TY - JOUR
AU - Illes, Peter
AB - Abstract Patch-clamp recordings indicated the presence of P2X7 receptors at neural progenitor cells (NPCs) in the subgranular zone of the dentate gyrus in hippocampal brain slices prepared from transgenic nestin reporter mice. The activation of these receptors caused inward current near the resting membrane potential of the NPCs, while P2Y1 receptor activation initiated outward current near the reversal potential of the P2X7 receptor current. Both receptors were identified by biophysical/pharmacological methods. When the brain slices were prepared from mice which underwent a pilocarpine-induced status epilepticus or when brain slices were incubated in pilocarpine-containing external medium, the sensitivity of P2X7 and P2Y1 receptors was invariably increased. Confocal microscopy confirmed the localization of P2X7 and P2Y1 receptor-immunopositivity at nestin-positive NPCs. A one-time status epilepticus in rats caused after a latency of about 5 days recurrent epileptic fits. The blockade of central P2X7 receptors increased the number of seizures and their severity. It is hypothesized that P2Y1 receptors after a status epilepticus may increase the ATP-induced proliferation/ectopic migration of NPCs; the P2X7 receptor-mediated necrosis/apoptosis might counteract these effects, which would otherwise lead to a chronic manifestation of recurrent epileptic fits. neural progenitor cells, P2X7 receptors, P2Y1 receptors, status epilepticus, subgranular zone Introduction The main neurogenic regions (niches) of the adult mammalian brain are the subependymal zones of the lateral ventricles also called subventricular zones (SVZ) and the subgranular zone (SGZ) of the dentate gyrus (DG) of the hippocampus (Mu et al. 2010; Ming and Song 2011; Urban and Guillemot 2014). The newborn neuronal cells originate from adult neural progenitor cells (NPCs), which are defined by their ability to self-replicate and differentiate into multiple neural lineages, including neurons, astrocytes, and oligodendrocytes. Recent data revealed significant differences between the brains of laboratory rodents and humans with regard to the extent and magnitude of neurogenesis; whereas neurogenesis is substantial in the human DG, it appears to be either absent in the human SVZ or is channeled towards the generation of neurons that migrate to the striatum rather than to the olfactory bulb (Sanai et al. 2011; Wang et al. 2011; Jessberger and Gage 2014). The SGZ of the rodent hippocampus, where the adult progenitors of the DG are found, lines the hilar side of the granule cell layer of the dentate gyrus. The developmental stages of these NPCs from radial glia-like progenitors (Type 1 cells) to transit-amplifying cells (Type 2 cells) and eventually neuroblasts, which mature to granule neurons of the dentate gyrus, are largely analogous to their counterparts in the SVZ (Parent 2007; Zhao et al. 2008). In view of the proven significance of the hippocampus for cognitive functions such as learning and memory, it was of special interest to find out that stimulation of SGZ neurogenesis with behavioral interventions such as exercise or environmental enrichment is associated with better performance on certain hippocampal learning tasks (Bruel-Jungerman et al. 2005; van Praag et al. 2005). It has been suggested that this is due to the increased synaptic integration of newborn neurons in the adult hippocampus, offering an expanded capacity for plasticity of shaping the existing circuitry in response to experience throughout life (Laplagne et al. 2006; Ge et al. 2008). However, pathological events (seizures, ischemia, and brain injury) may also result in transiently increased neurogenesis (Parent et al. 1997; Liu et al. 1998; Dash et al. 2001). In particular, the consequences of status epilepticus (SE) and the resulting extensive hippocampal remodeling, including reorganization of mossy fibers, dispersion of the granule cell layer and the appearance of granule cells in ectopic hilar regions has extensively been investigated (Jessberger et al. 2007; Kuruba et al. 2009; Kokaia 2011). Apparently, SE leads to increased NPC proliferation starting within a day after seizures and sustained for many months afterwards (Shapiro et al. 2007; Varodayan et al. 2009) accompanied by recurrent chronic epileptic fits (Bouilleret et al. 1999). During epileptic seizures, large amounts of nucleotides are released into the extracellular space because of metabolic limitation of the neurons and glial cells (Dale and Frenguelli 2009). These nucleotides activate a range of ionotropic (P2X) and metabotropic (P2Y) receptor classes (Burnstock et al. 2011). Certain subtypes of either receptor class, especially the P2X7 and P2Y1 receptors occur at cultured embryonic or adult NPCs and appear to regulate their functions causing in the first line necrosis/apoptosis (P2X7; Ulrich and Illes 2014) as well as proliferation/differentiation/migration (P2Y1; Illes et al. 2013). We asked ourselves, whether in situ SGZ NPCs are endowed with functional P2X7 and P2Y1 receptors and whether these receptors participate in the chronic manifestation of acute epileptic seizures. We concluded on the basis of our present electrophysiological, immunohistochemical, and behavioral study that nucleotides might induce via the activation of hypersensitive P2X7 receptors the necrosis/apoptosis of excess proliferating SGZ NPCs and thereby decrease the chances of the chronic manifestation of a one-time SE. Materials and Methods Experimental Animals Brain slices were prepared from mice of either sex (10–20 days or 8–10 weeks old) overexpressing green fluorescent protein under the control of the nestin [Tg(nestin/EGFP] or p2rX7 [Tg(P2X7/EGFP] genes, which were gifts of Dr Helmut Kettenmann, Berlin, Germany and Dr Teresa Miras-Portugal, Madrid, Spain, respectively. In addition, we used adult male Wistar rats (8–10 weeks old). All experiments carried out in Leipzig on mice or in Sao Paulo on rats were in accordance with the national guidelines for the use of animals in biomedical research. Induction of Seizures Seizures were induced in Tg(nestin/EGFP) mice by the application of pilocarpine hydrochloride (300 mg/kg i.p.), 30 min following the injection of scopolamine methyl bromide (1 mg/kg i.p.). Some mice were injected with kainic acid (30 mg/kg i.p.) instead of pilocarpine. Control mice obtained instead of scopolamine/kainic acid the respective volume of saline (0.9% NaCl). The seizures were evaluated using a modified version of the Racine scale (Shibley and Smith 2002). Mice which obtained SE or saline were 5 h later treated with midazolam (20 mg/kg i.p.). Control mice and mice that reached stages 4 and 5 of the Racine scale were sacrificed 24 h after saline-, pilocarpine-, or kainic acid-treatment for either electrophysiological or immunohistochemical investigations. For intracerebroventricular (i.c.v.) infusion, rats were anesthetized with ketamine (90 mg/kg i.p) and xylazine (10 mg/kg i.p.), and fixed to a stereotaxic apparatus for bilateral microcannula (12 mm in length and 0.55 mm in diameter) implantation. Microcannulas were positioned in the lateral ventricles according to the atlas of Paxinos and Watson (2006) (stereotactic coordinates: anterior–posterior, −0.08 mm; medial-lateral, ±0.14 mm; depth, −0.3 mm, posterior to bregma), and fixed to the skull with dental cement. Ten days later, rats underwent pilocarpine-induced epilepsy. Seizures were induced in rats by using a similar procedure as that used in mice. Following 5 h of the onset of SE caused by i.p. pilocarpine (370 mg/kg, i.p.), behavioral manifestations were alleviated with diazepam (1 mg/kg s.c.) and pentobarbital (30 mg/kg i.p.). Control rats received the same doses of diazepam and pentobarbital, 5 h after saline injection. Brain Slice Preparation, Whole-Cell Patch-Clamp Recordings, Drug Application Protocols Coronal hippocampal slices of Tg(nestin/EGFP) mice were prepared and superfused in an organ bath with oxygenated (95% O2 + 5% CO2) artificial cerebrospinal fluid (aCSF; 3 mL/min, room temperature) of the following composition (in mM): NaCl 126, KCl 2.5, CaCl2 2.4, MgCl2 1.3, NaH2PO4 1.2, NaHCO3 25, and glucose 11; pH 7.4. Neuroblasts were investigated in the rostral migratory stream (RMS); for these experiments corticostriatal slices including the RMS were prepared (Wang et al. 2003). To create low divalent cationic (low X2+) solution, MgCl2 was omitted from the medium and the CaCl2 concentration was decreased to 0.5 mM. Cells were visualized with an upright interference contrast microscope and 40× water immersion objective (Axioskope FS, Carl Zeiss). Patch pipettes were filled with intracellular solution of the following composition (in mM): K-gluconic acid 140, NaCl 10, MgCl2 1, HEPES 10, EGTA 11, Mg-ATP 1.5, Li-GTP 0.3; pH 7.2 adjusted with KOH. Membrane currents from neuroblasts were recorded by means of amphotericin B-perforated patches (Rae et al. 1991). Pipettes (4–7 MΩ resistances) were pulled by a horizontal micropipette puller (P-97; Sutter Instruments) from borosilicate capillaries. The resting membrane potential (Vm) of all cells was measured in the current-clamp mode of the patch-clamp amplifier (Multiclamp 700A; Molecular Devices) immediately after establishing whole-cell access. Current-step protocols, starting with −80 pA hyperpolarizing current and continuing with hyper- and then depolarizing current steps in 40 pA increments of 500 ms duration were applied. Afterwards, recording was continued in the voltage-clamp mode of the amplifier, the holding potential was set to −80 mV, and voltage steps of 500 ms duration were applied in the range of −100 to +50 mV, in 5 mV increments. Membrane capacitance (Cm), resistance (Rm), and the access resistance (Ra) were monitored before and during individual experiments. Cells, where the Ra values varied >20%, were discarded. When P2Y receptor ligands were investigated, the holding potential was stepped during agonist application to −30 mV for 60 s. pClamp 10.2 software (Molecular Devices) was used to store the recorded data for offline analysis/filtering and to trigger the fast application system. Agonists and antagonists