Supersensitivity of p2x7 receptors in cerebrocortical cell cultures after invitro ischemia

Journal of Neurochemistry, 2005, 95, 1421–1437 Supersensitivity of P2X7 receptors in cerebrocortical cell culturesafter in vitro ischemia Kerstin Wirkner,* Attila Ko¨falvi, ,1 Wolfgang Fischer,* Albrecht Gu¨nther,*,à Heike Franke,*Helke Gro¨ger-Arndt,*,§ Wolfgang No¨renberg,* Emı´lia Madara´sz, E. Sylvester Vizi, Dietmar Schneider,à Bea´ta Sperla´gh and Peter Illes*,§ *Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary àDepartment of Neurology, University of Leipzig, Leipzig, Germany §Interdisciplinary Center for Clinical Research of the Medical Faculty, University of Leipzig, Leipzig, Germany Neuronally enriched primary cerebrocortical cultures were (mIPSCs) after ischemia than after normoxia. Brilliant Blue exposed to glucose-free medium saturated with argon G almost abolished the effect of BzATP in normoxic neu- (in vitro ischemia) instead of oxygen (normoxia). Ischemia rons. Since neither the amplitude of mIPSCs nor that of the did not alter P2X7 receptor mRNA, although serum depri- muscimol-induced inward currents was affected by BzATP, vation clearly increased it. Accordingly, P2X7 receptor it is assumed that BzATP acts at presynaptic P2X7 recep- immunoreactivity (IR) of microtubuline-associated protein 2 tors. Finally, P2X7 receptors did not enhance the intracel- (MAP2)-IR neurons or of glial fibrillary acidic protein lular free Ca2+ concentration either in proximal dendrites or (GFAP)-IR astrocytes was not affected; serum deprivation in astrocytes, irrespective of the normoxic or ischemic pre- augmented the P2X7 receptor IR only in the astrocytic, but incubation conditions. Hence, facilitatory P2X7 receptors not the neuronal cell population. However, ischemia mark- may be situated at the axon terminals of GABAergic non- edly increased the ATP- and 2¢-3¢-O-(4-benzoylbenzoyl)- pyramidal neurons. When compared with normoxia, ische- mia appears to markedly increase P2X7 receptor-mediated previously incorporated [3H]GABA. Both Brilliant Blue G and GABA release, which may limit the severity of the ischemic oxidized ATP inhibited the release of [3H]GABA caused by damage. At the same time we did not find an accompanying ATP application; the Brilliant Blue G-sensitive, P2X7 recep- enhancement of P2X7 mRNA or protein expression, sug- tor-mediated fraction, was much larger after ischemia than gesting that receptors may become hypersensitive because after normoxia. Whereas ischemic stimulation failed to alter of an increased efficiency of their transduction pathways.
the amplitude of ATP- and BzATP-induced small inward Keywords: ATP, cortical cell culture, ischemia, P2X7 recep- currents recorded from a subset of non-pyramidal neurons, tor, receptor up-regulation.
BzATP caused a more pronounced increase in the fre- J. Neurochem. (2005) 95, 1421–1437.
High concentrations of ATP have been reported to activate acertain subtype of the ionotropic P2X receptor (P2X7; Abbreviations used: ACSF, artiﬁcial cerebrospinal ﬂuid; AP-5, Surprenant et al. 1996; Khakh et al. 2001). This receptor D(–)-amino-5-phosphonopentanoic acid; ATP, adenosine 5¢-triphosphate;BzATP, 2¢-3¢-O-(4-benzoylbenzoyl)-adenosine 5¢-triphosphate; CNQX, Received April 4, 2005; revised manuscript received July 29, 2005; accepted August 1, 2005.
CT values, cycle-threshold values; DIV, days in vitro; DPCPX, Address correspondence and reprint requests to Dr Peter Illes, Rudolf- 8-cyclopentyl-1,3-dipropylxanthine; GFAP, glial ﬁbrillary acidic protein; Boehm-Institute of Pharmacology and Toxicology, University of Leip- IR, immunoreactivity; MAP2, microtubule-associated protein 2; MCAO, zig, D-04107 Leipzig, Germany. E-mail: [email protected] middle cerebral artery occlusion; mIPSC, miniature inhibitory post- 1The present address of Attila Ko¨falvi is Center for Neuroscience synaptic current; MRS 2179, 2¢-deoxy-N6-methyladenosine 3¢,5¢- of Coimbra, Institute of Biochemistry, University of Coimbra, 504 bisphosphate; NMDA, N-methyl-D-aspartate; oxiATP, oxidized ATP; Coimbra, Portugal.
2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 1421–1437 K. Wirkner et al.
allows the passage of small cations on immediate activation receptor-mediated release of GABA without a major change by agonists; long-lasting activation, however, leads to a in the number of these receptors. It is concluded that P2X7 progressive dilation of the ion channel and formation of a receptors situated at the terminals of GABAergic neurons large pore (permeable to organic molecules and dyes), and may be responsible for a limitation of the ischemic neuronal membrane blebbing (Virginio et al. 1999; North 2002).
damage in the cell culture system.
The P2X7 receptor is thought to be expressed predomin- antly on antigen-presenting immune cells and epithelia(Rassendren et al. 1997). Astrocytes and Mu¨ller cells of Materials and methods the retina have also been described to bear P2X7 receptors,which possibly mediate inﬂammation and subsequent pro- Preparation of cortical cell cultures liferation (Pannicke et al. 2000; Gendron et al. 2003). In Cell cultures were prepared from rat fetuses at gestational day 16 addition, these astrocytic receptors release glutamate (Duan and grown as described earlier (Gu¨nther et al. 2002; Reinhardt et al.
2003). In short, a cell suspension made with a 1:1 mixture of et al. 2003) and GABA (Pannicke et al. 2000) participating Dulbecco's modiﬁed Eagle's medium and Nutrient F12 supplemen- in astrocyte–neuron communication. More recently, P2X7 ted with 20% fetal calf serum, 2.2 mM L-glutamine, 15 mM HEPES, receptors were identiﬁed on neurons of the central and 50 lg/mL gentamicin, and 30 mM D-glucose was seeded into poly- peripheral nervous system by single cell RT–PCR, immu- L-lysine-coated polystyrol dishes or on glass coverslips at a density nohistochemistry and functional investigations (Deuchars of 5 · 105 cells per dish/coverslip and cultured at 37C in a et al. 2001; Hu et al. 2001; Sperla´gh et al. 2002; Miras- humidiﬁed atmosphere containing 5% CO2/95% air. After 5 days of Portugal et al. 2003; Allgaier et al. 2004; Wang et al. 2004).
cultivation, the medium was replaced by Neurobasal medium Unfortunately, the value of antibodies in identifying this supplemented with 0.5 mM glutamine, serum-free supplement B27 neuronal target were questioned by the demonstration of a (v/v 50/1) and 50 lg/mL gentamicin. At 5 to 10 days later the pseudo-immunoreactivity (IR) for P2X neurobasal medium was removed and the cell cultures were rinsed hippocampus of P 2X = mice (Sim et al. 2004; Kukley twice with artiﬁcial cerebrospinal ﬂuid (ACSF) (composition in et al. 2004), although the P2X M: NaCl 140; KCl 5; MgCl2 2, CaCl2 2; HEPES 10; glucose 11; 7 receptor-mediated transmitter pH 7.4 adjusted with NaOH). Cultures were used thereby on release disappeared in the knockout animals (Papp et al.
days 10–15 (10–15 DIV) for most experiments. Exceptions were fura-2 measurements, where culturing until day 5 and from day 7 In support of the assumption that cerebral ischemia onward was identical to that described earlier, but in between aggravates brain injury via the efﬂux of ATP, both intrastri- cytosine b-arabino-furanoside (10 lM) was added for 24 h.
atally injected ATP (Ryu et al. 2002) and middle cerebralartery occlusion (MCAO; Kharlamov et al. 2002) caused Release experiments lesions, which were prevented by the application of the The [3H]GABA release experiments were carried out with slight wide-range P2 receptor antagonists suramin and pyridoxal- modiﬁcations of previous experimental protocols (Sperla´gh et al.
2002). The neurobasal medium in each culture dish was replaced Mechanical (Franke et al. 2001) or ischemic injury (Collo with 1 mL HEPES buffer of the following composition (in mM):NaCl 135; KCl 5; MgSO et al. 1997; Franke et al. 2004) has been shown to lead to the 4 0.6; CaCl2 1; glucose 6; HEPES 10; pH 7.3; saturated with O expression of previously absent P2X 2. When the experiment was performed under normal conditions, the culture dishes were kept under microglia, astrocytes and neurons. Further, a large number O2-saturated atmosphere for 1 h, then the buffer was replaced of investigations in cell culture preparations strongly suggest with 1 mL of the same buffer containing 0.5 lCi 4-amino-n- that P2 receptors are involved in ischemic neuronal damage [2,3-3H]butyric acid ([3H]GABA, speciﬁc activity 86.0 Ci/mmol; (Volonte et al. 1999; Cavaliere et al. 2001). More speciﬁc- Amersham Pharmacia, Braunschweig, Germany) and b-alanine ally, oxygen/glucose deprivation was shown to up-regulate (1 mM) for 20 min. b-Alanine has been described to prevent tritium uptake into glial cells but not neurons (Iversen and Kelly 1975).
