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W2099206674 abstract "Sepsis-associated encephalopathy (SAE) is a frequent but poorly understood neurological complication in sepsis that negatively influences survival. Here we present clinical and experimental evidence that this brain dysfunction may be related to altered neurotransmission produced by inflammatory mediators. Compared with septic patients, SAE patients had higher interleukin-1β (IL-1β) plasma levels; interestingly, these levels decreased once the encephalopathy was resolved. A putative IL-1β effect on type A γ-aminobutyric acid receptors (GABAARs), which mediate fast synaptic transmission in most cerebral inhibitory synapses in mammals, was investigated in cultured hippocampal neurons and in Xenopus oocytes expressing native or foreign rat brain GABAARs, respectively. Confocal images in both cell types revealed that IL-1β increases recruitment of GABAARs to the cell surface. Moreover, brief applications of IL-1β to voltage-clamped oocytes yielded a delayed potentiation of the GABA-elicited chloride currents (IGABA); this effect was suppressed by IL-1ra, the natural IL-1 receptor (IL-1RI) antagonist. Western blot analysis combined with IGABA recording and confocal images of GABAA Rs in oocytes showed that IL-1β stimulates the IL-1RI-dependent phosphatidylinositol 3-kinase activation and the consequent facilitation of phospho-Akt-mediated insertion of GABAARs into the cell surface. The interruption of this signaling pathway by specific phosphatidylinositol 3-kinase or Akt inhibitors suppresses the cytokine-mediated effects on GABAAR, whereas activation of the conditionally active form of Akt1 (myr-Akt1.ER*) with 4-hydroxytamoxifen reproduces the effects. These findings point to a previously unrecognized signaling pathway that connects IL-1β with increased “GABAergic tone.” We propose that through this mechanism IL-1β might alter synaptic strength at central GABAergic synapses and so contribute to the cognitive dysfunction observed in SAE. Sepsis-associated encephalopathy (SAE) is a frequent but poorly understood neurological complication in sepsis that negatively influences survival. Here we present clinical and experimental evidence that this brain dysfunction may be related to altered neurotransmission produced by inflammatory mediators. Compared with septic patients, SAE patients had higher interleukin-1β (IL-1β) plasma levels; interestingly, these levels decreased once the encephalopathy was resolved. A putative IL-1β effect on type A γ-aminobutyric acid receptors (GABAARs), which mediate fast synaptic transmission in most cerebral inhibitory synapses in mammals, was investigated in cultured hippocampal neurons and in Xenopus oocytes expressing native or foreign rat brain GABAARs, respectively. Confocal images in both cell types revealed that IL-1β increases recruitment of GABAARs to the cell surface. Moreover, brief applications of IL-1β to voltage-clamped oocytes yielded a delayed potentiation of the GABA-elicited chloride currents (IGABA); this effect was suppressed by IL-1ra, the natural IL-1 receptor (IL-1RI) antagonist. Western blot analysis combined with IGABA recording and confocal images of GABAA Rs in oocytes showed that IL-1β stimulates the IL-1RI-dependent phosphatidylinositol 3-kinase activation and the consequent facilitation of phospho-Akt-mediated insertion of GABAARs into the cell surface. The interruption of this signaling pathway by specific phosphatidylinositol 3-kinase or Akt inhibitors suppresses the cytokine-mediated effects on GABAAR, whereas activation of the conditionally active form of Akt1 (myr-Akt1.ER*) with 4-hydroxytamoxifen reproduces the effects. These findings point to a previously unrecognized signaling pathway that connects IL-1β with increased “GABAergic tone.” We propose that through this mechanism IL-1β might alter synaptic strength at central GABAergic