were pressure injected locally by means of a computer-controlled DAD-12 superfusion system (Adams and List). Behavioral Experiments AZ10606120, a negative allosteric modulator of P2X7 receptors, was dissolved in 0.9% saline. Two microliters of drug solution were applied at increasing concentrations (1 µL/min flow rate) into both lateral cerebral ventricles, 30 min after injecting pilocarpine, or in control experiments saline (see Chapter “Induction of seizures”). Brilliant Blue G (BBG) was also dissolved in 0.9% saline and was applied intraperitoneally, 30 min after injecting pilocarpine (Pilo), or in control experiments saline. Four groups of rats were studied in the experiments with AZ 10606120 (n = 6 animals per each group): 1) saline + saline group: rats received saline (i.c.v.), 30 min after intraperitoneal saline injection; 2) saline + AZ group: rats received 3 μg of AZ10606120 (i.c.v.), 30 min after intraperitoneal (i.p.) saline injection; 3) Pilo + saline group: rats received saline (i.c.v.), 30 min after pilocarpine (i.p.) injection; 4) Pilo + AZ group: rats received 3 μg of AZ10606120 (i.c.v.), 30 min after pilocarpine (i.p.) injection. A slightly modified procedure was adapted in the experiments with Brilliant Blue G (n = 6 animals per each group): 1) saline + saline group: rats received saline (i.p.); 30 min later i.p. saline was injected again and this injection was repeated the following 4 days each day once. 2) Saline + BBG group: rats received saline (i.p.); 30 min later 50 mg/kg of BBG (i.p.) was injected and this injection was repeated the following 4 days each day once. 3) Pilo + saline group: rats received pilocarpine (i.p.); 30 min later i.p. saline was injected and this injection was repeated the following 4 days each day once. 4) Pilo + BBG group: rats received pilocarpine (i.p.); 30 min later BBG (50 mg/kg; i.p.) was injected and this injection was repeated each day once for the following 4 days. Rats injected with saline instead of pilocarpine (i.p.) and subsequently with saline i.c.v. or i.p. (group 1) exhibited no spontaneous seizures. The results obtained in rats receiving pilocarpine (i.p.) and subsequently saline i.c.v. or i.p. (group 2) did not depend on the route of saline application and were therefore pooled (n = 12 animals). Forty-eight hours after the onset of SE, animals from the Pilo + saline, Pilo + AZ, and Pilo + BBG groups were placed in a video monitoring room, where they were observed for 28 days, 24 h per day. A video recording system (VC 16E 480C, Intelbras) was used to register seizures. The behavioral parameters analyzed were the 1) latency to the first spontaneous seizure, 2) the number of seizures and 3) the severity of seizures estimated by their distribution according to the different stages of the Racine scale. Immunohistochemistry Tg(nestin-EGFP) mice were transcardially perfused under deep CO2-anesthesia with 4% paraformaldehyde (PFA) solution in PBS (pH 7.4), 24 h after pilocarpine-induced status epilepticus. Untreated Tg(nestin-EGFP) mice were used as controls. Their brains were removed and 50 µm thick hippocampal slices were cut at the next day. The slices were incubated with rabbit anti-P2X7 (1:500; Alomone), in blocking solution for 24 h at 4°C. After washing, the slices were incubated with the corresponding secondary antibody, biotinylated donkey antirabbit IgG (1:65; Vector Laboratories) for 2 h at room temperature. For the detection of the P2X7 receptor antibodies, the streptavidin/biotin technique (ABC-Kit Vectastain®1:50; Vector Laboratories) and the 3,3′-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich, 0.07%) reaction were used. The immunoreactivity was imaged using a light microscope (Axiovison; Zeiss). Control experiments were carried out without the primary antibody or by preadsorption of the antibody with the immunizing peptide. Immunofluorescence Labeling and Fluoro Jade-B Staining Tg(nestin-EGFP) mice were handled 24 h after pilocarpine-induced SE (or after saline injection), as described above. Brain slices were incubated with an antibody mixture of mouse anti-EGFP (1:100; Clontech), goat anti-GFAP (glial fibrillary acidic protein; 1:300; Santa Cruz), and rabbit anti-P2X7 (1:500; Alomone), or rabbit anti-P2Y1 (1:800; Alomone), together with Triton X-100 and 5% fetal calf serum (FCS) in Tris-buffered saline (TBS) for 48 h at 4°C. For the simultaneous visualization of the different primary antibodies, mixtures of secondary antibodies specific for the appropriate species IgG (rabbit, mouse, and goat) were applied. Carbocyanin (Cy)2- (1:400), Cy3- (1:800), and Cy5- (1:200)-conjugated donkey IgGs (from Jackson ImmunoResearch) diluted in 0.3% Triton X-100 and 5% FCS in TBS were applied, respectively, for 2 h at room temperature. Cell nuclei were stained with Hoechst 33 342 (Molecular Probes). Control experiments were performed without primary antibodies or by preadsorption of the antibody with the immunizing peptides. The triple immunofluorescence was investigated by a confocal laser scanning microscope (LSM 510 Meta; Carl Zeiss) using excitation wavelengths of 488 nm (argon; yellow–green Cy2-immunofluorescence), 543 nm (helium/neon; red Cy3-immunofluorescence), and 633 nm (helium/neon2; blue Cy5-labeling). All immunohistochemistry experiments were repeated on at least 5 mice. Rats subjected to an i.p. pilocarpine- or saline injection, received AZ10606120 30 min later, or again saline i.c.v. Then, after a further 5 h they obtained diazepam (1 mg/kg s.c.) and pentobarbital (30 mg/kg i.p.). After 24 h of i.p. pilocarpine or saline application (n = 6 per group), these rats were transcardially perfused with 4% paraformaldehyde (PFA) solution in PBS (pH 7.4). 40 µm thick coronal slices of the hippocampus were stained according to standard protocols with Fluoro Jade-B (FJ-B) for further analysis. FJ-B labels degenerating neurons with a green fluorescent color. Photomicrographs were made with a fluorescence microscope (Olympus BX51); images were captured by Image-Pro software (Media-Cybernetics) and analyzed in Image J (NIH). Materials The following drugs were used: kainic acid (Abcam); pentobarbital sodium (Abbot); (N-[2-[[2-[(2-hydroxyethyl)amino]ethyl]amino]-5-quinolinyl]-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide dihydrochloride (AZ10606120, Astra-Zeneca); midazolam (Ratiopharm); diazepam (Santisa); 2′(3′)-O-(4-benzoylbenzoyl)adenosine-5′-triphosphate tri(triethylammonium) salt (BzATP), adenosine 5′-O-(2-thiophosphate) trilithium salt (ADP-β-S), adenosine 5′-triphosphate disodium salt hydrate (ATP), Brilliant Blue G (BBG), N-methyl-d-aspartic acid (NMDA), pilocarpine hydrochloride, scopolamine methyl bromide, uridine 5′-diphosphate sodium salt (UDP), uridine 5′-triphosphate sodium salt (UTP) (Sigma–Aldrich); (S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (S-AMPA), 2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate tetrasodium salt (MRS 2179), N,N″-1,4-butanediylbis[N′-(3-isothiocyanatophenyl)thiourea (MRS 2578), 3-[[5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl]methyl]pyridine hydrochloride (A-438079), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), gabazine, D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5), muscimol, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid tetrasodium salt (PPADS), tetrodotoxin (TTX) (Tocris Bioscience). The pH value of the ATP-containing aCSF superfused onto the cells under investigation was adjusted by NaOH to 7.4. Sterile saline (0.9% NaCl) was used as a vehicle for drug administration and placebo. Statistics Concentration–response curves were fitted using the Hill equation: I=ImaxCnHEC50×CnH where I is the peak current produced by agonist, Imax is the maximal current at saturating agonist concentration, nH is the Hill slope value, and EC50 is the concentration of agonist needed to produce 50% of Imax (OriginPro 8G; OriginLab). Means ± SEM are given throughout. Multiple comparisons between electrophysiological data were performed by one way analysis of variance (ANOVA) followed by the Holm–Sidak test. Two datasets were compared using the Student t-test. Comparison with a theoretical value of zero was made by using the Mann–Whitney test. Behavioral data (latency to the first seizure and the number of seizures) were shown as box plots (median, first and third quartiles, maximum, and minimum values). Statistical significance was determined by the unpaired Student's t-test. The number of seizures in different stages of the Racine scale was analyzed by two-way ANOVA followed by the Bonferroni t-test. In all cases, a probability level of 0.05 was considered to be statistically significant. Results Electrophysiological Characterization of NPCs, Neuroblasts, and Granule Cells In the SGZ, NPCs were identified on the basis of their EGFP-fluorescence (Supplementary Fig. 1F,G). These cells were considered to be non-neuronal because of their failure to spike in response to depolarizing current injection (Supplementary Fig. 1A,B). In contrast, granule cells in the nearby DG fired a single action potential or series of action potentials depending on their state of maturation (Supplementary Fig. 1Da,Ea; Pedroni et al. 2014). Only those NPCs were chosen for recording, which exhibited strong EGFP-fluorescence (Mignone et al. 2004). In the present experiments, voltage-clamp protocols identified two subclasses of EGFP-bright NPCs: Type I cells of the SGZ exhibited nearly linear I/V curves and thereby passive membrane properties (Supplementary Fig. 1Ab,c), while Type II cells were outwardly rectifying (Supplementary Fig. 1Bb,c). Moreover, Type 2 cells had lower Vm and Cm values and higher Rm and Rs values than Type 1 cells (Supplementary Table 1). Because of the small size and fragility of neuroblasts, we decided to use the perforated patch technique to record the membrane currents of these cells. Interestingly, hyperpolarizing current pulses elicited in all neuroblasts unexpectedly large voltage deflections (Supplementary Fig. 1Ca), which, however, correspond to their outwardly rectifying properties and expectedly high membrane resistances (Supplementary Fig. 1Cb,c). Immature and mature granule cells had much