7 receptor IR in primary cultures of cerebellar granule neurons (Cavaliere et al. 2002) and in CA1 pyramidal After incubation with [3H]GABA, the cells were covered with an neurons of organotypic hippocampal cultures (Cavaliere 80 lm-pore nylon mesh for physical protection from the super- et al. 2004). It was hypothesized that after ischemia, the fusion, and were then subjected to a subsequent 15-min washoutwith HEPES buffer at a rate of 2 mL/min. Upon termination of the excessive release of the excitotoxic ATP up-regulates P2X7 washout, 1-min samples were collected from the efﬂuent. When receptors and thereby facilitates the cellular necrosis caused experiments were performed under ischemic conditions, the by metabolic limitation.
neurobasal medium was replaced with glucose-supplemented and The aim of the present study was to investigate in O2-saturated HEPES buffer, and the dishes were kept under O2- neuronally enriched primary cortical cultures, whether an saturated atmosphere as described above, but only for 30 min. Then, ischemic stimulus of relatively short duration increases P2X7 glucose was substituted with equimolar saccharose, and O2 was receptor-mediated functions, and whether such an effect replaced with argon in the atmosphere and in the solutions. From correlates with a corresponding increase in P2X this time onward, all solutions were glucose-free and argon- IR. We found that ischemia markedly increases the P2X saturated. Thus, the second 30 min of pre-incubation, the 20 min 2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 1421–1437 Neuronal P2X7 receptors and ischemia 1423 of incubation with the isotope and the 15 min of washout yielded was prepared by omitting 11 mM glucose from the standard ACSF and 65 min of ischemic pre-treatment before the sample collection. All by increasing the content of NaCl to 151 mM.
experiments were carried out at room temperature. To minimize the Incubation was performed according to two different time- formation of GABA metabolites, all solutions contained aminooxy- schedules: The ﬁrst 60-min incubation was always in normoxic and acetic acid (100 lM) in normal and ischemic conditions as well. This glucose-containing medium followed by a 30 (or in some 65-min ischemic stimulus caused a somewhat larger decrease of the experiments 60)-min incubation period with either normoxic and ATP/ADP ratio from 6.1 ± 0.9 (n ¼ 5) to 0.39 ± 0.01 (n ¼ 6; glucose-containing medium (normoxia) or hypoxic and glucose-free p < 0.05; by 90%) than a 30-min ischemic stimulus (60% medium (ischemia). Immediately after the total incubation period of depression of the ATP/ADP ratio) used for immunocytochemistry, 90 (or 120) min, the cells were incubated with normal ACSF electrophysiology and Ca2+ microﬂuorimetry (see below).
solution before and during electrophysiological recording or loading During the sample collection period, two kinds of protocol were with fura-2 and subsequent microﬂuorimetry. In some of the applied as described previously (Sperla´gh et al. 2002). According to experiments, before electrophysiology and fura-2 microﬂuorimetry, the ﬁrst protocol, the normoxic and ischemic cell cultures were there was no 90-min pre-incubation in a desiccator (normal challenged with increasing concentrations of ATP or BzATP in each condition). It has previously been shown that a 30-min in vitro experiment, for 1 min every 5–6 min. Of the P2X7 receptor ischemia (hypoxia and glucose-deﬁciency) markedly depressed the antagonists, Brilliant Blue G (1 lM) and PPADS (30 lM) were ATP/ADP ratio (by 60%) in comparison with normoxic controls present in all solutions used, from the beginning of pre-incubation.
(Gu¨nther et al. 2002; Reinhardt et al. 2003).
Oxidized ATP (oxiATP; 300 lM) was present only during the95-min incubation period; however, this time was sufﬁcient for the Whole-cell patch-clamp recordings antagonist to irreversibly inhibit P2X7 receptors.
Membrane currents were recorded in the whole-cell conﬁguration of Concentration–response curves were ﬁtted using the following the patch-clamp method at room temperature (20–22C) using an logistic function (SigmaPlot, SPSS, Erkrath, Germany), Axopatch 200 B ampliﬁer (Axon Instruments, Union City, CA,USA) (Allgaier et al. 2004). The pipette solution contained (in mM): E ¼ Emin þ ðEmax EminÞ½1 þ ðEC50 þ AÞn; CsCl 140, MgCl2 1, CaCl2 1, HEPES 10, EGTA 10, MgATP 1.5,LiGTP 0.3, N-methyl-lidocaine iodide (QX-314) 2; pH adjusted to where E is the steady-state effect produced, A is the agonist 7.3 with CsOH. The holding potential was )60 mV. For current- concentration, Emax and Emin are the maximal and minimal effects, clamp measurements, CsCl was replaced in the pipette solution by respectively, n is the Hill coefﬁcient, and EC50 is the concentration equimolar KCl and the pH was adjusted with KOH instead of of agonist producing 50% of Emax.
CsOH. Currents were ﬁltered at 2–5 kHz with the inbuilt lowpass- According to the second protocol, with 10-min delay, the ﬁlter of the patch-clamp ampliﬁer. Data acquisition and analysis ischemic cell cultures were stimulated with ATP (3 mM) twice (S1 were performed computer-controlled using pClamp 8.0 software and S2). Six minutes before S2, various drugs, i.e. tetrodotoxin (Axon Instruments; sampling rate, 3–10 kHz). An ACSF of known (1 lM), D(–)-amino-5-phosphonopentanoic acid (AP-5, 50 lM) plus composition (see above) was used as the external recording solution.
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 lM), Reactive GABAA receptor-mediated miniature inhibitory postsynaptic pot- Blue 2 (10 lM) plus 2¢-deoxy-N6-methyladenosine 3¢,5¢-bisphos- entials (mIPSCs) were measured at )60 mV, in the presence of AP-5 phate (MRS 2179, 10 lM), and 8-cyclopentyl-1,3-dipropylxanthine (50 lM), CNQX (10 lM) and tetrodotoxin (0.5 lM) in the external (DPCPX, 0.25 lM) plus 8-(3-chlorostyryl)caffeine (10 lM) were medium. mIPSCs were analysed using commercially available applied. The latter two compounds were dissolved in ethanol, software (MiniAnalysis 4.3; Synaptosoft, Decatur, GA, USA).
whereas all the other drugs were dissolved in water. The ﬁnal ATP (1–10 mM), 2¢-3¢-O-(4-benzoylbenzoyl)-adenosine 5¢-tri- concentration of ethanol (0.25%) had no effect on the [3H]GABA phosphate (BzATP; 30–300 lM), Brilliant Blue G (0.3 lM) and muscimol (10 lM) were dissolved in ACSF; the pH of the agonist The release of [3H]GABA was calculated as percentage of the solutions in this and all subsequent experiments was balanced to 7.3.
amount of radioactivity in the neurons at the sample collection time When we measured effects on the holding current, ATP and BzATP (fractional release; FR%) (Sperla´gh et al. 2002). It was found that were applied by a pressurized fast-ﬂow superfusion system (DAD- [3H]GABA represents the majority (95%) of total tritium efﬂux.
12; Adams and List, Westbury, NY, USA) for 1 s each, separated bydrug-free intervals of 3 min. In experiments with muscimol, the Incubation conditions for electrophysiology, Ca2+ GABAA agonist was applied for 2 s every 3 min; BzATP was microfluorimetry, immunocytochemistry, and real-time superfused for 6 min, immediately after ﬁnishing the second polymerase chain reaction application of muscimol. The same superfusion system was used A culture dish was rapidly transferred to 500 mL of ACSF (see above) to apply BzATP and Brilliant Blue G, when effects on the mIPSC and incubated for 90 min at 37C. The control solution was saturated amplitude and frequency were measured. Mean mIPSC amplitudes with 100% oxygen (pO2, 710–740 mmHg; pH, 7.3). The oxygen and frequencies were calculated during a control period of 3 min saturation was controlled by a Clark type electrode via an oxymeter and during the last 3 min of the subsequent application of BzATP (StrathKelvin instruments, Glasgow, Scotland). A condition termed for 5 min. In separate experiments, BzATP, Brilliant Blue G, or ‘in vitro ischemia' was achieved by placing the dishes into glucose- BzATP plus Brilliant Blue G was applied for two subsequent 5-min free ACSF separated from the environmental air in a desiccator and periods each. The evaluation times lasted again for 3 min as carefully gassed with 100% argon (pO2, 1–2 mmHg) for at least mentioned above. The mean amplitude and frequency of mIPSCs 30 min before starting the incubation period. A glucose-free medium were normalized with respect to the control values recorded during 2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 1421–1437 K. Wirkner et al.
the ﬁrst 3-min period in drug-free ACSF. The changes were mixture volume consisting of 10 lL of 2 · QuantiTect SYBR expressed as percentage potentiation of the time-matching controls Green PCR Master Mix (Qiagen, containing HotStar Taq DNA recorded in drug-free ACSF for 15 min in total. Amplitude polymerase, PCR-Buffer, dNTP mix and SYBR Green I), 2 lL histograms were binned in 2-pA intervals.
reverse and forward primers each at 0.5 lM, 2-lL samples of cDNAand 4 lL RNase-free water. The reactions comprised 15 min of Fura-2 microfluorimetry initial denaturation at 95C, 45 cycles of denaturation (94C for Cell cultures, after the 30-min incubation procedure in normoxic or 30 s), annealing (40C for 20 s), and extension (72C for 10 s). All ischemic ACSF (see above), were washed in superfusion medium reactions were made in duplicate with no template control.