synapses and so contribute to the cognitive dysfunction observed in SAE. Acute impairment of mental function is often the first manifestation of sepsis. This condition is best defined as “sepsis-associated encephalopathy” (SAE) 4The abbreviations used are: SAE, sepsis-associated encephalopathy; a.u., arbitrary units; CSF, cerebrospinal fluid; 4-HT, 4-hydroxytamoxifen; ER, estrogen receptor; GABA, γ-aminobutyric acid; GABAAR, GABA type A receptor; IL-1ra, IL-1 receptor antagonist; IL-1RI, IL-1 receptor type-I; myr-Akt1.ER*, conditionally active form of Akt1; PB, phosphate buffer; PBS, phosphate-buffered saline; PI3K, phosphatidylinositol 3-kinase; SAPS, simplified acute physiology scoring; TNFR-I + II, TNF receptor type I and II; PKC, protein kinase C; LPS, lipopolysaccharide. 4The abbreviations used are: SAE, sepsis-associated encephalopathy; a.u., arbitrary units; CSF, cerebrospinal fluid; 4-HT, 4-hydroxytamoxifen; ER, estrogen receptor; GABA, γ-aminobutyric acid; GABAAR, GABA type A receptor; IL-1ra, IL-1 receptor antagonist; IL-1RI, IL-1 receptor type-I; myr-Akt1.ER*, conditionally active form of Akt1; PB, phosphate buffer; PBS, phosphate-buffered saline; PI3K, phosphatidylinositol 3-kinase; SAPS, simplified acute physiology scoring; TNFR-I + II, TNF receptor type I and II; PKC, protein kinase C; LPS, lipopolysaccharide. in order to stress the absence of direct infection of the central nervous system. The syndrome is characterized by a marked decrease in cerebral activity resulting in confusion, somnolence, and disorientation and decreased consciousness (1Papadopoulos M.C. Davies D.C. Moss R.F. Tighe D. Bennett E.D. Crit. Care Med. 2000; 28: 3019-3024Crossref PubMed Scopus (237) Google Scholar). As in other kinds of metabolic encephalopathy, diagnosis of this condition is dependent on the exclusion of all other possible causes of brain dysfunction (2Wilson J.X. Young G.B. Can. J. Neurol. Sci. 2003; 30: 98-105Crossref PubMed Scopus (128) Google Scholar). The exact incidence of SAE is unknown. Sprung et al. (3Sprung C.L. Peduzzi P.N. Shatney C.H. Schein R.M. Wilson M.F. Sheagren J.N. Hinshaw L.B. Crit. Care Med. 1990; 18: 801-806Crossref PubMed Scopus (344) Google Scholar) reported that 23% of a large series of 1333 septic patients enrolled in the Veterans Affairs Systemic Sepsis Study developed encephalopathy. The same authors found a significantly higher mortality (49%) in the SAE group compared with the group of patients with normal mental status (26%). A direct relation between the severity of cerebral dysfunction and increased mortality in septic patients was also reported in other studies (2Wilson J.X. Young G.B. Can. J. Neurol. Sci. 2003; 30: 98-105Crossref PubMed Scopus (128) Google Scholar, 4Eidelman L.A. Putterman D. Putterman C. Sprung C.L. J. Am. Med. Assoc. 1996; 275: 470-473Crossref PubMed Google Scholar).The pathophysiology of SAE remains poorly understood and is probably multifactorial. Damage to endothelial cells and breakdown of the blood-brain barrier mediated by cytokines and reactive oxygen species occur at an early stage of sepsis and can alter cerebral blood flow (5Bolton C.F. Young G.B. Zochodne D.W. Ann. Neurol. 1993; 33: 94-100Crossref PubMed Scopus (313) Google Scholar). Perimicrovessel edema, disruption of astrocyte end-feet, and neuronal injury are observed in septic encephalopathic pigs with fecal peritonitis (6Davies D.C. J. Anat. 2002; 200: 639-646Crossref PubMed Scopus (253) Google Scholar), but limited data are available from human studies. An imbalance between branched chain and aromatic amino acids and reduced plasma and cerebrospinal fluid concentrations of ascorbate, probably due to excessive oxidative stress, has been reported in some patients with SAE (7Basler T. Meier-Hellmann A. Bredle D. Reinhart K. Intensive Care Med. 2002; 28: 293-298Crossref PubMed Scopus (81) Google Scholar, 8Voigt K. Kontush A. Stuerenburg H.J. Muench-Harrach D. Hansen H.C. Kunze K. Free Radic. Res. 2002; 36: 735-739Crossref PubMed Scopus (48) Google Scholar).We have demonstrated previously a significant association between the intensity of the inflammatory response and outcome in patients with sepsis (9Arnalich F. López J. Codoceo R. Jiménez M. Madero R. Montiel C. J. Infect. Dis. 1999; 180: 908-911Crossref PubMed Scopus (159) Google Scholar, 10Arnalich F. García-Palomero E. López J. Jiménez M. Madero R. Renart J. Vázquez J.J. Montiel C. Infect. Immun. 