higher Rm values than Type 1 NPCs, which were in the range of the Rm values determined for Type 2 NPCs (Supplementary Table 1). Immature granule cells fired only a single action potential in response to strong depolarizing current injection, whereas mature granule cells had considerably lower firing thresholds and reacted with series of action potentials to depolarization (Supplementary Fig. 1Da,Ea). Whole-cell patch-clamp recordings-documented corresponding action currents to depolarizing voltage steps (Supplementary Fig. 1Db,Eb). P2X7 Receptor-Mediated Current Responses in NPCs, Neuroblasts, and Granule Cells We locally applied by means of a fast superfusion system identical concentrations of the general P2 receptor agonist ATP (1 mM; Fig. 1Aa) or the prototypic P2X7 receptor agonist Bz-ATP (300 µM; Fig. 1Ab), both in the presence of normal aCSF, and aCSF containing no Mg2+ and a low concentration of Ca2+ (0.5 mM; low X2+ aCSF). Agonist application was for 10 s every 2 min. As compared with normal aCSF, the ATP- and Bz-ATP-induced current responses greatly increased in low X2+ aCSF (Fig. 1Ac). Although the membrane parameters of Type 1 and 2 NPCS significantly differed from each other (Supplementary Table 1), all cells responded to the purinergic agonists with similar current amplitudes and the results obtained were therefore pooled. Furthermore, in this series of experiments and in all further ones, we never detected cells which were insensitive to ATP/Bz-ATP in low X2+ medium. Because of the potentiation of the nucleotide effects in low X2+ aCSF, all further electrophysiological experiments were carried out under these conditions, except when explicitly stated. Figure 1. Open in new tabDownload slide Open in new tabDownload slide ATP- and BzATP-induced currents of NPCs; pharmacological and biophysical characterization. Individual agonist concentrations were applied every 2 min for 10 s. The holding potential in the whole-cell recording mode was −80 mV. (A) Representative tracings from individual cells stimulated by ATP (1 mM; a) and Bz-ATP (300 µM; b), in normal or low X2+ bath medium, respectively. Percentage mean ± SEM increase of the fourth current amplitude in comparison with the second current amplitude in a series of applications (c). *P < 0.05; statistically significant difference from 100% (filled column). The number of experiments is indicated in each column. (B) Concentration–response relationships for ATP (100–30 000 µM; a) and Bz-ATP (30–3000 µM; b) in a low X2+ bath medium; representative single cells. Mean ± SEM current responses to Bz-ATP and ATP of the indicated number of cells (c). (C) Current responses to six consecutive applications of BzATP (300 µM) at different holding potentials (−90; −60; −30; 0; 30; 60 mV). Representative recordings from 1 NPC (a). Mean ± SEM of the peak current amplitudes of 8 cells are plotted at each holding potential (b); the reversal potential is near 0 mV, indicating the existence of a nonselective cationic current. (D) Gradual depression of BzATP (300 µM)-induced currents in the presence of increasing concentrations of A-438079 (1; 10 µM). Representative recording from 1 NPC (a). Percentage mean ± SEM decrease of the current amplitudes from the second (no antagonist; 100%) to the fourth (1 µM) and sixth (10 µM) application of Bz-ATP (300 µM) in the presence of the indicated concentrations of A-438079 (b). *P < 0.05; statistically significant difference from 100%. (E) Effect of Bz-ATP (300 µM) at an NPC in the whole-cell mode and at an excised patch prepared by slowly withdrawing the patch pipette from the same cell. Representative recording from 1 NPC (a). Mean ± SEM of Bz-ATP (300 µM)-induced currents (b). *P < 0.05; statistically significant difference between the 2 columns. (F) Stability of Bz-ATP (300 µM) current responses in the absence and the presence of an antagonist cocktail (AP-5, 50 µM; CNQX, 20 µM; gabazine, 10 µM; TTX, 0.5 µM). Representative recording from a single cell in the presence and the absence of this antagonist cocktail (a). Percentage mean ± SEM of the change from the second (control) to the fourth (antagonist cocktail) and sixth (washout) current amplitude (b). *P < 0.05; statistically significant differences between the second and third column. (G) Excitatory (NMDA, 100 µM; AMPA, 100 µM) and inhibitory (muscimol, 100 µM) amino acids caused inward currents in the NPCs; Bz-ATP (300 µM) had a similar effect. Representative recordings from a single cell (a) and mean ± SEM of the agonist-induced current amplitudes (b). The number of cells is indicated in each column. Figure 1. Open in new tabDownload slide Open in new tabDownload slide ATP- and BzATP-induced currents of NPCs; pharmacological and biophysical characterization. Individual agonist concentrations were applied every 2 min for 10 s. The holding potential in the whole-cell recording mode was −80 mV. (A) Representative tracings from individual cells stimulated by ATP (1 mM; a) and Bz-ATP (300 µM; b), in normal or low X2+ bath medium, respectively. Percentage mean ± SEM increase of the fourth current amplitude in comparison with the second current amplitude in a series of applications (c). *P < 0.05; statistically significant difference from 100% (filled column). The number of experiments is indicated in each column. (B) Concentration–response relationships for ATP (100–30 000 µM; a) and Bz-ATP (30–3000 µM; b) in a low X2+ bath medium; representative single cells. Mean ± SEM current responses to Bz-ATP and ATP of the indicated number of cells (c). (C) Current responses to six consecutive applications of BzATP (300 µM) at different holding potentials (−90; −60; −30; 0; 30; 60 mV). Representative recordings from 1 NPC (a). Mean ± SEM of the peak current amplitudes of 8 cells are plotted at each holding potential (b); the reversal potential is near 0 mV, indicating the existence of a nonselective cationic current. (D) Gradual depression of BzATP (300 µM)-induced currents in the presence of increasing concentrations of A-438079 (1; 10 µM). Representative recording from 1 NPC (a). Percentage mean ± SEM decrease of the current amplitudes from the second (no antagonist; 100%) to the fourth (1 µM) and sixth (10 µM) application of Bz-ATP (300 µM) in the presence of the indicated concentrations of A-438079 (b). *P < 0.05; statistically significant difference from 100%. (E) Effect of Bz-ATP (300 µM) at an NPC in the whole-cell mode and at an excised patch prepared by slowly withdrawing the patch pipette from the same cell. Representative recording from 1 NPC (a). Mean ± SEM of Bz-ATP (300 µM)-induced currents (b). *P < 0.05; statistically significant difference between the 2 columns. (F) Stability of Bz-ATP (300 µM) current responses in the absence and the presence of an antagonist cocktail (AP-5, 50 µM; CNQX, 20 µM; gabazine, 10 µM; TTX, 0.5 µM). Representative recording from a single cell in the presence and the absence of this antagonist cocktail (a). Percentage mean ± SEM of the change from the second (control) to the fourth (antagonist cocktail) and sixth (washout) current amplitude (b). *P < 0.05; statistically significant differences between the second and third column. (G) Excitatory (NMDA, 100 µM; AMPA, 100 µM) and inhibitory (muscimol, 100 µM) amino acids caused inward currents in the NPCs; Bz-ATP (300 µM) had a similar effect. Representative recordings from a single cell (a) and mean ± SEM of the agonist-induced current amplitudes (b). The number of cells is indicated in each column. Increasing concentrations of ATP (100–30 000 µM; Fig. 1Ba) and Bz-ATP (30–3000 µM; Fig. 1Bb) were superfused (10 s application, 2-min intervals) onto NPCs in order to construct concentration–response curves (Fig. 1Bc). Although the concentration–response curves did not reach a clear maximum for either agonist, we did not investigate the effects of concentrations higher than 3 mM for Bz-ATP and 30 mM for ATP. Our aim was to avoid possible cell-damage of the NPCs which could be demonstrated, albeit on a longer time-scale, in cultured SVZ NPCs in previous experiments (Messemer et al. 2013). Due to the lack of an unequivocal maximal effect, the generated Hill plots for ATP (Imax = −1225.9 ± 459.3 pA, EC50 = 1009.7 ± 531.0 µM, nH = 1.8 ± 0.3; n = 6) and Bz-ATP (Imax = −1297.5 ± 77.8 pA, EC50 = 7423.3 ± 1354.5 µM, nH = 1.3 ± 0.1; n = 7) are only of limited significance. Nonetheless, it can be concluded that in accordance with our expectations, Bz-ATP appears to be more efficient than ATP and a low X2+ medium (see above) markedly potentiates the nucleotide effects. Application of Bz-ATP (300 µM) at various holding potentials (Fig. 1Ca) and the resulting I/V curve (Fig. 1Cb) indicated a reversal potential of the current responses at −1.2 ± 6.0 mV (n = 6). This value did not differ in a statistically significant manner from the reversal potential of nonselective cationic currents at 0 mV (P > 0.05). A further argument for the involvement of P2X7 receptors was supplied by the use of the highly selective antagonist A-438079 (Fig. 1D). In fact, increasing concentrations of A-438079 (1, 10 µM) gradually decreased the effect of Bz-ATP (300 µM). It is interesting to note that this effect was completely reversible on washout. A remaining question to be answered is, whether the P2X7 receptor is located at the SGZ NPCs themselves or at neighboring cells (neurons, glial cells, endothelium) which release after stimulation by ATP a nonidentified signaling molecule/transmitter onto NPCs. After determining the Bz-ATP (300 µM)-induced current (Fig. 1Ea), we slowly withdrew the patch pipette to terminate the whole-cell recording condition and to separate the excised patch from the surrounding cells. However, when the same concentration of Bz-ATP was superfused around the tip of the patch pipette, the current response only decreased but was in no way abolished (Fig. 1Ea,b). Of course, it cannot be excluded that in some cases the NPC was only detached from its surrounding tissue rather than preparing an excised patch. Another approach was also utilized to show that the effect of Bz-ATP (300 µM) did not depend on a P2X7 receptor-mediated release of a signaling molecule/transmitter. In