

97

a strong candidate, as it is a well-known microglial chemoattractant (Davalos et al., 2005). Importantly, ATP is released by apoptotic cells to attract microglia (Elliott et al., 2009) but it is also released during seizures, either from neurons or from astrocytes (Dale and Frenguelli, 2009; Santiago et al., 2011). In the extracellular space ATP is rapidly degraded by ectonucleotidases to adenosine diphosphate (ADP) and adenosine monophosphate (AMP) (Zimmermann, 1999). Therefore, direct methods for measuring extracellular ATP such as the luciferin-luciferase assay (Crouch et al., 1993) or extracellular measurements with microelectrode biosensors (Heinrich et al., 2012; Llaudet et al., 2003) are very complicated to perform in a mouse brain in vivo. Thus, we resorted to indirectly determine the action of ATP released during seizures on microglia in vitro. For this purpose we used acute hippocampal slices and organotypic hippocampal slices and treated them with a seizure inducing epileptogenic cocktail. The cocktail contained high K+ _{which increases the extracellular amount}

of K+ _{inducing cell depolarization (Rutecki et al., 1985), and low Mg}2+_{, a physiological blocker of}

NMDA channels, which avoids their inactivation and induces seizures (Traub et al., 1994). The cocktail also contained 4-aminopyridine (4-AP), a non-selective blocker of voltage-dependent potassium channels, which acts inhibiting the uptake of extracellular potassium by astrocytes (Luhmann et al., 2000) (Figure 17 and Figure 18).

First, we assessed whether the seizures induced by the epileptogenic cocktail were affecting microglia via ATP. Microglia senses ATP via a plethora of receptors such as P2X ionotropic receptors and P2Y metabotropic receptors (Domercq et al., 2013). Thus, we used brilliant blue G (BBG), a broad purinergic P2X receptor antagonist to assess whether the seizure induced release of ATP was directly affecting microglia. For this purpose we used PND20-30 acute hippocampal slices treated with either epileptogenic cocktailin the presence or absence of BBG (Figure 17). We recorded field recordings to register seizures and assessed microglial currents by patch-clamp. We observed that the cocktail induced a depolarizing response in microglia with a latency of 11 ± 2min (Figure 17A-D). Importantly, this result is in accordance with data showing that after K+ induced depolarization the maximum ATP release is reached in approximately 9min in rat hippocampal slices (Heinrich et al., 2012). Furthermore, we observed that when BBG was applied alongside the cocktail the large inward currents in microglia were partially blocked (Figure 17A-D), indicating a cationic current through ATP regulated channels. To assess if BBG was altering the cocktail-induced seizures, we recorded the field potential before and after BBG and found that BBG did not alter the frequency or amplitude of the epileptic discharges (Figure 17E-G). Due to the complexity and workload involved, the complete pharmacology to assess the individual effect of all ATP, ADP

98

and adenosine receptors on microglial phagocytosis will be completed in the future. Overall, this data demonstrated that microglia sense seizures via ATP.

Figure 17. Seizures trigger ATP-mediated microglial activation. (A), Experimental design used to induce seizures in acute hippocampal slices with an epileptogenic cocktail that included high K+, low Mg2+ and 4-aminopyridine (4-AP) in ACSF, in the presence or absence of the broad P2X receptor antagonist BBG (5M). (B) Seizure activity was recorded in CA1, where it had higher amplitude than in the DG. Top, extracellular recording (mV) and bottom, simultaneous microglia patch clamp recording (pA) before and after seizure induction in the absence or in the presence of BBG. (C) Patch-clamp currents (in pA) induced in microglia by the seizure activity after the epileptogenic cocktail in the absence (control, n=18 cells) or presence of the P2X antagonist BBG (n=11 cells). (D) Latency (in minutes) of the currents induced in microglia by the seizure activity. (E) Extracellular recording of the seizure activity induced by the epileptogenic cocktail before and after the purinergic antagonist BBG was added in acute hippocampal slices. (F) Spike amplitude (in mV) induced by the epileptogenic cocktail. (G) Spike frequency (in Hz) induced by the epileptogenic cocktail. Bars represent mean ± SEM. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001 by Student´s t-test (B).

Finally, we determined whether the cocktail induced electrophysiological microglial response would correlate with a phagocytosis impairment. We treated hippocampal organotypic slices with the epileptogenic cocktail for 1h (Figure 18) and found that apoptotic cell numbers increased (Figure 18B). Similarly to what we observed in MTLE, the Ph index decreased (Figure 18C), while both the Ph capacity (Figure 18D, E) and microglia (Figure 18F) remained unchanged. Consequently, the Ph/A coupling was lost in the cocktail treated slices (Figure 18G). Thus, this data showed that seizures affected microglia via ATP, impairing microglial phagocytosis. These results further confirmed our in vivo data indicating that MTLE

99

seizures per se impaired phagocytosis and pointed towards ATP as the mediator of this phagocytosis impairment.

Figure 18. Microglial phagocytosis is acutely impaired in vitro due to seizures induced by a proepileptogenic coktail. (A) Experimental design and representative images of the DG of hippocampal organotypic slices treated with ACSF (control, n=3) or an epileptogenic cocktail (high K+, low Mg2+, 4-AP; n=3) for 1h. Normal or apoptotic (pyknotic/karyorrhectic) nuclear morphology was visualized with DAPI (white), microglia by the transgenic expression of fms-EGFP (cyan), and membrane permeability (characteristic of necrotic cells) by PI (red). High magnification inserts show details of phagocytosed apoptotic cells in the two conditions. Arrows, phagocytosed cells; arrowheads, non-phagocytosed cell. (B) Number of dead apoptotic cells in 200000µm3 of the DG in organotypic slices treated with the epileptogenic cocktail. (C) Ph index in organotypic slices (% of apoptotic cells phagocytosed) treated with the epileptogenic cocktail. Note that the Ph index in ACSF-treated slices is higher than in organotypic culture media-treated slices (Figure 7). (D) Weighted Ph capacity of microglia (in parts per unit, ppu). (E) Histogram showing the Ph capacity of microglia (in % of cells). (F) Number of microglial cells. (G) Ph/A coupling (in fold-change) in organotypic slices treated with the epileptogenic cocktail. Bars represent mean ± SEM. * indicates p<0.05 and ** p<0.01 by Student´s t-test. Scale bars=30µm.