2000; 68: 1942-1945Crossref PubMed Scopus (224) Google Scholar, 11Arnalich F. López-Maderuelo D. Codoceo R. López J. Solís-Garrido L.M. Capiscol C. Fernandez-Capitán C. Madero R. Montiel C. Clin. Exp. Immunol. 2002; 127: 331-336Crossref PubMed Scopus (104) Google Scholar). Because of that observation, we hypothesized that one or more of the inflammatory mediators could play a key role in the origin of this type of encephalopathy. Our hypothesis is based on three previous observations as follows: (i) inflammatory stimuli increase the levels of mediators and their receptors in rodent brain and raise the level of circulating mediators that cross into the central nervous system (12Breder C.D. Dinarello C.A. Saper C.B. Science. 1988; 240: 321-324Crossref PubMed Scopus (672) Google Scholar, 13Banks W.A. Kastin A.J. J. Neuroimmunol. 1997; 79: 22-28Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 14Sternberg E.M. J. Clin. Investig. 1997; 100: 2641-2647Crossref PubMed Scopus (297) Google Scholar); (ii) 30–50% of melanoma and renal carcinoma patients receiving high subcutaneous IL-2 doses as a coadjuvant treatment for their cancer developed confusion and delirium (15Rosenberg S.A. Lotze M.T. Yang J.C. Linehan W.M. Seipp C. Calabro S. Karp S.E. Sherry R.M. Steinberg S. White D.E. J. Clin. Oncol. 1989; 7: 1863-1874Crossref PubMed Scopus (352) Google Scholar); and (iii) the effects of intracerebral injection of IL-1β in rodents mimic the electroencephalographic changes and soporific effects of SAE (16Krueger J.M. Walter J. Dinarello C.A. Wolff S.M. Chedid L. Am. J. Physiol. 1984; 246: R994-R999PubMed Google Scholar, 17Dunn A.J. Swiergiel A.H. Ann. N. Y. Acad. Sci. 1998; 840: 577-585Crossref PubMed Scopus (68) Google Scholar).Cerebral synaptic activity is decreased in SAE and because type A γ-aminobutyric acid receptors (GABAARs) regulate fast synaptic transmission in most cerebral inhibitory synapses in mammals, we suspected that this receptor would be a good target for inflammatory mediators. In fact, increased GABAergic neurotransmission has been reported in patients with hepatic encephalopathy (18Jones E.A. Weissenborn K. J. Neurol. Neurosurg. Psychiatry. 1997; 63: 279-293Crossref PubMed Scopus (111) Google Scholar).Therefore, the first phase of this study analyzed the plasma levels of different cytokines in septic patients with and without encephalopathy. In light of the strong association that we observed between the IL-1β plasma levels and neurological affectation, we designed a second experimental phase using cultured rat hippocampal neurons and Xenopus oocytes injected with rat brain mRNA to study the following: 1) how cytokine affects GABAARs in these two very different cell types, and 2) the mechanism behind this putative interaction. Different experimental approaches are used in this study, including immunocytochemistry, confocal microscopy, molecular biology, and pharmacological, electrophysiological, and biochemical techniques.EXPERIMENTAL PROCEDURESPatients—Over a 1-year period, a total of 75 consecutive patients admitted to our medical ward in a European university hospital with a diagnosis of sepsis were screened daily for the onset of acute mental impairment. Our institutional review board approved the study, and informed written consent was obtained from the patient and/or his/her guardian (family member). Sepsis was defined according to standardized international