fact, the Bz-ATP-induced current was not altered in the presence of a cocktail containing antagonists for NMDA receptors (AP-5; 50 µM), non-NMDA receptors (CNQX; 20 µM), GABAA receptors (gabazine; 10 µM), and a blocker of Na+-dependent action potentials (TTX; 0.5 µM) (Fig. 1Fa,b). In this way, we aimed at excluding the effects of glutamate and GABA via their ionotropic receptors, as well as effects via the action potential-induced release of further neuronal transmitters. In view of the stability of the current amplitudes caused by Bz-ATP (300 µM) in the presence of such a cocktail (Fig. 1Fa,b; percentage change of the 5th current in comparison with the 2nd current was 14.9 ± 6.3%, n = 6, P > 0.05), it can be concluded that Bz-ATP acts directly at P2X7 receptors localized at the NPC plasma membrane. Of course, it was important to find out, whether the assumedly blocked receptors are actually present at NPCs. For this purpose we applied NMDA, AMPA, and muscimol (100 µM each) in random order to stimulate NMDA, non-NMDA, and GABAA receptors, respectively (Fig. 1Ga). All three agonists induced current responses that did not differ either from each other or from the Bz-ATP (300 µM)-induced current when compared by ANOVA (Fig. 1Gb). However, under these conditions, Bz-ATP caused a more rapidly declining current than the other agonists and the effect of muscimol appeared to be smaller than that of Bz-ATP when calculated by the Student t-test (P < 0.05). In neuroblasts (Supplementary Fig. 2Aa,b; normal, −7.6 ± 0.8 pA; low X2+, −22.8 ± 2.3 pA; n = 6), Bz-ATP at 300 µM caused currents of lower amplitude than in NPCs (e.g., Fig. 1Ab; normal, −13.8 ± 3.6 pA; low X2+, 163.9 ± 14.6 pA; n = 6; P < 0.05 each). Two points of uncertainty have to be taken into consideration: 1) the perforated patch conditions are probably not immediately comparable with the whole-cell recording conditions and 2) the properties of SVZ neuroblasts used for this part of the study might not be completely identical with SGZ neuroblasts. Nonetheless, we conclude with reasonable safeness that the density of P2X7 receptors may decrease in the course of neuronal differentiation. Otherwise, neuroblasts behaved in all respects similarly to NPCS. A low X2+ medium strongly potentiated the Bz-ATP (300 µM) current, when compared with effects in a normal bath medium (Supplementary Fig. 2A) and A-438079 (10 µM) markedly depressed the current response to Bz-ATP (300 µM) (Supplementary Fig. 2Ba). Although an antagonist cocktail of the already described composition did not interfere in a statistically significant manner with the effect of Bz-ATP (300 µM), there appeared to be a small tendency for inhibition, which was fully reversible on washout (Supplementary Fig. 2Bb). Thus, most P2X7 receptors appear to be located at the neuroblasts themselves, rather than at the surrounding cells and there is not much room for astrocytic signaling as an executor of P2X7 receptor function. Eventually, hippocampal granule cells (without discriminating between immature and mature cell types) responded to Bz-ATP (300 µM) with very small amplitude currents, in spite of recording in a low X2+ medium (Supplementary Fig. 2C). Nevertheless, the rapid local superfusion of Bz-ATP caused an effect that clearly surmounted the minuscule artifact elicited by the superfusion of drug-free medium. The failure of both an antagonist cocktail of the above mentioned composition (Supplementary Fig. 2D) and of A-438079 (10 µM) (Supplementary Fig. 2E) to depress the Bz-ATP (300 µM)-induced responses exclude the presence of P2X7 receptors at granule cells. In order to confirm the results obtained by the highly selective P2X7 receptor antagonist A-438079, we repeated these experiments using a nonselective P2X/Y antagonist PPADS (10 µM); in this latter case, we again did not observe an inhibitory effect (13.8 ± 8.1% potentiation; n = 5; P > 0.05). Thus, the slight and reversible, but statistically nonsignificant depression of Bz-ATP currents by both an antagonist cocktail and A-438079 might be artifacts, simply due to the change of solutions by local superfusion. Pilocarpine- and Kainic Acid-Induced Seizures Potentiate P2X7 Receptor Sensitivity of NPCs and Astrocytes The following experiments were designed to address the question, whether long-lasting and strong epileptic seizures alter the sensitivity of P2X7 receptors at SGZ NPCs towards their agonist ATP. For this purpose, a standard protocol was adapted (see, e.g., Fig. 2A) to evoke Bz-ATP (300 µM) current responses in normal and low X2+ bath medium. Hippocampal slices were prepared either from untreated Tg(nestin/EGFP) mice or from identical mice which were treated with pilocarpine or kainic acid, as described in the Methods Section. Pilocarpine (300 mg/kg i.p.) and kainic acid (30 mg/kg i.p.) both induced equally strong SE which was evaluated on a modified Racine scale (stages 4–5; Shibley and Smith 2002). Figure 2. Open in new tabDownload slide Effects of pilocarpine and kainic acid on Bz-ATP-induced currents in NPCs. (A) The i.p. injection of pilocarpine (300 mg/kg) caused status epilepticus in Tg(nestin/EGFP) mice. When hippocampal brain slices were prepared 24 h later from these animals, the current responses of their NPCs to Bz-ATP (300 µM) were largely increased both in a normal and a low X2+ bath medium. Representative experiments for a control NPC (a) and an NPC from pilocarpine-injected mice (b). Mean ± SEM of experiments in slices obtained from normal and pilocarpine-injected mice, respectively (c). The percentage changes of the Bz-ATP currents are shown in (e). Control current responses were designated as 100%. (d,f) A 1 h incubation with pilocarpine (300 µM)-containing bath medium also increased the Bz-ATP (300 µM) currents in NPCs; cells were patched in brain slices incubated in pilocarpine-free and pilocarpine-containing bath solutions, respectively. The number of cells is indicated. The data are presented similarly as for the pilocarpine injection. *P < 0.05; statistically significant differences from IBz-ATP of NPCs determined without pilocarpine injection or incubation, in a normal and low X2+ bath medium, respectively. §P < 0.05; statistically significant difference from measurements in a normal bath medium. (B) The i.p. injection of kainic acid (30 mg/kg) caused status epilepticus in Tg(nestin/EGFP) mice. When hippocampal brain slices were prepared 24 h later from these animals, the current responses of their NPCs to Bz-ATP (300 µM) were largely increased both in a normal and a low X2+ bath medium. Representative recordings from a control NPC (a) and an NPC from a kainate-injected mouse (b). Mean ± SEM of experiments in slices obtained from normal and kainate-injected mice (c). The percentage changes of the Bz-ATP currents are shown in (e). Control current responses were designated as 100%. (d,f) A 1 h incubation with kainate (10 µM)-containing bath medium also increased the Bz-ATP (300 µM) currents in NPCs; cells were patched in brain slices incubated in kainate-free and kainate-containing bath solution, respectively. The data are presented similarly as for the pilocarpine injection. The number of cells is indicated in each column. *P < 0.05; statistically significant difference from IBz-ATP of NPCs determined without kainate injection, in a low X2+ bath medium. Figure 2. Open in new tabDownload slide Effects of pilocarpine and kainic acid on Bz-ATP-induced currents in NPCs. (A) The i.p. injection of pilocarpine (300 mg/kg) caused status epilepticus in Tg(nestin/EGFP) mice. When hippocampal brain slices were prepared 24 h later from these animals, the current responses of their NPCs to Bz-ATP (300 µM) were largely increased both in a normal and a low X2+ bath medium. Representative experiments for a control NPC (a) and an NPC from pilocarpine-injected mice (b). Mean ± SEM of experiments in slices obtained from normal and pilocarpine-injected mice, respectively (c). The percentage changes of the Bz-ATP currents are shown in (e). Control current responses were designated as 100%. (d,f) A 1 h incubation with pilocarpine (300 µM)-containing bath medium also increased the Bz-ATP (300 µM) currents in NPCs; cells were patched in brain slices incubated in pilocarpine-free and pilocarpine-containing bath solutions, respectively. The number of cells is indicated. The data are presented similarly as for the pilocarpine injection. *P < 0.05; statistically significant differences from IBz-ATP of NPCs determined without pilocarpine injection or incubation, in a normal and low X2+ bath medium, respectively. §P < 0.05; statistically significant difference from measurements in a normal bath medium. (B) The i.p. injection of kainic acid (30 mg/kg) caused status epilepticus in Tg(nestin/EGFP) mice. When hippocampal brain slices were prepared 24 h later from these animals, the current responses of their NPCs to Bz-ATP (300 µM) were largely increased both in a normal and a low X2+ bath medium. Representative recordings from a control NPC (a) and an NPC from a kainate-injected mouse (b). Mean ± SEM of experiments in slices obtained from normal and kainate-injected mice (c). The percentage changes of the Bz-ATP currents are shown in (e). Control current responses were designated as 100%. (d,f) A 1 h incubation with kainate (10 µM)-containing bath medium also increased the Bz-ATP (300 µM) currents in NPCs; cells were patched in brain slices incubated in kainate-free and kainate-containing bath solution, respectively. The data are presented similarly as for the pilocarpine injection. The number of cells is indicated in each column. *P < 0.05; statistically significant difference from IBz-ATP of NPCs determined without kainate injection, in a low X2+ bath medium. In preparations taken from pilocarpine-treated mice, the Bz-ATP (300 µM)-induced current measured both in a normal and a low X2+ bath medium was much larger than in preparations taken from untreated mice (Fig. 2Aa–c). Calculations of the percentage increase showed that potentiation in a normal medium was even more pronounced than potentiation in a low X2+ medium (Fig. 2Ae). Then, we turned from epileptic animals to animals whose hippocampal slices were incubated for 1 h in pilocarpine (10 µM)-containing bath medium (Fig. 2Ad,f). Under these conditions, the sensitivity increase to Bz-ATP (300 µM) could be reproduced, although the percentage potentiation in a normal bath medium no longer surmounted that measured in a low X2+ medium. In order to confirm that the seizure activity itself rather than the incubation by pilocarpine causes the potentiation of Bz-ATP-induced current amplitudes in NPCs, we performed two series of experiments. 