(composition in mM: NaCl 133; KCl 4.8; KH2PO4 1.2; CaCl2 1.3; Ampliﬁcation, data acquisition and analyses were carried out by HEPES 10; glucose 10; pH 7.4; room temperature) and loaded in Light Cycler instrument (Roche Diagnostics, Mannheim, Germany) this solution with fura-2 acetoxymethyl ester (5 lM, for 30 min).
using Light Cycler 5.3.2 software (Roche). The identity of the PCR After loading, all cultures were washed again to remove products was conﬁrmed by sequencing. The sequence of P2X7 extracellular traces of the dye. The coverslips were then placed primers were as follows: forward 5¢-TGTCCCTATCTCTCCACG- on the stage of an inverted microscope with epiﬂuorescence optics ACTCAC-3¢ and reverse 5¢-ATTTCCACACTGGCACCAACTC- (Diaphot 200; Nikon, Du¨sseldorf, Germany). Throughout the GG-3¢ (GenBank accession number NM011027). The ampliﬁcation experiments, the cells were continuously superfused at 0.8 mL/min with this pair of oligonucleotides yielded a 119 bp fragment.
by means of a roller pump. A high external K+-containing medium To determine the suitable endogenous reference gene, we (50 mM, Na+ replaced by an equimolar amount of K+; 3 s examined cycle-threshold (CT) values of several endogenous superfusion), and ATP or BzATP (300 lM each; 10 s superfusion) reference genes, such as 18S rRNA, ribosomal protein L28, and were applied directly to single cells by pressure every 12 min, b-actin. The results indicate that 18S rRNA expression offers using a fast-ﬂow superfusion system (DAD12, Adams and List).
superior consistency during normoxic and ischemic conditions. The Cyclopiazonic acid (10 lM), a Ca2+-free solution (with 1 mM primer sequences for the housekeeping gene 18S rRNA were as EGTA), Brilliant Blue G (1 lM) or PPADS (30 lM) were follows: forward 5¢-TCAAGAACGAAAGTCGGAGGTT-3¢ and superfused 10 min before and during the next pressure application reverse 5¢-GGTCATCTAAGGGCATCACAG-3¢. The CT values of ATP or BzATP.
for P2X7 were normalized with respect to the CT values for 18S Fluorescence ratio measurements were made on single, morpho- logically identiﬁed multipolar non-pyramidal neurones (over the cellsomata or proximal dendrites) and astrocytes with a dual wavelength Immunofluorescence and confocal microscopy spectrometer (alternating excitation at 340/380 nm). Fura-2 ﬂuores- P2X7 receptor IR in cultured cortical neurons was detected with cence was measured at 510/520 nm by a microscope photometer polyclonal rabbit anti-P2X7 antibodies raised against highly puriﬁed attached to a photomultiplier detection system (Ratiomaster System; peptides corresponding to amino acids 576–595 of the carboxyl PTI, Wedel, Germany). The agonist-induced rise of [Ca2+]i was terminus of the cloned rat P2X7 receptor. The cultures were washed deﬁned as the peak increase in the D ﬂuorescence ratio (i.e. the twice for 5 min in Hank's buffered saline solution and ﬁxed in ice- ﬂuorescence ratio 340/380 nm in response to the agonist minus the cold methanol. For permeabilization and blocking, the cells were basal ﬂuorescence ratio). Data acquisition and analysis were pre-treated with 0.1% Triton X-100 and 5% fetal calf serum in Tris- performed computer-controlled by using commercially available buffered saline (0.05 M, pH 7.6) for 30 min. Then, the cultures were software (FeliX, Version 1.1; PTI). Calibration of [Ca2+]i was incubated with an antibody mixture of the mouse microtubule- performed by determining Ca2+-saturated fura-2 signals (Rmax) in associated protein 2 (MAP2, 1:1000) and/or mouse glial ﬁbrillary the presence of 10 lM ionomycin (Mg2+-free buffer), and Ca2+-free acidic protein (GFAP, 1:1000) and the rabbit P2X7 (1:600) receptor signals (Rmin) in the presence of 25 mM EGTA (Ca2+-free buffer), antibody, in combination with Cy2-conjugated goat anti-mouse IgG (1:400), Cy3-conjugated goat anti-rabbit IgG (1:1000), and K+ (50 mM for 3 s) evoked a D ﬂuorescence ratio of 2.76 ± 0.26, Cy5-conjugated streptavidin (1:800). For the GABA-immunnoﬂuo- which corresponds to a mean maximum elevation of [Ca2+]i from rescence study, the cultures were ﬁxed with paraformaldehyde (4%)/ basal 75.1 ± 4.1 nM to 845.4 ± 93.4 nM (n ¼ 12 cells).
glutaraldehyde (0.25%) for 10 min at 4C followed by washing andblocking as described above. The cell cultures were incubated with RNA isolation and real-time polymerase chain reaction using the antibody mixture of rabbit anti-P2X7 and mouse anti-GABA SYBR-Green chemistry (1:100) and the respective Cy2/Cy3-labeled secondary antibodies.
Total RNA was prepared from 5 · 105 cultured cells using the After intensive washing and mounting on slide glasses, all stained RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the sections were dehydrated in a series of graded ethanol, processed manufacturer's directions. The amount of RNA was quantiﬁed by through n-butylacetate and covered with entellan (Merck, Darm- measuring the absorbance at 260 nm and 280 nm in a spectropho- stadt, Germany). Control experiments were carried out without tometer (Eppendorf, Wesseling, Germany). The quality of the the primary P2X7 receptor antibody or by pre-adsorption of the extracted RNA was visualized on a 1% agarose gel after antibody with the immunizing peptides. The immunoﬂuorescence was investigated by a confocal laser scanning microscope (LSM A two-step reverse transcription was performed by using 510, Zeiss, Oberkochen, Germany) at an excitation wavelength of SuperScript II (Life Technologies, Eggenstein, Germany). cDNA 633 nm (helium/neon2, blue Cy5-immunoﬂuorescence), 543 nm was puriﬁed with QIAquick PCR Puriﬁcation Kit (Qiagen). The (helium/neon1, red Cy3-immunoﬂuorescence), and 488 nm (argon, SYBR Green real-time PCR assay was carried out in 20 lL PCR 2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 1421–1437 Neuronal P2X7 receptors and ischemia 1425 For statistical evaluation, 50 GABA-, MAP2- or GFAP-immu- apoptotic stimulus, enhances the transcription of P2X7 noreactive cells were counted in each culture dish after each receptors already within a period of 1–2 h. However, treatment schedule (untreated, normoxia, ischemia). Then cells ischemia failed to further increase the P2X7 mRNA over with double IR for P2X7 plus GABA, MAP2 or GFAP were also the normoxic values during the same time periods.
counted and expressed as a percentage of the total cell population Next, we utilized immunocytochemistry and confocal laser scanning microscopy to characterize the prominent cell typesin the cortical cultures investigated. Previous studies revealed Materials and drugsThe following antibodies and conjugated markers were used: mouse that approximately 80–90% of neurons were GABA-immu- monoclonal anti-GABA (Clone GB-69; Sigma-Aldrich, Taufkir- noreactive (Fischer et al. 2002). In fact, many untreated chen, Germany), rabbit anti-P2X neurons were labeled for GABA IR and a considerable 7 receptor subtype (intracellular C-terminus binding, Lot #: AN-03; Alomone Laboratories, Jerusa- population was co-labeled for GABA- and P2X7 receptor IR lem, Israel); mouse anti-MAP2 (Chemikon, Temecula, CA, USA); (Fig. 1b, i, ii and iv). An antibody raised against a C-terminal Cy2-conjugated goat anti-mouse IgG, Cy3-conjugated goat anti- epitope of the P2X7 receptor was used in all experiments.
rabbit IgG (Jackson Immuno Research, Baltimore, USA).
Three different types of neurons (pyramidal, multipolar and The following drugs were used: adenosine 5¢-triphosphate bipolar) were morphologically identiﬁed. Especially the disodium salt (ATP), AP-5, aminooxyacetic acid, BzATP, Coomas- relatively small bipolar cell bodies co-expressed the GABA- CNQX, DPCPX, cyclopiazonic acid, EGTA, MRS 2179, cytosine 7 IR with a high incidence (Fig. 1b, ii); larger multipolar neurons less often exhibited co-expression, b-D-arabino-furanoside, fura-2 acetoxymethyl ester (Fura-2/AM),muscimol, whereas the assumedly glutamatergic pyramidal neurons L-glutamine, N-methyl-D-aspartate (NMDA), oxiATP, PPADS, Reactive Blue 2, tetrodotoxin (all from Sigma-Aldrich, usually did not stain for GABA (Fig. 1b, i). These ﬁndings Taufkirchen, Germany); baﬁlomycin, N-methyl-lidocaine iodide are in partial contradiction to the ﬁnding that in the rat (QX-314; Tocris, Bristol, U.K); 4-amino-n-[2,3-3H]butyric acid CNS the majority of the P2X7 IR was localized at non- ([3H]GABA, speciﬁc activity, 86.0 Ci/mmol; Amersham Pharmacia GABAergic neurons (Deuchars et al. 2001; Sperla´gh et al.
Biotech, Buckinghamshire, UK); Dulbecco's modiﬁed Eagle's 2002; Atkinson et al. 2004). A possible reason for this medium, Dulbecco's medium Nutrient F12, Neurobasal medium, discrepancy is that tissue damage during the culturing gentamycine, trypsine (Life Technologies, Karlsruhe, Germany); procedure may markedly up-regulate P2X7 receptor expres- fetal calf serum (Seromed, Berlin, Germany). All chemicals were of sion in comparison with the in vivo conditions.
analytical grade.