1) When instead of 1 h, pilocarpine (10 µM) was in contact with the NPCs for 2–4 min only, the Bz-ATP (300 µM)-induced current responses (low X2+ medium: −170.3 ± 58.5 pA; n = 7) did not change (9.6 ± 17.6% inhibition; P > 0.05). 2) Sodium-dependent action potentials were blocked by tetrodotoxin (TTX) and thereby also the pilocarpine-induced epileptic discharges in the brain slices. When hippocampal slices were incubated for 1 h in pilocarpine (10 µM)-containing aCSF, Bz-ATP (300 µM) caused in the NPCs a current of 248.8 ± 18.6 pA amplitude (low X2+ medium; n = 12). However, when such slices were incubated for the same time in an aCSF containing pilocarpine (10 µM)-plus TTX (0.3 µM)-containing medium, the Bz-ATP current was decreased by approximately 70% (74.3 ± 4.8 pA; n = 8; P < 0.05). Thus, pilocarpine itself failed to affect the Bz-ATP current, but most likely induced repetitive, epilepsy-like spike discharges which sensitized the P2X7 receptor to its prototypic agonist. Another pharmacological stimulus to elicit epileptic fits was kainic acid. The outcome of these experiments was not really unequivocal. On the one hand, NPCs of kainic acid-treated mice acquired increased sensitivity to Bz-ATP (300 µM) on the verge of statistical significance (Fig. 2Ba–c), but on the other hand there was no marked difference between the potentiation of the Bz-ATP effects in normal and low X2+ bath medium (Fig. 4Be). Experiments carried out after kainic acid (10 µM)-incubation for 1 h, yielded a tendency of Bz-ATP (300 µM) to cause larger current amplitudes in NPCs of these hippocampal slices compared with nonincubated NPCs, but this difference did not reach the limit of statistical significance (Fig. 2Bd,f). In conclusion, pilocarpine but not kainic acid induced a robust potentiation of Bz-ATP effects in postepileptic NPCs. Since in neurogenic niches astrocytes and NPCs are densely packed one beside the other one, it was of interest to find out whether SE increases the sensitivity of P2X7 receptors only at NPCs or at NPCs and astrocytes simultaneously. Astrocytes of Tg(nestin/EGFP) mice were identified by searching for nonfluorescent cells in the SGZ of the DG. These cells did not fire action potentials in response to the injection of depolarizing current and exhibited I/V characteristics typical for astrocytes and NPCs. Incubation with both pilocarpine (10 µM; 1 h) and kainic acid (10 µM; 1 h) equally facilitated the low X2+ Bz-ATP currents of astrocytes when compared with their aCSF incubated counterparts (Supplementary Fig. 3A). In another series of experiments, we recorded transmembrane currents from fluorescent cells in hippocampal slices of Tg(P2X7/EGFP) mice (Supplementary Fig. 3B). Out of 12 non-neuronal cells in total, 5 cells responded to Bz-ATP (300 µM) with small currents (−23.9 ± 5.5 pA) and another 7 with large currents (−376.9 ± 62.4 pA; P < 0.05). There was no intermediate response whatsoever in between these two families of current responses. Therefore, we concluded that in the SGZ, both astrocytes (Oliveira et al. 2011) and NPCs possess P2X7 receptors. Because all previous experiments were carried out in hippocampal slices of juvenile (10–20 days old) mice, we investigated whether hippocampal slices of more mature (8–10 weeks old) mice react in a similar manner as their juvenile counterparts. In fact, NPCs of older mice also responded to Bz-ATP (300 μM) with current amplitudes (−209.4 ± 62.2 pA; n = 7) which were depressed by the selective P2X7 receptor antagonist A-438079 (10 μM) by 63.2 ± 7.7% (P < 0.05). Moreover, incubation with pilocarpine (10 μM) for 1 h increased the Bz-ATP (300 μM)-induced currents in a low X2+ bath medium by 120.5 ± 16.9% (n = 6; P < 0.05) in comparison with the current amplitudes recorded under the same conditions but without pilocarpine incubation. P2X7 Receptor-Mediated Current Responses in Astrocytes, but not GABAergic or Glutamatergic Interneurons of the Hilus Hippocampi Neurons of the hilus hippocampi belong to the glutamatergic (mossy cells) and GABAergic classes (spiny and aspiny neurons, basket cells) of interneurons (Lubke et al. 1998; Hofmann et al. 2006). Basket cells are located within 10–20 μm from the SGZ; we selected mossy cells and spiny and aspiny neurons by patching cells farther away from this cell layer. The above mentioned three cell types can be differentiated by morphological and electrophysiological criteria from each other. Mossy cells are larger than other types of hilar cells, have a whole-cell capacitance of >200 pF, display spontaneous excitatory postsynaptic currents (sEPSCs), only few action potentials in response to depolarizing current injection, and very small after-hyperpolarization following an action potential. In a first approach, we identified astrocytes based on the lack of action potentials in response to depolarizing current injection (Fig. 3A, left panel). Then, mossy cells (Fig. 3C, left panel) were discriminated from spiny/aspiny neurons by only a single action potential induced by depolarizing current injection (Fig. 3B, left panel). The sEPSCs were relatively rare and had small amplitudes, most probably because our low X2+ bath medium blocked the release of glutamate. Figure 3. Open in new tabDownload slide Effects of amino acid transmitters and Bz-ATP on astrocytes and different types of interneurons in the hilus hippocampi. GABAergic (spiny/aspiny) and glutamatergic (GLUergic; mossy) neurons were identified by their size, their whole-cell capacitance and their firing pattern after the injection of depolarizing current into the cells (for further information see Results section). Hyper- and depolarizing current pulses of increasing amplitudes were delivered at the resting membrane potential (left panels; see Methods Section). Recording of agonist-induced currents are shown in an astrocyte (A), as well as a GABAergic (B) and a glutamatergic (C) neuron at a holding potential of −80 mV (middle panels). AMPA (100 μM), Bz-ATP (300 μM) and muscimol (100 μM) was superfused in a low X2+ bath medium for 10 s every 3 min. The mean current amplitude for two subsequent applications was averaged for statistical evaluation (mean ± SEM; right panels). In contrast to astrocytes, neurons did not respond to Bz-ATP; the statistical significance between the astrocytic and neuronal currents is indicated by an asterisk (P < 0.05). The number of cells is indicated in each column. Figure 3. Open in new tabDownload slide Effects of amino acid transmitters and Bz-ATP on astrocytes and different types of interneurons in the hilus hippocampi. GABAergic (spiny/aspiny) and glutamatergic (GLUergic; mossy) neurons were identified by their size, their whole-cell capacitance and their firing pattern after the injection of depolarizing current into the cells (for further information see Results section). Hyper- and depolarizing current pulses of increasing amplitudes were delivered at the resting membrane potential (left panels; see Methods Section). Recording of agonist-induced currents are shown in an astrocyte (A), as well as a GABAergic (B) and a glutamatergic (C) neuron at a holding potential of −80 mV (middle panels). AMPA (100 μM), Bz-ATP (300 μM) and muscimol (100 μM) was superfused in a low X2+ bath medium for 10 s every 3 min. The mean current amplitude for two subsequent applications was averaged for statistical evaluation (mean ± SEM; right panels). In contrast to astrocytes, neurons did not respond to Bz-ATP; the statistical significance between the astrocytic and neuronal currents is indicated by an asterisk (P < 0.05). The number of cells is indicated in each column. According to expectations, astrocytes responded to the local superfusion of AMPA (100 μM), Bz-ATP (300 μM) and muscimol (100 μM) with inward current, confirming the presence of AMPA, P2X7, and GABAA receptors (Fig. 3A–C, middle and right panels; Lalo et al. 2011). Agonist application was for 10 s every 3 min; the mean current amplitude for two subsequent applications of the same agonist was averaged in the course of statistical evaluation. Mossy cells on the one hand and spiny/aspiny neurons on the other, also reacted to AMPA and muscimol, but did not exhibit inward current in response to Bz-ATP. Mossy cells activate directly granule cells, but indirectly also inhibitory neurons; they have been described to be very vulnerable and their impeded function can lead to hyperexcitability of hippocampal circuits (Scharfman and Bernstein 2015). In contrast, spiny/aspiny neurons supply a direct inhibitory input to granule cells, the lack of which could also lead to hyperexcitability. In view of the “brake function” of both glutamatergic and GABAergic neurons in the hilus hippocampi and the absence of functional neuronal P2X7 receptors on both types of neurons, their direct or indirect participation in the induction of spontaneous epileptic fits after a one-time SE in infancy or early adulthood is most unlikely. P2Y Receptor-Mediated Current Responses in NPCs; Pilocarpine-Induced Seizures Potentiate P2Y Receptor Sensitivity NPCs were described to possess not only P2X7, but also P2Y1,2,4 receptors (Illes et al. 2013; Burnstock and Dale 2015). In a first approach, we tested ADP-β-S (P2Y1,12,13), UDP (P2Y6), and UTP (P2Y2,4) with the indicated selectivity for rodent P2Y receptors (von Kügelgen 2006). At a holding potential of −30 mV, SGZ NPCs responded to ADP-β-S (300 µM), UDP (1000 µM), and UTP (1000 µM) with outward currents (Fig. 4Aa,b). We applied muscimol (100 µM) as a control agonist, which activates GABAA receptors and therefore opens Cl− channels to induce inward current at −30 mV. The outward current response to all three nucleotides was abolished after replacing all K-gluconic acid in the pipette solution by equimolar CsCl (Fig. 4B). This procedure prevents the outward flow of K+ and therefore identified the nucleotide effects as being due to the activation of a potassium channel. In a next step, we excluded the involvement of a Ca2+-dependent potassium channel, because an increase of free intracellular Ca2+ due to less buffering by EGTA (decrease in the pipette solution from 11 to 0.2 mM) failed to influence the nucleotide-induced currents (compare Fig. 4Ab with C). Figure 4. Open in new tabDownload slide Physiological and pharmacological characterization of P2Y receptors on NPCs. The holding potential was changed from −80 to −30 mV during the application of agonists. (A) ADP-β-S (300 µM)-, UDP (1000 µM)- and UTP (1000 µM)-induced outward and muscimol (100 µM)-induced inward currents in NPCs. Representative recordings (a) and mean ± SEM of the current amplitudes (b). (B) The replacement of K+ by equimolar Cs+ in the pipette solution almost abolished the effects of ADP-β-S, UDP, and UTP, but increased that of muscimol. *P < 0.05; statistically significant changes from the respective current amplitude in (Ab). (C) An increase of the free Ca2+ concentration in the pipette solution by decreasing the buffering capacity of EGTA (0.2 mM instead of the usual 11 mM) failed to change the nucleotide-induced current amplitudes. (D) MRS 2179 (30 µM) depressed the ADP-β-S (300 µM) or UDP (1000 µM)-induced current amplitudes; MRS2578 (30 µM) also depressed the effect of UDP. Representative recording from a single cell indicates the inhibitory action of MRS 2578 (a). The percentage mean ± SEM change of IADP-β-S and IUDP was evaluated at the fifth current response in the presence of antagonists in relation to the second current response, before antagonist application (b,c). (E) The i.p. injection of pilocarpine (300 mg/kg) caused status epilepticus in Tg(nestin/EGFP) mice. When hippocampal brain slices were prepared 24 h later from these animals, the current responses of their NPCs to ADP-β-S (300 µM) were largely increased in a normal bath medium (a). The percentage mean ± SEM changes of the ADP-β-S currents are shown in (b). Control current responses were designated as 100%. A 1 h incubation with pilocarpine (10 µM)-containing bath medium also increased the ADP-β-S (300 µM) currents in NPCs (b); cells were patched in brain slices incubated in pilocarpine-free (control; 100%) and pilocarpine-containing bath solution, respectively. The number of cells is indicated in each column. *P < 0.05; statistically significant difference from the control amplitudes (100%). Figure 4. Open in new tabDownload slide Physiological and pharmacological characterization of P2Y receptors on NPCs. The holding potential was changed from −80 to −30 mV during the application of agonists. (A) ADP-β-S (300 µM)-, UDP (1000 µM)- and UTP (1000 µM)-induced outward and muscimol (100 µM)-induced inward currents in NPCs. Representative recordings (a) and mean ± SEM of the current amplitudes (b). (B) The replacement of K+ by equimolar Cs+ in the pipette solution almost abolished the effects of ADP-β-S, UDP, and UTP, but increased that of muscimol. *P < 0.05; statistically significant changes from the respective current amplitude in (Ab). (C) An increase of the free Ca2+ concentration in the pipette solution by decreasing the buffering capacity of EGTA (0.2 mM instead of the usual 11 mM) failed to change the nucleotide-induced current amplitudes. (D) MRS 2179 (30 µM) depressed the ADP-β-S (300 µM) or UDP (1000 µM)-induced current amplitudes; MRS2578 (30 µM) also depressed the effect of UDP. Representative recording from a single cell indicates the inhibitory action of MRS 2578 (a). The percentage mean ± SEM change of IADP-β-S and IUDP was evaluated at the fifth current response in the presence of antagonists in relation to the second current response, before antagonist application (b,c). (E) The i.p. injection of pilocarpine (300 mg/kg) caused status epilepticus in Tg(nestin/EGFP) mice. When hippocampal brain slices were prepared 24 h later from these animals, the current responses of their NPCs to ADP-β-S (300 µM) were largely increased in a normal bath medium (a). The percentage mean ± SEM changes of the ADP-β-S currents are shown in (b). Control current responses were designated as 100%. A 1 h incubation with pilocarpine (10 µM)-containing bath medium also increased the ADP-β-S (300 µM) currents in NPCs (b); cells were patched in brain slices incubated in pilocarpine-free (control; 100%) and pilocarpine-containing bath solution, respectively. The number of cells is indicated in each column. *P < 0.05; statistically significant difference from the control amplitudes (100%). Next, we characterized the receptors involved in K+ current generation by pharmacological means. The effects of ADP-β-S (300 µM) and UDP (1000 µM) were both inhibited by the highly selective P2Y1 receptor antagonist MRS2179 (30 µM; Fig. 4Db,c). In addition, the selective P2Y6 receptor antagonist MRS 2578 (30 µM) strongly depressed the UDP effect (Fig. 4Da). The antagonism by MRS2179 and MRS2578 were reversible on washout. It was interesting to find out, whether the demonstrated sensitivity increase of P2X7 receptors at NPCs prepared from mice undergoing pilocarpine-induced SE is accompanied by a sensitivity increase of P2Y1 receptors. In fact, both pilocarpine (300 mg/kg i.p.) injection and pilocarpine incubation (10 µM for 1 h) caused a clear-cut potentiation of the ADP-β-S (300 µM) current (Fig. 4Ea,b). Increase of P2X7- and P2Y1 Receptor Immunoreactivities at NPCs after Pilocarpine-Induced Status Epilepticus In a first series of experiments, we monitored in light microscopic snapshots P2X7 receptor-labeling in the SGZ of pilocarpine-injected and uninjected Tg(nestin/EGFP) mice (Fig. 5A,B). In the DG, subgranular zone and hilus hippocampi of untreated mice, there was only a faint P2X7 receptor-immunoreactivity (IR; Fig. 5Aa,b), whereas 24 h after pilocarpine injection, in the same region, the P2X7 receptor-IR was increased (Fig. 5Ba,b; arrows). Figure 5. Open in new tabDownload slide Increase of P2X7 receptor-immunoreactivity in the hippocampus of Tg(nestin/EGFP) mice under control conditions and after pilocarpine-induced status epilepticus. Light microscopic pictures show faint P2X7-immunoreactivity (IR) in the dentate gyrus, subgranular zone and hilus hippocampi of an uninjected mouse (A) and its moderate increase 24 h after pilocarpine (50 mg/kg, i.p.)-induced status epilepticus (B). The boxed regions are shown by higher magnification in (Ab,Bb). Examples for accumulations of P2X7-immunopositivity are indicated by arrows. Scale bars for each panel are shown. Representative snapshots obtained from 5 to 6 animals per group each. Figure 5. Open in new tabDownload slide Increase of P2X7 receptor-immunoreactivity in the hippocampus of Tg(nestin/EGFP) mice under control conditions and after pilocarpine-induced status epilepticus. Light microscopic pictures show faint P2X7-immunoreactivity (IR) in the dentate gyrus, subgranular zone and hilus hippocampi of an uninjected mouse (A) and its moderate increase 24 h after pilocarpine (50 mg/kg, i.p.)-induced status epilepticus (B). The boxed regions are shown by higher magnification in (Ab,Bb). Examples for accumulations of P2X7-immunopositivity are indicated by arrows. Scale bars for each panel are shown. Representative snapshots obtained from 5 to 6 animals per group each. Subsequently, a confocal laser scanning microscope was used to evaluate immunohistochemical images. We prepared hippocampal slices both from pilocarpine-injected and uninjected Tg(nestin/EGFP) mice, and stained them for EGFP (in this case a marker of nestin) and for P2X7/P2Y1 receptors (Fig. 6). P2X7 and P2Y1-IRs were present at EGFP-immunopositive cells both with (Fig. 6A,B) and without (Fig. 6C) epileptic seizures, suggesting that NPCs express these two receptor-types under both conditions although at variable densities (see below). Single or double immunopositivity for EGFP and P2X7 receptors are shown in Figure 6A,Ca,b. P2Y1 receptor-IR was also present at EGFP-immunopositive cells; single and double immunopositivity for EGFP and P2Y1 receptors are shown in Figure 6B,Ca,b. Regions of higher expression of P2X7 or P2Y1-IRs are indicated by asterisks on the cell bodies and arrows at their processes, respectively. Apparently only few cells out of a larger population (see staining of many cell nuclei by Hoechst 33 342 in Fig. 6Aa,Ba) exhibited stem cell-like characteristics and carried at the same time P2X7 or P2Y1 receptors. Thus, in the SGZ of the hippocampus, P2X7 and P2Y1 receptor-immunopositive cells occurred both in pilocarpine-injected and uninjected mice. However, staining for these receptors appeared to be more pronounced in the epileptic mice than in their control counterparts (compare Fig. 6Ca,b with Ab,c for P2X7 receptors and Fig. 6Cc,d with Bb,c for P2Y1 receptors). Figure 6. Open in new tabDownload slide Localization of P2X7- and P2Y1-immunoreactivities at hippocampal NPCs of Tg(nestin/EGFP) mice. Confocal laser scanning microscopic pictures from the dentate gyrus and subgranular zone of such mice under control conditions (C) and 24 h after pilocarpine (50 mg/kg, i.p.)-induced status epilepticus (A,B). Localization of EGFP- (marker of nestin; green) and P2X7-immunoreactivities (IRs; red) (Aa–e; Ca,b). Localization of EGFP- (marker of nestin; green) and P2Y1-IRs (red) (Ba–e; Cc,d). Hoechst 33 342 (blue) is used to label the cell nuclei. Note that (Ab,c) and (Ad,e) show two different areas of the SGZ in the same mouse, just as (Bb,c) and (Bd,e). Scale bars for each panel are indicated. Representative snapshots obtained from 5 to 7 animals per group each. Figure 6. Open in new tabDownload slide Localization of P2X7- and P2Y1-immunoreactivities at hippocampal NPCs of Tg(nestin/EGFP) mice. Confocal laser scanning microscopic pictures from the dentate gyrus and subgranular zone of such mice under control conditions (C) and 24 h after pilocarpine (50 mg/kg, i.p.)