Of course a co-localization of the P2X7- and GABA IR is not sufﬁcient to decide whether the P2X 7 IR is situated on Means ± SEM of n determinations are shown. Multiple compari- neurons or astrocytes, because both cell-types may synthesize sons with the control value were made by one-way analysis of GABA. To ﬁnd out which cells express the P2X7 IR, cortical variance (ANOVA) followed by the Kruskal–Wallis analysis and the cultures were labeled with the neuronal marker MAP2 and the Bonferroni's t-test. Two values were compared with the Dunnett's astrocytic marker GFAP (Fig. 1b, iii). The P2X7 receptor IR test or the Student's t-test as appropriate. Cumulative probability was co-localized both with MAP2 and GFAP IR on neurons plots of mIPSCs were constructed for amplitudes and interevent and astrocytes, respectively. A statistical evaluation of these intervals and compared using the Kolmogorov–Smirnov test. A data revealed that whereas a high percentage of untreated probability level of 0.05 was considered as the limit of signiﬁcance.
GABA- and MAP2-immunopositive cells co-stained also forP2X7 IR, only a low percentage of untreated GFAP-immu-nopositive cells exhibited also P2X7 IR (Fig. 1b, iv). Further, both normoxic and ischemic pre-incubation for 30 min failedto alter the number of cells that co-expressed P2X7 IR with Effect of in vitro ischemia on P2X7 receptor mRNA and GABA or MAP2 IR. In contrast, the number of cells immunoreactivity in cortical neurons and astrocytes kept co-expressing P2X7 and GFAP immunopositivity was simi- larly enhanced both after normoxic and ischemic pre- First, we investigated whether the P2X7 receptor mRNA in treatment (Fig. 1b, iv). It is suggested that, in GFAP- cortical cell cultures is up-regulated by pre-incubation immunopositive astrocytes, serum deprivation increased in ischemic, glucose-free ACSF saturated with argon, in P2X7 mRNA (see above) and its transcription to the comparison with pre-incubation in normoxic, glucose-con- corresponding receptor protein, within a period of 1–2 h.
taining ACSF (Fig. 1a). Control measurements were made in However, ischemia failed to further enhance the P2X7 mRNA the normal serum-containing culturing medium. Both norm- over the normoxic values during the same time periods.
oxic and ischemic ACSF time-dependently decreased the When cell cultures were incubated with Tris-buffered normalized cycle-threshold values indicating an increase of saline instead of the primary antibody, or with primary P2X7 receptor mRNA in real-time PCR determinations. We antibody-serum that had been pre-absorbed with peptide assume that serum deprivation, which is a well-known 2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 1421–1437 K. Wirkner et al.
Normalized cycle- as % of GABA, 7 MAP2 or GFAP 20P2X Fig. 1 Transcription and translation of P2X7 receptors in cortical cell (MAP2) and glial fibrillary acidic protein (GFAP) IR. Confocal laser cultures of rats. (a) Increased synthesis of P2X7 receptor mRNA by scanning microphotographs. (b, i) Co-localization of GABA (Cy2) serum deprivation but not by ischemic pre-incubation for 1 or 2 h. The and P2X7 (CY3) IR at the cell body of one multipolar cell. (b, ii) Co- cycle-threshold (CT) values normalized with respect to the endog- localization of GABA- and P2X7 IR at a number of bipolar cells. Many enous reference gene 18S rRNA, were used as a measure of mRNA cellular processes co-express GABA- and P2X7 IR both in (b, i) and levels. Note that the transcription of the P2X7 receptor is time- (b, ii). Scale bars in (b, i) and (b, ii), 20 lm. (b, iii) Co-localization of dependently increased by a replacement of the culturing medium by MAP2 (Cy2), P2X7 (Cy3), and GFAP (Cy5) IR on neurons and ACSF, irrespective of its glucose-content or its saturation by oxygen astrocytes, as well as on cell bodies and processes/fibers. MAP2 and or argon. Untreated (culturing medium; empty columns), normoxic GFAP were used as neuronal and astrocytic markers, respectively.
(ACSF plus oxygen; grey columns) and ischemic (glucose-free ACSF Scale bar, 50 lm. (b, iv) Cells with double-immunoreactivity for P2X7 plus argon; black columns) cells both in (a) and (b). Means ± SEM of plus GABA, MAP2 or GFAP expressed as a percentage of the total 7–15 experiments. *p < 0.05; statistically significant differences from cell population of 50 counted cells. Normoxic and ischemic pre- untreated controls. (b) Immunocytochemical characterization of the incubation was applied for 30 min. Means ± SEM of 50 cells from prominent cell-types present in untreated cultures and co-localization five culture dishes each. *p < 0.05; statistically significant difference of P2X7 receptor IR with GABA, microtubule-associated protein 2 from untreated control cells in the respective set of columns.
2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 1421–1437 Neuronal P2X7 receptors and ischemia 1427 antigen for 1 h before use (1 lg of peptide per 1 lg of at 10 mM (Fig. 2a, ii). The induction of a stimulatory BzATP antibody), no immunoﬂuorescence with either of the control action by ischemia and the shift in the potency of ATP to the procedures was observed.
higher concentration range indicate the recruitment of P2X7-like receptor activity under ischemic conditions (but see ATP- as well as 2¢-3¢-O-(4-benzoylbenzoyl)-ATP induced below). Apparently, ATP stimulated non-P2X7 receptors at stimulation of [3H]GABA release from cortical cell lower concentrations but became more selective to the P2X7 cultures and interaction with Brilliant Blue G, oxidized receptor at higher concentrations (Ralevic and Burnstock ATP and pyridoxal-phosphate-6-azophenyl-2¢, 4¢-disulfonic acid Then, we investigated the inhibitory activities of the two As previous studies revealed that approximately 80–90% of P2X7-receptor selective antagonists oxidized ATP (300 lM) neurons in cerebrocortical cultures were GABA-immunore- and Brilliant Blue G (1 lM), as well as that of the non- active (Fischer et al. 2002), storage pools for GABA were selective P2X7 receptor antagonist PPADS (30 lM), on the labeled by pre-incubation with [3H]GABA (see Methods).
effect of ATP. All these compounds markedly depressed the After 15 min of washout, the spontaneous tritium efﬂux in ATP (0.01–10 mM)-induced [3H]GABA release both in the ﬁrst 1-min sample was 0.180 ± 0.005% (n ¼ 11) of the normoxic (Fig. 2b, i) and ischemic (Fig. 2b, ii) superfusion total tritium content in the cell culture. This value remained media. It is noteworthy that Brilliant Blue G abolished a fairly constant until the end of the experiment. Subsequent to much larger fraction of the response to ATP under ischemic stimulation with 1-min superfusions of ATP (0.03–10 mM), a than under normoxic conditions, indicating that the P2X7 concentration-dependent elevation in the basal [3H]GABA receptor-mediated fraction is selectively increased during outﬂow was observed with an EC50 value of 340 lM ischemia. Neither antagonist altered the resting release (data (Fig. 2a, i and ii). The action of ATP was reversible upon not shown). Finally, Brilliant Blue G (1 lM), a selective washout (Fig. 2a, i). At 1 mM, the effect of ATP reached its P2X7 receptor antagonist, about halved the BzATP-induced plateau, and no further increase of the evoked [3H]GABA [3H]GABA release after ischemic stimulation (Fig. 2c) release was observed for 3, 6, and 10 mM of ATP (Fig. 2a, suggesting that BzATP equally activates P2X7 and non- i and ii). The relatively selective P2X7 receptor agonist P2X7 receptors at all concentrations used.
BzATP (0.1–1 mM; Ralevic and Burnstock, 1998), had no Next, we examined the possible involvement of various effect on the [3H]GABA release (Fig. 2a, ii).
transmitter systems in the effect of ATP to release After ischemic pre-treatment, the basal [3H]GABA release [3H]GABA under ischemic conditions. In these experi- in the ﬁrst 1-min sample was 0.188 ± 0.008% (n ¼ 13, ments, the cultures were stimulated with ATP (3 mM) twice, p > 0.05), and thereby not signiﬁcantly different from that with 10-min intervals. The net tritium release evoked by the measured in a normoxic ACSF. ATP (0.3–30 mM) showed a ﬁrst ATP challenge (S1) was reproducible upon a subse- lower potency but increased efﬁcacy to release [3H]GABA quent identical stimulus (S2), resulting in an S2/S1 ratio of under these conditions (Fig. 2a, i and ii). The smallest ATP 0.831 ± 0.053% (n ¼ 6; Figs 3a and b). Tetrodotoxin concentration that evoked reproducible [3H]GABA release (1 lM), a blocker of sodium-dependent action potentials (0.6 mM) was 10-times higher than under normoxic was without effect on the S2/S1 ratio, conﬁrming that ATP conditions. The effect of ATP reached its maximum at does not release by propagated action potentials an 10 mM with an EC50 value of 4.3 mM (Fig. 2a, ii). The unknown transmitter from neighboring neurons to cause maximum response to ATP in an ischemic medium was [3H]GABA release. The combined blockade of ionotropic two-times higher than under normoxic condition.
glutamate receptors of the non-NMDA- (CNQX; 10 lM) As ATP had a lower potency in ischemia than in normoxia, and NMDA-types (AP-5; 50 lM) were also ineffective.
it is possible that the ability of the cultured cells to release Similarly, the combined application of the non-selective [3H]GABA under energy deprivation is diminished. In an P2Y receptor antagonist Reactive Blue 2 (10 lM) and the attempt to compare the release ability of these cells under P2Y1 receptor selective antagonist MRS 2179 (10 lM) had normoxic and ischemic conditions, culture dishes were no effect; P2Y1 receptors were described to abundantly depolarized with a 25 mM K+-buffer (equimolar replacement occur on cortical neurons (Mora´n-Jime´nez and Matute of 20 mM NaCl with KCl) for 1 min, resulting in a 2000). Finally, the blockade of A1 and A2A adenosine 0.216 ± 0.044% evoked [3H]GABA release in normoxia receptors by DPCPX (0.25 lM) and 8-(3-chlorostyryl)caf- and 0.227 ± 0.055% in ischemia (n ¼ 4 each; p > 0.05).
feine (10 lM), respectively, did not alter the response to Hence, the ability of the cell cultures to release [3H]GABA ATP (Fig. 3b). Hence, ionotropic glutamate receptors, P2Y upon stimulation by K+ were unaffected by the ischemic receptors and adenosine receptors neither mediate the conditions. When the ischemic cultures were challenged with stimulated [3H]GABA release by ATP under ischemic BzATP, this agonist was one order of magnitude more potent conditions nor contribute to it. It appears likely that P2X7 than ATP (EC50 ¼ 120 lM), although its maximum effect (at receptors situated at the GABA neurons themselves directly 1 mM) amounted to only 40% of the maximum ATP effect trigger [3H]GABA release.