-induced status epilepticus (A,B). Localization of EGFP- (marker of nestin; green) and P2X7-immunoreactivities (IRs; red) (Aa–e; Ca,b). Localization of EGFP- (marker of nestin; green) and P2Y1-IRs (red) (Ba–e; Cc,d). Hoechst 33 342 (blue) is used to label the cell nuclei. Note that (Ab,c) and (Ad,e) show two different areas of the SGZ in the same mouse, just as (Bb,c) and (Bd,e). Scale bars for each panel are indicated. Representative snapshots obtained from 5 to 7 animals per group each. It has to be noted that not all NPCs possessed P2X7 receptor-IR and on the reverse some non-NPCs (possibly astrocytes or even neurons; Illes et al. 2012) also stained for these receptors. Hence, we consider it important to emphasize that our electrophysiological measurements excluded the presence of functional P2X7 receptors at hilar mossy cells as well as spiny/aspiny interneurons. Moreover, the immunoreactivity for P2Y1 receptors showed a still wider dispersal in the hippocampus than that for P2X7 receptors. Once again the astrocytic and neuronal localization of P2Y1 receptors might explain this finding (Franke et al. 2012; Guzman and Gerevich 2016). In view of the widespread distribution of P2Y1 receptor-IR in the hippocampus it is difficult to exclude the functional interference by many different types of neurons and astrocytes bearing this receptor with the normal hippocampal circuitry. Thus, indirect effects may arise by such processes. However, we consider it much more likely that P2Y1 receptors located at the NPCs themselves directly modulate the proliferation/migration of NPCs and thereby promote the ectopic localization and aberrant function of freshly generated granule cells (see Discussion). Finally, we searched for ectopic cells located outside of the SGZ (Supplementary Fig. 4). This figure is arranged in a basically similar manner as Figure 6, and shows the colocalization of P2X7-IR with EGFP (marks nestin-immunopositive cells) and GFAP (marks astrocytes/Type 1 NPCs). Open arrowheads point to a Type 1 NPC (EGFP/GFAP/P2X7), while the closed arrowheads point to a Type 2 NPC (EGFP/P2X7). Af and Bf show the boxed areas in Ad and Bd, respectively, at higher magnification. Prevention by P2X7 Receptor Blockade of the Status Epilepticus-Induced Neuronal Degeneration in CA3 Pyramidal Cells and the Hilus Hippocampi of Rats In this series of experiments, we used rats rather than mice, because the implantation of cannulas to their lateral brain ventricles is considerably easier, and in addition, they are the standard species for investigating pilocarpine-induced SE. Moreover, we injected AZ10606120 i.c.v., a negative allosteric modulator of P2X7 receptors, instead of the competitive antagonist A-438079. It was expected that AZ10606120 will cause a longer lasting and unsurmountable inhibition of the endogenously released, epilepsy-related high local ATP concentrations. Fluoro Jade-B (FJ-B) was used to label degenerating neurons in hippocampal brain slices. In our SE model, AZ10606120 (1, 2, 3 µg) applied 30 min after SE, concentration-dependently prevented the labeling by FJ-B of pyramidal neurons in the CA1 and CA3 layers (Supplementary Fig. 5). There was no comparable labeling, when saline was injected instead of pilocarpine (Supplementary Fig. 5Ab,Bb). After having shown that the highest concentration of AZ10606120 (3 µg i.c.v.) selectively interacts with the pilocarpine-induced FJ-B labeling in the two hippocampal areas studied, we re-investigated the effect of AZ10606120 in the CA1 and CA3 areas in comparison with the hilus hippocampi (Fig. 7). We found that this negative allosteric modulator almost completely prevented the degeneration of the dispersed hilar neurons, when injected 30 min after SE (Fig. 7Ad, right panel). A reduction of fluorescence was observed in CA1/CA3 as well (Fig. 7Ad, middle and left panels). Once again, FJ-B did not label neurons after the combination of i.c.v. AZ10606120 with i.p. saline (Fig. 7Ab), whereas i.c.v. saline combined with pilocarpine i.p. resulted in a marked neuronal labeling with FJ-B (Fig. 7Ac). Figure 7. Open in new tabDownload slide Prevention by P2X7 receptor blockade of the status epilepticus-induced neuronal degeneration in the hippocampus of the rat; such a receptor blockade increases the spontaneous seizure activity after a status epilepticus. Brain slices for fluorescence light microscopy were prepared 24 h after pilocarpine (370 mg/kg, i.p.)-induced status epilepticus and were stained with Fluoro Jade-B; behavioral measurements were made between 2 and 30 days after status epilepticus. (A) Boxed regions in (a) indicate the locations of the fluorescence light microscopic snapshots in (b–d) for CA1 (left panels), CA3 (middle panels), and hilus hippocampi (right panels). I.p. injection of saline instead of pilocarpine was followed by i.c.v. application of AZ10606120 (AZ; 3 µg) (b). I.p. pilocarpine injection was followed by i.c.v. application of saline (c) or AZ10606120 (d). Note that within each panel two pictures are shown at different magnification (see scale bars). Representative snapshots obtained from 6 animals per group each. (B) The latency time of spontaneous seizures following a status epilepticus caused by pilocarpine followed by the i.c.v. application of saline or AZ10606120 (3 µg; a). Increase of the number of seizure attacks (b) and the number of seizure attacks reaching the indicated intensity on a Racine scale (c) by the same treatments. Box plots (median, first and third quartiles, maximum, and minimum values) of measurements on six rats per group. *P < 0.05; statistically significant differences from the respective “Pilo + saline” group and from the “Pilo + AZ” group, as indicated. Figure 7. Open in new tabDownload slide Prevention by P2X7 receptor blockade of the status epilepticus-induced neuronal degeneration in the hippocampus of the rat; such a receptor blockade increases the spontaneous seizure activity after a status epilepticus. Brain slices for fluorescence light microscopy were prepared 24 h after pilocarpine (370 mg/kg, i.p.)-induced status epilepticus and were stained with Fluoro Jade-B; behavioral measurements were made between 2 and 30 days after status epilepticus. (A) Boxed regions in (a) indicate the locations of the fluorescence light microscopic snapshots in (b–d) for CA1 (left panels), CA3 (middle panels), and hilus hippocampi (right panels). I.p. injection of saline instead of pilocarpine was followed by i.c.v. application of AZ10606120 (AZ; 3 µg) (b). I.p. pilocarpine injection was followed by i.c.v. application of saline (c) or AZ10606120 (d). Note that within each panel two pictures are shown at different magnification (see scale bars). Representative snapshots obtained from 6 animals per group each. (B) The latency time of spontaneous seizures following a status epilepticus caused by pilocarpine followed by the i.c.v. application of saline or AZ10606120 (3 µg; a). Increase of the number of seizure attacks (b) and the number of seizure attacks reaching the indicated intensity on a Racine scale (c) by the same treatments. Box plots (median, first and third quartiles, maximum, and minimum values) of measurements on six rats per group. *P < 0.05; statistically significant differences from the respective “Pilo + saline” group and from the “Pilo + AZ” group, as indicated. P2X7 Receptor Blockade Causes Increased Spontaneous Seizure Activity After a Status Epilepticus in Rats Two days after a pilocarpine-induced SE, we started to video-observe rats for 28 further days. We noticed that, when a few days had elapsed, spontaneous SE-like seizure attacks manifested at irregular intervals. The latency time of these spontaneous seizures after the SE caused by pilocarpine (Fig. 7Ba) did not change after the i.c.v. application of AZ10606120 (3 µg), but both the number of seizure attacks (Fig. 7Bb) and the number of seizure attacks reaching a certain intensity (Racine stages 2–4; Fig. 7Bc) were increased in comparison with the i.c.v. saline-treated controls. Moreover, the largest increase in the number of seizure attacks was observed for stage 2. In order to strengthen the above findings, we injected intraperitoneally the blood–brain permeable P2X7 receptor antagonist BBG (50 mg/kg; Ryu and McLarnon 2008; Arbeloa et al. 2012). BBG (i.p.) had an effect comparable with that of AZ10606120 (i.c.v.) on all parameters of pilocarpine-dependent spontaneous seizures (Supplementary Fig. 6). Discussion Ample evidence indicates that P2X7 and P2Y1 receptor-mRNA and protein are present at embryonic and adult proliferating NPCs and participate in various cellular functions (Illes et al. 2013; Glaser et al. 2014; Ulrich and Illes 2014); however, the pathophysiological significance of these receptors is still insufficiently clarified. P2X7 receptors have been reported to stimulate both necrosis/apoptosis (Delarasse et al. 2009; Messemer et al. 2013) and neuronal differentiation (Tsao et al. 2013; Glaser et al. 2014) of cultured, proliferating NPCs. Culturing NPCs in the presence of high concentrations of growth factors, may change their characteristics, making it difficult to draw firm conclusions regarding their in situ properties. We therefore recorded membrane currents from NPCs in hippocampal DG/SGZ brain slice preparations obtained from Tg(nestin/EGFP) mice. Both ATP and Bz-ATP-induced currents were largely potentiated in a low X2+ bath medium. It has previously been shown that Ca2+ affects the receptor function by acting as an allosteric modulator (Yan et al. 2011) of P2X7 receptors, rather than by altering the permeability dynamics of the ion channel (Khakh and Lester 1999). Moreover, Bz-ATP was more potent than ATP, in perfect agreement with the described effects of these nucleotides at recombinant P2X7 receptors (Sperlagh et al. 2006; Sperlagh and Illes 2014). The Bz-ATP-induced current reversed near 0 mV and was thereby certainly due to the opening of cationic P2X receptor channels. Moreover, the highly selective antagonist A-438079 concentration-dependently decreased the Bz-ATP currents. These data, in combination with detailed investigations on cultured NPCs bear out the presence of a P2X7 receptor (Messemer et al. 2013). In these earlier studies we tested ATP, Bz-ATP and α,β-meATP as