2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 1421–1437 K. Wirkner et al.
Fig. 2 Change in ATP- and 2¢-3¢-O-(4-benzoylbenzoyl)-ATP (BzATP)- of ATP or BzATP under normoxic conditions. (b) Concentration– induced transmitter release from cortical cell cultures of rats after response curves of ATP for the fractional rate of release of [3H]GABA ischemic stimulation; interaction with P2X7 receptor antagonists. (a, i) in normoxic and ischemic cell cultures. (b, i) Interaction of the P2X7 Concentration-dependent release by ATP and BzATP of [3H]GABA receptor antagonists oxidized ATP (oxiATP), Brilliant Blue G (BBG), release; experimental protocol documenting the effect of ATP under and pyridoxal-phosphate-6-azophenyl-2¢,4¢-disulfonic acid (PPADS) normoxia and ischemia. After 30 min of pre-incubation, a subsequent with ATP under normoxic conditions. s, ATP alone; ,, ATP + oxiATP 20-min incubation with [3H]GABA, and a subsequent 15-min wash- 300 lM; n, ATP + BBG 1 lM; e, ATP + PPADS 30 lM. Mean- out, 1-min samples were collected from the effluent. The cell s ± SEM of 6–12 experiments. (b, ii) Interaction of the P2X7 receptor cultures were challenged with increasing concentrations of ATP, antagonists oxiATP, BBG, and PPADS with ATP under ischemic indicated by arrows. FR% denotes the fractional release percentages.
conditions. d, ATP alone; ., ATP + oxiATP 300 lM; m, ATP + BBG Means ± SEM of 11–13 experiments. (a, ii) Concentration–response 1 lM; r, ATP + PPADS 30 lM. The experimental protocol and the curves of ATP and BzATP for the fractional rate of release of evaluation procedure was identical to that shown in Fig. 1(b, i).
[3H]GABA in normoxic and ischemic cell cultures. The agonist-induced Means ± SEM of 4–13 experiments. *p < 0.05; statistically significant [3H]GABA release is calculated by the area under the curve (AUC) difference from the effect of ATP in the absence of antagonists. (c) method from experiments similar to those shown in (a). s, ATP in Concentration–response curves of BzATP for the fractional release of normoxia; e, BzATP in normoxia; d, ATP in ischemia; r, BzATP in [3H]GABA in ischemic cell cultures. r, BzATP; m, BzATP + BBG.
ischemia. Means ± SEM of 7–13 experiments. ATP and BzATP Means ± SEM of 6–8 experiments. *p < 0.05; statistically significant caused a larger maximum release in ischemic than in normoxic difference from the effect of BzATP alone.
cultures. *p < 0.05; statistically significant difference from the effect 2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 1421–1437 Neuronal P2X7 receptors and ischemia 1429 excluded up to this stage. Therefore, we utilized electro- physiological techniques in an attempt to discriminate between the neuronal (mostly quantal) and astrocytic (mostly non-quantal) release of GABA.
In a ﬁrst series of experiments, a 140 mM KCl-containing pipette solution was used to record the membrane potential of visually identiﬁed non-pyramidal neurons, which were found to react to depolarizing current injection with action potentials (resting membrane potential, )74.8 ± 3.1 mV, n ¼ 5). In all following experiments, a 140 mM CsCl-containing pipette solution was used. BzATP (300 lM) caused only negligible inward current (3.8 ± 1.6 pA), in spite of a marked response to the ionotropic glutamate receptor agonist NMDA (30 lM;230.2 ± 37.8 pA; n ¼ 7 each), which was co-applied with glycine (10 lM) and tetrodotoxin (0.3 lM). Hence, the verylow sensitivity of the cortical neurons both to BzATP (300 lM)and ATP (1–10 mM; < 50 pA; not shown) itself appears to exclude the presence of a considerable P2X7 receptor popu- lation in untreated neurons. In addition, neither normoxic nor ischemic pre-incubation of the culture system for 30–60 min caused facilitation of the ATP- or BzATP-induced current amplitudes. As both ATP and BzATP caused only small currentamplitudes if any, we did not investigate the interaction of these agonists with P2X7 receptor antagonists.
In the usual 10–15-day-old cortical cultures, individual neurons became synaptically interconnected. The ACSFcontained tetrodotoxin (0.5 lM), which abolishes sodium- Fig. 3 Characterization of ATP-induced release of [3H]GABA in dependent action potentials but fails to alter spontaneous ischemically treated cortical cell cultures of rats. (a) Experimental pro- quantal transmitter release. At a holding potential of tocol for investigating the mode of action of ATP to release [3H]GABA in )60 mV, both excitatory glutamate receptor agonists and ischemically treated cultures. Three and 13 min after starting to collect samples, 1-min ATP (3 mM) perfusions were used as indicated by the A receptor agonists induce inward currents. Therefore, CNQX (10 lM) and AP-5 (50 lM) were added to pharma- 1, S2), resulting in a comparable tritium outflow. (b) The presynaptic effect of ATP is independent from Na+-dependent action cologically isolate GABAA receptor-mediated mIPSCs from potentials, and the activation of ionotropic glutamate receptors, as well contaminating NMDA and non-NMDA receptor-mediated as adenosine and P2Y receptors. Tetrodotoxin (TTX; 1 lM), 6-cyano-7- excitatory mEPSCs.
nitroquinoxaline-2,3-dione (CNQX; 10 lM) plus D(–)-amino-5-phos- The control frequency (1.9 ± 0.3 Hz) and amplitude phonopentanoic acid (AP-5; 50 lM), Reactive Blue 2 (RB2; 10 lM) plus (15.6 ± 1.2 pA; n ¼ 42 each) of mIPSCs, respectively, could 2¢-deoxy-N6-methyladenosine 3¢,5¢-bisphosphate (MRS; 10 lM), and 8- be pooled from all experiments, as there was no difference cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.25 lM) plus 8-(3-chloro- between these parameters of normoxically and ischemically styryl)caffeine (CSC; 10 lM) were all applied 6 min before S2.
treated cells. The frequency of mIPSCs slightly decreased Means ± SEM of the S2/S1 ratios of 4–6 experiments.
after the ﬁrst 5 min and then remained stable both afternormoxic (5 min, )8.4 ± 0.9%; 10 min, )9.1 ± 1.0% changefrom 0 min; n ¼ 5) and ischemic (5 min, 10.9 ± 2.3%; 10 min, )9.2 ± 5.5% change from 0 min; n ¼ 4) incubation.
currents and increase in the frequency of miniature It is noteworthy that the frequency increase in the normoxic inhibitory postsynaptic potentials of cortical neurons; cultures by BzATP (300 lM) was also stable over 10 min interaction with Brilliant Blue G (n ¼ 5), and Brilliant Blue G (0.3 lM) did not alter the Although in the neuronally enriched cultures used by us, mIPSC frequency when given alone (n ¼ 6).
astrocytes are only a minor fraction of the total cell number, The GABAA receptor antagonist bicuculline (10 lM) abol- and in addition b-alanin (1 mM) was included into the ACSF ished the mIPSC amplitudes measured after ischemic pre- solution to prevent tritium uptake into glial cells (Iversen and incubation; this effect was reversible on washout (Fig. 4a, i; Kelly 1975; see Materials and methods), the possibility that n ¼ 3). The thereby identiﬁed spontaneous quantal release of ATP and BzATP release GABA from astrocytes (Pannicke GABA was reversibly increased by BzATP (300 lM) applica- et al. 2000; Illes and Ribeiro 2004) could not be completely tion for 5 min (Fig. 4a, ii). The inter-event interval distribution 2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 1421–1437

K. Wirkner et al.
of this cell was shifted towards the left, reﬂecting an increase of (Fig. 4b, i and ii). Finally, neither the frequency nor the mIPSC frequency by BzATP (Fig. 4a, iii). Figure 4(a, iv) amplitude of mIPSCs changed within 5 min of superfusion shows that the number of events at each mIPSC amplitude was with medium, irrespective of whether the pre-incubation was augmented by BzATP. As the mean mIPSC amplitude was not normoxic or ischemic (Fig. 4b, i and ii). The percentage altered, the data are compatible with a frequency increase potentiation of the mIPSC frequency by BzATP (300 lM), in + Bicuculline 10 µM mIPSC amplitude (pA) Inter-event interval (ms) Fig. 4 Modulation by 2¢-3¢-O-(4-benzoylbenzoyl)-ATP (BzATP) of before (filled bars) and after the application of BzATP (empty bars).