agonists, PPADS, TNP-ATP, and 5-BDBD as antagonists and ivermectin, Zn2+ and H+ as allosteric modulators. Still more importantly, neither ATP nor Bz-ATP caused any current response in cells from P2X7 deficient mice, whereas these agonists were fully active in the background wild-type animals (see Messemer et al. 2013). In view of the pertinent communication between astrocytes and NPCs in stem cell niches (Cao et al. 2013; Boccazzi et al. 2014), the question arises, whether the ATP/BzATP effects are indirect, targeting originally astrocytes and transmitted to NPCs by the release of a possible glial signaling molecule (glutamate, Duan et al. 2003; GABA, Wirkner et al. 2005; ATP, Suadicani et al. 2006). Arguments for a direct effect of Bz-ATP at the NPC plasma membrane were obtained by a 2-fold approach: 1) the nucleotide agonist continued to induce current responses at excised patches, which were no longer in physical contact with the surrounding SGZ tissue; 2) a mixture of blockers for ionotropic glutamate (NMDA, AMPA) and GABA (GABAA) receptors as well as propagated action potentials (TTX) failed to decrease the Bz-ATP-induced currents. It seems to be a universal rule found in different types of species and brain regions that in late embryonic, early postnatal development, the first functional synapses are GABAergic, followed only subsequently by a glutamatergic input (Ben-Ari 2001). At the early developmental stage, GABA acts as an excitatory transmitter due to an increased [Cl−]i level and the resulting depolarizing GABA action. Our experiments showed at the ionic gradient determined by the intra- and extracellular concentration of Cl− in the patch solution and bath medium, respectively, an inwardly directed current response to the GABAA receptor agonist muscimol (100 µM); equal concentrations of Bz-ATP, NMDA, and AMPA (100 µM each) also induced inward currents. Hence, in SGZ slices prepared from 10- to 20-day-old Tg(nestin/EGFP) mice, ionotropic P2X7, GABAA, NMDA, and AMPA receptors are all present. In agreement with these findings, excitatory GABAA receptors have been shown to exist in NPCs of the SVZ (Stewart et al. 2002) and SGZ (Wang et al. 2005); a continuous GABA release from neuroblasts has even been suggested to provide a feedback mechanism to control the proliferation of NPCs by activating GABAA receptors (Liu et al. 2005). We have shown that all these receptors, including those of the P2X7 subtype, persist at the age of 8–10 weeks at SGZ NPCs. Extracellular purines and pyrimidines play major roles during embryogenesis, organogenesis, and postnatal development in vertebrates (Burnstock and Dale 2015). Among others, nucleotides are early signaling molecules and neurotransmitters, involved in the regulation of NPC development and differentiation. According to this line of reasoning, we searched for functional P2X7 receptors at neuroblasts of the RMS and granule neurons of the DG. The RMS is a channel, the wall of which consists of astrocytes; this channel is densely packed by neuroblasts (Liu et al. 2005). Our experiments with Bz-ATP alone, or Bz-ATP in combination with A-438079 or an antagonist cocktail suggest that neuroblasts possess P2X7 receptors themselves. In contrast, a similar experimental procedure indicates that granule cells of the DG are devoid of P2X7 receptors, in agreement with the suggestion that neuronal precursors transiently express these receptors, losing them during the maturation process (Sperlagh et al. 2006; Rubini et al. 2014). The temporal lobe epilepsy syndrome can be modeled in rodents by chemical induction of status epilepticus using kainic acid or pilocarpine (Turski et al. 1983; Engel et al. 2012). After chemically induced epilepsy in rodents, the P2X7 receptor-IR is upregulated in granule neurons and microglia, paralleled by astroglial loss (Kim et al. 2009; Jimenez-Pacheco et al. 2013). The immediate effect of P2X7 receptor antagonists applied together with kainic acid to cause status epilepticus, was attenuation of the seizures and in consequence amelioration of the histological damage in neurons and astrocytes (for example Engel et al. 2012). In the present experiments, we noticed an increase of P2X7 receptor sensitivity after pilocarpine- or kainic acid-induced seizures at NPCs both in normal and low X2+ bath medium. The sensitivity increase was more pronounced with pilocarpine, than when kainic acid was used as an epileptogenic agent. This may be due to the suggested interaction between the P2X7 receptor-pannexin-1 complex and M1 receptor-mediated seizure activity (Kim and Kang 2011). P2Y receptors, including P2Y1 have been repeatedly shown to promote proliferation of cultured adult and embryonic NPCs; the methods used were the determination of cell number (Mishra et al. 2006), immunohistochemistry experiments done with the proliferation marker KI-67 (Rubini et al. 2009), and the measurement of bromodeoxyuridine incorporation into these cells (Pearson et al. 2005). The extension of such in vitro experiments to the in situ (brain slice) and in vivo (whole animal) situation unequivocally supported the involvement of P2Y1 receptors in proliferation (Suyama et al. 2012; Cao et al. 2013). A further NPC function regulated by P2Y1 receptors is migration over short or longer distances (Scemes et al. 2003; Liu et al. 2008). Although a direct correlation between P2Y1 receptor expression at NPCs and their proliferation/migration after status epilepticus has hitherto not been investigated, we believe that such a correlation might exist. In an attempt to prove the presence of functional P2Y receptors at NPCs we applied a series of P2Y receptor agonists (ADP-β-S, UTP, UDP) at a holding potential of −30 mV to unmask outward K+ currents which are obscured at the normal holding potential of −80 mV. Both ADP-β-S- and UDP-induced currents were inhibited by the selective P2Y1 receptor antagonist MRS2179. In the case of UDP the P2Y6 receptor antagonist MRS2578 was also active, indicating a possible release of ATP/ADP from NPCs or astrocytes by P2Y6 receptor activation. This ATP/ADP is then supposed to activate an outward K+ current of NPCs via P2Y1 receptor stimulation (for analogous results see Rubini et al. 2009). The main finding of these series of experiments was however that in vivo epileptic seizures increased the amplitude of the P2Y1 receptor-mediated currents. Our fluorescence microscopic experiments suggested that pilocarpine-induced SE led to the upregulation of a low-intensity P2X7 receptor-immunopositivity in the SGZ. Confocal laser microscopy showed a colocalization of P2X7 receptor-IR with nestin-EGFP. In these mice, nestin-EGFP was also colocalized with P2Y1 receptor-IR. Thus, immunohistochemistry proved that postepileptic NPCs possess protein for P2X7 and P2Y1 receptors. Furthermore, ectopic NPCs and astrocytes after SE were also endowed with these two receptor-types. Eventually, in spite of earlier reports that P2X7 receptor antagonists inhibited the acute behavioral manifestations of SE induced by injection of kainic acid into the nucleus amygdala (Engel et al. 2012), blockade of P2X7 receptors even facilitated the spontaneous epileptic fits following pilocarpine-induced SE. In fact, the negative allosteric modulator AZ10606120 injected into the lateral ventricle of rats (and also BBG applied intraperitoneally), probably protected hilar neurons against the injurious effects of SE and favored the survival, ectopic migration and neuronal differentiation of NPCs as well as their integration into pathological neuronal circuits. In consequence, we hypothesize that SE causes metabolic constraint to NPCs which in turn leads to a massive outflow of nucleotides onto the same NPCs. The subsequent stimulation of functionally overactive P2Y1 receptors produces proliferation and migration to ectopic sites in the hilus hippocampi. The accompanying stimulation of overactive P2X7 receptors, however, counterbalances this effect, resulting in apoptosis/necrosis of surplus NPCs, and preventing the chronic manifestation of a one-time epileptic fit. The postnatal development of laboratory rodents and humans occurs at different pace; the rodent hippocampus and specifically the dentate gyrus proliferates maximally around P8, is still developing at P20, and reaches mature-like structures in the first to second postnatal month (Avishai-Eliner et al. 2002). The same stage of maturation is achieved in humans only a number of years after birth. Therefore, our observations may have particular relevance for febrile seizures in children which are the most common form of pathological brain activity during development (Bender and Baram 2007; Dubé et al. 2007). Fever might promote neuronal excitability through a combination of several mechanisms, including hyperthermia per se, inflammatory cytokines and alkalosis, perhaps in the setting of genetic susceptibility. In consequence, prolonged febrile seizures might lead to limbic epilepsy via processes involving the above described purinergic mechanisms. Supplementary Material Supplementary material can be found at: http://www.cercor.oxfordjournals.org/. Funding This study was supported by grants of the Deutsche Forschungsgemeinschaft (IL 20/21-1; KI 677/4-2), the Sino-German Centre for the Promotion of Science (GZ 919), the Brazilian National Council for Scientific and Technological Development (CNPq) and Fapesp. The PhD fellowship of M.G.L.A. was financed by the CNPq. The scholarship of P.G. was financed by the Sino-German Centre. The scholarships of M.T.K. and J.L. were supplied by the Deutsche Akademische Austauschdienst (DAAD) and the China Scholarships Council (CSC), respectively. 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Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com
TI - Pilocarpine-Induced Status Epilepticus Increases the Sensitivity of P2X7 and P2Y1 Receptors to Nucleotides at Neural Progenitor Cells of the Juvenile Rodent Hippocampus
JO - Cerebral Cortex
DO - 10.1093/cercor/bhw178
DA - 2017-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/pilocarpine-induced-status-epilepticus-increases-the-sensitivity-of-aOiIcaZB0x
SP - 3568
EP - 3585
VL - 27
IS - 7
DP - DeepDyve
ER -