GABAA receptor-mediated mIPSCs in neurons of rat cortical cell cul- Data obtained in the cell shown in (a, ii) were further analyzed in (a, iii) tures after normoxic or ischemic pre-treatment. Miniature inhibitory and (a, iv). (b, i) Increase of the mean frequency (empty columns) but postsynaptic currents (mIPSCs) were recorded at a holding potential not amplitude (filled columns) by BzATP in ischemic cells (right set of of )60 mV. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 lM), columns). The changes were expressed as percentage potentiation of D(–)-amino-5-phosphonopentanoic acid (AP-5; 50 lM) and tetrodo- the time-matching controls recorded in drug-free ACSF (left set of toxin (TTX; 0.5 lM) were all present in the medium. Average values of bars). Only seven out of 10 cells responded to the effect of BzATP. (b, amplitude and frequency of mIPSCs were calculated during a control ii) Increase of the mean frequency (empty columns) but not amplitude period of 3 min and during the last 3 min of the subsequent application (filled columns) by BzATP in normoxic cells (right set of columns). The of BzATP (300 lM) for 10 min in total. (a, i) Consecutive traces changes were expressed as percentage potentiation of the time- showing typical mIPSCs before, during and 10 min after the applica- matching controls recorded in drug-free ACSF (left set of bars in b, i tion of bicuculline (10 lM) in normoxic cultures. (a, ii) Consecutive and b, ii; ischemia, n ¼ 4; normoxia, n ¼ 5). Only five out of 10 cells traces showing typical mIPSCs before, during and 10 min after the responded to the effect of BzATP. p < 0.05; statistically significant application of BzATP (300 lM) in ischemic cultures. (a, iii) Cumulative difference from zero. #p < 0.05; statistically significant difference from plots of amplitude distribution before (continuous line) and after the the effect of BzATP in ischemic cells.
application of BzATP (dotted line). (a, iv) The amplitude distribution 2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 1421–1437 Neuronal P2X7 receptors and ischemia 1431 comparison with the time-matching controls, documented a 1094.0 ± 243.1 pA marked increase in the case of seven out of 10 ischemic cells 1085.3 ± 293.4 pA (n ¼ 5; p > 0.05) 6 min after super- (Fig. 4b, i) and a less pronounced increase in the case of ﬁve fusion with BzATP (300 lM). Hence, BzATP-sensitive P2X7 out of 10 normoxic cells (Fig. 4b, ii). At the same time, BzATP receptors appear to be situated at many GABAergic nerve (300 lM) failed to alter the mean amplitude of mIPSCs when terminals innervating neighbouring cells in cortical cell compared with the time-matching control group.
cultures. It is unlikely that the enzymatic degradation of A 10-times lower concentration of BzATP (30 lM) also BzATP to Bz-adenosine, which is inactive at adenosine tended to increase the frequency of mIPSCs to a larger extent in receptors but may hetero-exchange with intracellular adeno- the ischemic cells (51.1 ± 13.5%; ﬁve out of nine neurons) sine (Kukley et al. 2004), results in the activation of than in the normoxic cells (18.9 ± 2.4%; four out of 13 facilitatory presynaptic A2A receptors (for 3[GABA] release neurons; p > 0.05), although this change did not reach the see previous section). In this case, the P2X7 receptor level of statistical signiﬁcance. In addition, there was an antagonistic Brilliant Blue G would certainly not be able to increase in the number of responsive cells in the ischemic abolish the effect of BzATP.
cultures versus the normoxic ones (55% vs. 31%; see above).
As the BzATP-induced facilitation of mIPSC frequency The effect of BzATP in a subset of neurons only indicates a absolutely depended on the presence of Ca2+ in the non-homogeneity of the total GABAergic cell population extracellular medium, we conclude that P2X7 receptor investigated. The rise time from 10% to 90% of the peak activation triggers an exocytotic, neuronal release of GABA.
current amplitude (s10)90%) and the time constant of decay The ﬁnding that BzATP failed to increase the outﬂow of (sdecay) were calculated to characterize mIPSCs. The time- [3H]GABA (sum of the quantal and non-quantal release), course of the mIPSCs, determined both in the normoxically although it did slightly increase the mIPSC frequency under (s10)90%, 4.2 ± 0.3 ms; sdecay, 34.2 ± 1.1 ms; n ¼ 5) and normoxic conditions, may indicate that BzATP mostly ischemically treated group of cells (s10)90%, 3.9 ± 0.3 ms; facilitates the quantal, exocytotic secretion of transmitter sdecay, 35.1 ± 1.3 ms; n ¼ 7), was not changed by BzATP (Vyskocil and Illes 1997) of GABA.
Next, we investigated whether BzATP facilitates the ATP- and 2¢-3¢-O-(4-benzoylbenzoyl)-ATP-induced quantal release of GABA via P2X7 receptor activation. In increase in [Ca2+]i of cortical neurons and astrocytes and fact, superfusion with the P2X7 receptor antagonist Brilliant interaction with Brilliant Blue G and pyridoxal- Blue G (0.3 lM) for 5 min did not alter the amplitude or frequency of mIPSCs recorded from normoxically pre- Although P2X7 receptor activation unequivocally stimula- treated neurons (n ¼ 5; Fig. 5a, i). However, Brilliant Blue ted GABA release, in neuronally enriched and ischemically G almost abolished the facilitatory effect of BzATP (300 lM; primed cortical cells, it is unclear whether the release of Fig. 5a, ii and iii). As only a fraction of cortical neurons GABA from the minor population of astrocytes is a Ca2+- responded to BzATP application (Fig. 4), only those cells dependent exocytotic process contributing to the neuronal were included into this evaluation, which responded to the release. To answer these questions, experiments were agonist with a frequency increase. In a further series of designed to measure the cytosolic-free Ca2+ concentration mIPSC measurements, superfusion with a Ca2+-free ACSF by the fura-2 technique; the ﬂuorescence ratio (340/ (no Ca2+, 1 mM EGTA) for 10 min in total, did not alter the 380 nm) was taken as a relative measure of free [Ca2+]i frequency (5 min, )6.4 ± 12.9%; 10 min, 5.5 ± 8.7%; n ¼ (for absolute values see the Materials and methods 7), but slightly decreased the amplitude (5 min, )20.0 ± 3.8%; 10 min, )26.4 ± 4.2%; n ¼ 7; p < 0.05), when compared The responses to high external K+ (50 mM; cell depolar- with the 0-min value determined in a Ca2+-containing ACSF.
ization and subsequent opening of voltage-gated Ca2+ When cortical cultures were superfused at ﬁrst for 5 min with channels), as well as to ATP (300 lM) and BzATP Ca2+-free ACSF and subsequently for another 5 min with (300 lM) were tested on neuronal somata and proximal BzATP (300 lM) in the continuing absence of Ca2+, there dendrites, as well as on astrocytes. We have chosen was no change either in the frequency (Ca2+-free ACSF, astrocyte-free areas for the determination of [Ca2+]i in )8.3 ± 8.7%; BzATP, )18.7 ± 6.7%; n ¼ 11; p > 0.05) or dendrites. Measurements were made either in untreated in the amplitude (Ca2+-free ACSF, 6.2 ± 8.6%; BzATP, cultures, or in normoxically/ischemically pre-incubated cul- 2.7 ± 11.1%; n ¼ 11; p > 0.05) of mIPSCs, compared with tures (Fig. 6). Whereas in untreated cultures, high external the 0-min values measured in Ca2+-containing ACSF.
K+ caused a pronounced [Ca2+]i signal in all neuronal cell Our experiments with mIPSCs suggest that BzATP and bodies tested, the responses in dendrites and astrocytes Brilliant Blue G interact at a presynaptic site of action. This became gradually smaller (Fig. 6a; Bennett et al. 2003). By idea was strengthened by the fact that BzATP failed to alter contrast, the ATP- and BzATP-induced [Ca2+]i responses current responses to the GABAA receptor agonist muscimol were very small in neuronal somata, but considerably larger (10 lM; Fig. 5b). The amplitude of muscimol-induced in dendrites and especially in astrocytes.
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BzATP-induced potentiation (%) Fig. 5 Modulation by 2¢-3¢-O-(4-benzoylbenzoyl)-ATP (BzATP) of showing typical experiments in two individual cells. (a, iii) Increase of GABAA receptor-mediated miniature inhibitory postsynaptic currents the mean frequency (empty columns) but not amplitude (filled col- (mIPSCs) in neurons of rat cortical cell cultures after normoxic pre- umns) of mIPSCs by BzATP in normoxic cells; no increase of mIPSC treatment and interaction with Brilliant Blue G (BBG); no effect of frequency in the presence of BBG (n ¼ 7; see a, ii). The changes were BzATP on the current response to muscimol. mIPSCs were recorded expressed as percentage potentiation of the time-matching controls at a holding potential of )60 mV. 6-Cyano-7-nitroquinoxaline-2,3-di- recorded in drug-free ACSF for 10 min and in ACSF containing BBG one (CNQX; 10 lM), D(–)-amino-5-phosphonopentanoic acid (AP-5; (0.3 lM) for an additional 5 min (n ¼ 5; see a, i). *p < 0.05; statistically 50 lM) and tetrodotoxin (TTX; 0.5 lM) were all present in the medium.
significant difference from zero. #p < 0.05; statistically significant dif- Average values of amplitude and frequency of mIPSCs were calcu- ference from the effect of BzATP in the absence of BBG. (b) Effect of lated during a control period of 3 min, during the last 3 min of the BzATP (300 lM) on the inward current induced by muscimol (10 lM) subsequent application BBG (0.3 lM) for 5 min, as well as during the locally superfused for 2 s with 3-min intervals. BzATP was superfused last 3 min of further superfusion with BBG alone or in combination with for 6 min during two consecutive muscimol applications. Typical BzATP (300 lM) for a further 5 min. (a, i and ii) Consecutive traces recording out of five similar ones.
In normoxic and ischemic cell cultures, the responses of (Figs 6a–c).
the neuronal cell bodies to high external K+ tended ATP-induced [Ca2+]i transients were clearly depressed by to decrease when compared with those of untreated the normoxic/ischemic pre-incubation in astrocytes. The preparations, although this change did not reach the level measurements on dendrites indicated no changes after 2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 1421–1437 Neuronal P2X7 receptors and ischemia 1433 In the following experiments, we tried to ﬁnd out which type of P2 receptor is situated at dendrites and astrocytes of cortical cultures. By deﬁnition, P2X receptors are ligand-gated cationicchannels and therefore allow the passage of Ca2+ from the external medium, whereas P2Y receptors may be coupled to Gproteins, which release Ca2+ from its intracellular stores viainositol 1,4,5-trisphosphate (Abbracchio and Burnstock 1994; Illes et al. 2000). Therefore, we roughly characterized the P2 receptors by measuring the increase of [Ca2+]i in proximal dendrites of cortical neurons and astrocytes in response to ATP (300 lM), both in the presence and absence of external Ca2+ orcyclopiazonic acid (Fig. 7a). The [Ca2+]i transients of the neuronal cell bodies were not evaluated, because of their very small amplitudes (Figs 6a–c). Cyclopiazonic acid inhibits the Ca2+-ATPase of the endoplasmatic reticulum and thereby depletes this intracellular Ca2+ pool. A Ca2+-free medium (plus1 mM EGTA) and cyclopiazonic acid (10 lM) both depressedthe ATP-evoked increase of [Ca2+]i, indicating that proximal dendrites and astrocytes possess a mixed P2X/P2Y receptor population (Fig. 7a).
As our primary goal was to prove or disprove an ischemia- ∆ fluorescence ratio (340/380 nm) 0.0 induced supersensitivity of P2X7 receptors in the investigated cells, we also utilized the P2X7 receptor preferential agonistBzATP (300 lM) in interaction with the selective and non-selective P2X 7 receptor antagonists Brilliant Blue G (1 lM) and PPADS (30 lM), respectively (Ralevic and Burnstock1998; Jiang et al. 2000; Fig. 7b). Brilliant Blue G failed todepress the increase of [Ca2+]i caused by either ATP or BzATP and both following normoxic and ischemic stimulation (Fig. 7b, i). By contrast, PPADS markedly inhibited [Ca2+]i responses to both agonists in dendrites as well as astrocytes (Fig. 7b, ii and iii). Although under normoxic conditionsPPADS depressed the response to ATP in astrocytes with a Fig. 6 Increase of [Ca2+]i caused by a high potassium-containing slightly lower efﬁciency than in the proximal dendrites, external medium, ATP or 2¢-3¢-O-(4-benzoylbenzoyl)-ATP (BzATP) in PPADS was absolutely equipotent in blocking agonist effects cell somata and proximal dendrites of neurons as well as in astro- cytes of cortical cell cultures of rats. Cells were loaded with the Ca2+ under normoxia and ischemia in neurons and astrocytes.
indicator fura-2/AM (5 lM). The fluorescence ratio (340/380 nm) was In conclusion, the measurement of [Ca2+]i transients in taken as a measure of [Ca2+]i. Cells were depolarized by a 50 mM proximal dendrites and astrocytic cell bodies failed to K+-containing extracellullar medium (empty columns) for 3 s every support the notion that P2X7 receptor activation initiates a 12 min. ATP (300 lM; hatched columns) or BzATP (300 lM; cross- Ca2+-dependent release of GABA from these cellular sites.
hatched columns) were applied for 10 s every 12 min. Responses of The quantal release of transmitters (e.g. glutamate) from non-treated controls (a, n ¼ 12) as well as normoxically (b, n ¼ 9) and astrocytes has been shown to be triggered by an increase of ischemically (c, n ¼ 11) pre-incubated cultures are shown. *p < 0.05; the intracellular Ca2+ concentration leading to the activation significant difference from ATP. There is no significant difference of the exocytotic machinery (Pasti et al. 2001; Nedergaard within the K+-, ATP- and BzATP-induced [Ca2+]i responses between et al. 2002). However, this was certainly not the case under (a) (b) and (c), with the exception of the ATP signals in the astrocytes the present experimental conditions.
of normoxically and ischemically incubated cultures, which are smaller than the respective signals under normal conditions (p < 0.05).
normoxic/ischemic pre-incubation. Finally, ischemia incomparision with normoxia did not alter the responses to A marked decline of intracellular ATP levels with a either ATP or BzATP in either cell-type. Hence, all these concomitant efﬂux of ATP into the extracellular space has results unequivocally argue against an up-regulation of P2 been shown to occur in the rat brain during the ﬁrst minutes receptor function in the somata and dendrites of neurons as after oxygen depletion in vivo (Hisanaga et al. 1986; Volonte well as in astrocytes.
et al. 2003). Cerebral ischemia also activated microglial cells 2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 1421–1437 K. Wirkner et al.
damage (Franke et al. 2004) of the rat brain likewise led to the expression of previously absent P2X7 receptors on astrocytes and neurons.
Useful model systems to study ischemic alterations under in vitro conditions are cortical cell cultures in which hypoxia was caused by substituting oxygen in the medium by argon (Gu¨nther et al. 2002; Reinhardt et al. 2003).
Argon has been reported to rapidly displace residual oxygen from the incubation medium and thereby to lead to Brilliant Blue G 1 µM a much faster decline of cellular oxygen levels than exposure to nitrogen (Kusumoto et al. 1996). The duration of the ischemic stimulus was 30–60 min, because already a 30-min period was shown previously to markedly decrease the ATP/ADP ratio regarded as a measure of the metabolic limitation (Gu¨nther et al. 2002; Reinhardt et al. 2003).
In previous in vivo experiments, 4 days after MCAO a marked up-regulation of P2X7 receptor expression was observed both by immunohistochemistry and western-blot analysis (Franke et al. 2004). On a ﬁrst glance, these results appear to contrast with our data demonstrating no change of 7 receptor IR by ischemia at either neurons or astrocytes.
We assume that the ischemic stimulus in the long term leads to the synthesis of new P2X7 receptors, although in the short term an increased functional response may prevail. Under in vivo conditions, cortical neurons and astrocytes of the intact brain were found to be devoid of P2X7 receptor IR Fig. 7 Characterization of P2 receptor types on proximal dendrites (Franke et al. 2004). Such an immunoreactivity was, how- and astrocytes by measurements of [Ca2+]i in cortical cell cultures of ever, present on cultured astrocytes kept in a serum- rats. Cells were loaded with the Ca2+ indicator fura-2/AM (5 lM).
containing medium and was up-regulated on subsequent The fluorescence ratio (340/380 nm) was taken as a measure of serum deprivation. It is suggested that the culturing proce- [Ca2+]i. (a) Effects of a Ca2+-free medium (containing 1 mM EGTA) or dures may initiate the synthesis of this ‘suicide receptor' as cyclopiazonic acid (CPA; 10 lM) on ATP-induced Ca2+ responses in an early apoptotic signal (North 2002).
dendrites (hatched columns) and astrocytes (cross-hatched columns).
The cultures were continuously superfused with physiological solution Recently, two publications described the presence of P2X7 and stimulated four times (S1–S4) with ATP (300 lM) for 10 s every receptor IR in the hippocampus of P 2X = mice, and 12 min (b, ii). A Ca2+-free medium or cyclopiazonic acid were super- therefore seriously questioned the validity of the assumedly fused for 10 min both before and during the stimulation with ATP (S3).
selective antibodies (Kukley et al. 2004; Sim et al. 2004).
Drug effects were evaluated as the percentage change of the ATP- However, P2X7 receptor IR was missing in the retinal induced signal at S3 versus the second control signal at S2. (b) Effect ganglion cell layer of P 2X = mice, when compared with of the P2X7 receptor antagonists Brilliant Blue G (1 lM; b, i) and their wild-type controls (Franke et al. 2005), suggesting a PPADS (30 lM; b, ii and iii) on the [Ca2+]i responses caused by ATP region-speciﬁc expression of immunoreactivity for neuronal P2X7 receptors in the brain of the knockouts. In addition, in (BzATP; 300 lM; dashed columns) in neuronal dendrites and astro- granule cells cultured from cerebella of P 2X = cytes. The pre-incubation condition (normoxia or ischemia) is indicated by the absence or presence of grey shading. Stimulation by ATP and 7-like receptors were found that keep some of BzATP, superfusion with the antagonists, and the evaluation of drug the properties of the genuine receptor (Sanchez-Nogueiro effects was identical to that described in (a). *p < 0.05; significant et al. 2005). These receptors may represent splice variants difference from the effect of ATP (300 lM) in dendrites after normoxic containing the epitope (e.g. Arg-576 to Tyr-595 in their incubation. Means ± SEM of n ¼ 4–7 experiments in each column.
C-terminus) against which antibodies were generated, but Typical recording made on a proximal dendrite from an ischemically being devoid of amino acid sequences responsible for treated cell culture (b, ii).
important receptor functions (Cheewatrakoolpong et al.
2005). Hence, the general denial to utilize antibodies for as well as increased the expression of P2X7 receptors on this identifying P2X7 receptor protein in the brain and the refusal cell type (Collo et al. 1997; Morigiwa et al. 2000). In to accept the existence of neuronal P2X7 receptors appears to addition, both mechanical (Franke et al. 2001) and ischemic be at the present time premature.
2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 1421–1437 Neuronal P2X7 receptors and ischemia 1435 The main ﬁnding of the present study is that neuronal preferential agonist BzATP were similar when measured after presynaptic P2X7 receptors exhibited an increased response normoxic and ischemic pre-incubation. By contrast, BzATP to ATP and BzATP after ischemic stimulation in spite of no increased the frequency of the GABAergic mIPSCs in a changes in P2X7 mRNA and IR. As our culturing conditions manner absolutely depending on external Ca2+; this suggests favored an enrichment of GABAergic interneuron-like cells that in the network formed by cultured interneurons, the (Fischer et al. 2002), we measured the release of previously spontaneous quantal release of the transmitter GABA is incorporated [3H]GABA in response to increasing concen- enhanced by presynaptic P2X7 receptors situated at the axon trations of ATP and BzATP mostly from this cell population.
terminals themselves. A presynaptic site of action of BzATP Although BzATP had no effect in normoxic cell cultures, it is favored by the change in mIPSC frequency without a became active in ischemically stimulated cell cultures. The corresponding change in amplitude and by the failure of maximum effect of ATP to release [3H]GABA was markedly BzATP to interfere with the postsynaptic effect of the potentiated by ischemia, although at the same time a GABAA receptor agonist muscimol.
rightward shift of the curve occurred. Because ATP is a ATP and BzATP induced similar increases in neuronal non-selective agonist at all P2X (and many P2Y) receptor (somatic/dendritic) and astrocytic [Ca2+]i transients after pre- types, and only high concentrations of the agonist activate incubation with normoxic medium or with hypoxic and P2X7 receptors, it appears that ischemia potentiates the P2X7 glucose-free medium. The strong depression of the [Ca2+]i receptor function, but depresses the effect of some other response to the two agonists by the non-selective P2X/P2Y release-enhancing P2 receptors. The non-selective antagonist receptor antagonist PPADS in the neuronal dendrites as well as PPADS and the selective P2X7 antagonists Brilliant Blue G in the astrocytes conﬁrmed the participation of P2 receptors.
and oxidized ATP, all inhibited the effect of ATP. The results However, the selective P2X7 receptor antagonist Brilliant Blue with Brilliant Blue G are especially convincing, as a much G did not alter the ATP and BzATP effects excluding the larger fraction of the release was abolished by the antagonist involvement of this receptor subtype. Therefore, these data do after the ischemic, in comparison with the normoxic pre- not support a Ca2+-dependent exocytotic GABA release incubation. It is noteworthy that Brilliant Blue G at 1 lM has initiated by P2X7 receptors situated at the proximal dendrites been described not to considerably interfere with any of the of neurons or at the cell bodies of astrocytes (see Results; Pasti recombinant P2X receptors expressed in HEK293 cells et al. 2001; Nedergaard et al. 2002).
(Jiang et al. 2000). In addition, Brilliant Blue G at this In conclusion, neuronal P2X7 receptors may release concentration did not alter the [Ca2+]i transients caused by glutamate (hippocampus, Deuchars et al. 2001; Sperla´gh ATP/BzATP either in dendrites or astrocytes (see below), et al. 2002) and, in consequence (Sperla´gh et al. 2002) or excluding the blockade of P2X receptors of the non-P2X7 independently of this effect (present study), GABA. It is subtypes by this antagonist.
necessary to emphasize that an unequivocal evidence for the Hence, the question arises, whether P2X7 receptors are involvement of the P2X7 receptor subtype in these processes situated at the GABAergic neuronal terminals themselves or is still lacking. Complicating factors are the unavailability of at neighboring neurons/astrocytes. The measurement of absolutely selective agonists, antagonists, and possibly also intrasynaptosomal [Ca2+]i transients in pinched off synaptic antibodies for P2X7 receptors. However, based on the terminals conﬁrmed an inﬂux of Ca2+ via P2X7 receptor multitude of experimental approaches used by us, we are channels (Lundy et al. 2002; Miras-Portugal et al. 2003).
quite conﬁdent that ATP or BzATP by acting on neuronal Alternative possibilities are that the release of GABA occurs P2X7 receptors in fact release GABA and that this effect is from astrocytes (Pannicke et al. 2000), or that neighboring greatly enhanced after a preceding ischemic stimulus. It is excitatory neurons bearing P2X7 receptors indirectly stimu- suggested that an augmentation of the excitatory glutamat- late the GABAergic neuronal activity. However, an indirect ergic system may facilitate the ischemia-induced neuronal mode of action by a transmitter or neuromodulator is most damage, whereas the augmentation of the inhibitory unlikely, as this compound does not act via ionotropic GABAergic system may be neuroprotective; the sum of glutamate receptors (CNQX, AP-5), P2Y receptors (Reactive these two opposing inﬂuences may determine the infarct size.
Blue 2, MRS 2179) or A1 and A2A receptors (DPCPX, CSC), The existence of a massive GABAergic innervation in the as proved by the respective antagonists. An action potential- parietal cortex may explain the ﬁnding that in P2X7 receptor- dependent release of a transmitter from neighbouring neu- deﬁcient mice or after the application of P2X7 receptor rons could be discarded, as tetrodotoxin failed to depress the antagonists, MCAO caused an infarct size of similar increase in the ATP-induced [3H]GABA release after ische- magnitude than in untreated wild-type mice (Le Feuvre mia. Based on these data as a whole, a direct stimulation of et al. 2003). However, after mechanical damage to the spinal GABAergic nerve terminals by their P2X7 receptor popula- cord, P2X7 receptor antagonists signiﬁcantly improved both tion is the most likely mechanism of action.
functional recovery and diminished cell death in the The membrane currents caused by high concentrations of peritraumatic zone (Wang et al. 2004). This may be due to the non-selective agonist ATP or of the P2X7 receptor the scarce GABAergic innervation of the spinal cord.
2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 1421–1437 K. Wirkner et al.
Franke H., Grosche J., Scha¨dlich H., Kru¨gel U., Allgaier C. and Illes P.
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2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 1421–1437

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CHIMIE NOUVELLE N° 117 - décembre 2014 Joséphine K. NTUMBA(1)(2), Kalulu M. TABA(2), Raphaël ROBIETTE(1)*(1) Institut de la Matière Condensée et des Nanosciences (IMCN), Université catholique de Louvain, Place Louis Pasteur 1 bte L4.01.02, B-1348 Louvain-la-Neuve, Belgique. Tel: +32 (0)10 47 91 76, Fax: +32 (0)10 47 42 68, [email protected] http://www.uclouvain.be/raphael.robiette(2) Département de Chimie, Université de Kinshasa, P. O. Box 190, Kinshasa

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Dear CM/PLM Professional, Please find enclosed DSD's newsletter, addressing news in CM, PLM, ERP and Process Improvements that we thought might interest you. We would appreciate receiving comments and suggestions to make this newsletter more helpful and interesting. We encourage you to send us news you hear that might be of interest to others of the CM community. Subscription to this newsletter is free-of-charge. To subscribe to our mailing list please send us an e-mail with SUBSCRIBE in the subject. If you do not want to receive further issues please send us an e-mail with REMOVE in the subject. CAD AND DESIGN TOOLS 1. ANSYS Reports First Quarter 2016 Financial Results ANSYS, Inc. today announced its financial results for the first quarter of 2016. The Company reported GAAP and non-GAAP revenue growth in constant currency of 6% and 5%, respectively, and GAAP and non-GAAP diluted earnings per share of $0.63 and $0.77, respectively, for the quarter. Recurring revenue, which is comprised of lease license and maintenance revenue, totaled 78% of revenue for the first quarter . 2. Gerber Technology's Digital Solution Links Data, Smart Machines to Drive Mass Production and Mass Customization Gerber Technology is showcasing its Digital Solution at the Texprocess Americas show in Atlanta. Apparel and industrial companies are under significant competitive pressures to design, develop and produce their products faster and more efficiently while ensuring they get the right products to market at the right time and at the right price. Whether producing volume for the masses or customizing small lots, companies are struggling because of an inability to move data from process to process and machine to machine. At Texprocess, Gerber will demonstrate each step of its Digital Solution to illustrate how companies can network their software and smart machines to form an end-to-end solution to help meet the workflow challenges of mass production and mass customization. 3. FARO Reports First Quarter 2016 Financial Results FARO Technologies, Inc. (FARO) today announced its financial results for the first quarter ended March 31, 2016. Sales for the quarter ended March 31, 2016 were $75.7 million, up 8.3% compared with $69.9 million in the first quarter last year. Excluding approximately $0.9 million of unfavorable foreign exchange impacts, first quarter sales would have increased approximately 10% as compared with the first quarter of 2015. The Company's sales growth was driven primarily by higher metrology sales within the Americas and Asia-Pacific regions. Gross margin for the quarter was 56.3% compared with 56.6% in the prior year period with product and service margins remaining relatively consistent with the prior year period. Operating income for the quarter was $4.3 million compared with $1.9 million in the prior year period, reflecting increased sales volume partially offset by a modest increase in operating expenses. 4. Cadence Expands OrCAD Solution to Address Flex and Rigid-Flex Design Challenges for IoT, Wearables and Mobile Devices Today at CDNLive EMEA, Cadence Design Systems, Inc. announced the OrCAD 17.2-2016 release with new capabilities for OrCAD® Capture, PSpice® Designer and PCB Designer that address challenges with flex and rigid-flex design as well as mixed-signal simulation complexities