criteria (19Levy M.M. Fink M.P. Marshall J.C. Abraham E. Angus D. Cook D. Cohen J. Opal S.M. Vincent J.L. Ramsay G. Crit. Care Med. 2003; 31: 1250-1256Crossref PubMed Scopus (4611) Google Scholar), and SAE was defined as acute alteration of mental status (i.e. inattention, disorganized thinking, and altered level of consciousness) secondary to sepsis. Patients with SAE were included in the study if neurological symptoms had begun within 12 h of admission, and they did not meet the following exclusion criteria: 1) alcoholism or having received medications known to cause mental confusion at admission to hospital; 2) a history consistent with significant memory impairment before hospitalization, i.e. dementia, psychosis, or neurological disease; 3) metabolic encephalopathy because of renal or liver failure, complete respiratory failure (oxygen saturation of <90% on room air and arterial PCO2 ≥ 45 mm Hg), or electrolytic disturbance; and/or 4) immunosuppression because of poorly controlled diabetes or treatment with oral corticosteroids or cytotoxic drugs within the previous month. The diagnosis of SAE was established by ruling out other possible causes of encephalopathy through standard procedures, including blood examinations as well as cranial computed tomography scanning and electroencephalographic recordings (20Young G.B. Bolton C.F. Archibald Y.M. Austin T.W. Wells G.A. J. Clin. Neurophysiol. 1992; 9: 145-152Crossref PubMed Scopus (244) Google Scholar). Twenty one patients with SAE were finally entered in the study. Encephalopathy was scored daily by two classic neuropsychological tests: the Glasgow Coma Scale (21Teasdale G. Jennet B. Lancet. 1974; 2: 81-84Abstract PubMed Scopus (9350) Google Scholar) and the Reaction Level Scale (22Starmark J.E. Stalhammar D. Holmgren E. Rosander B. J. Neurosurg. 1988; 69: 699-706Crossref PubMed Scopus (160) Google Scholar) until the neuropsychological symptoms had completely cleared. For each patient with encephalopathy and enrolled in this study, two other septic patients who had normal results on their neuropsychological tests were included as controls; both groups of patients have similar ages, duration of fever before the collection of blood samples, and severity of disease. The Folstein Mini-Mental State Examination (23Folstein M.F. Folstein S.E. McHugh P.R. J. Psychiatr. Res. 1975; 12: 189-198Crossref PubMed Scopus (70416) Google Scholar) was performed to exclude dementia in the control group (score ≥24) and to estimate base-line cognitive performance in patients after SAE had resolved. Cerebrospinal fluid (CSF) was analyzed in patients whose encephalopathy persisted beyond the first 48 h after study enrollment to exclude a direct infection of the brain. The simplified acute physiology scoring (SAPS) II system (24Auriant I. Vinatier I. Thaler F. Tourneur M. Loirat P. Crit. Care Med. 1998; 26: 1368-1371Crossref PubMed Scopus (51) Google Scholar) was used to assess illness severity for each patient at the time of study entry and at 48 h after entering the study.A venous blood sample of 10 ml was obtained within 24 h after severe alterations in mental status had been noted (day 1), repeated 48 h later (day 3), and again on the day the encephalopathy resolved completely. Resolution was confirmed by normal performance in all neuropsychological tests. CSF samples (1 ml) were obtained from the 10 patients who remained encephalopathic 48 h after entering the study. A cell count of <5/ml and a protein level of <0.45 g/liter were considered normal. TNF-α, IL-1β, and IL-6 levels were determined by an enzyme-linked immunoassay (Medgenix Diagnostic, Fleurus, Belgium) as described elsewhere (10Arnalich F. García-Palomero E. López J. Jiménez M. Madero R. Renart J. Vázquez J.J. Montiel C. Infect. Immun. 2000; 68: 1942-1945Crossref PubMed Scopus (224) Google Scholar, 11Arnalich F. López-Maderuelo D. Codoceo R. López J. Solís-Garrido L.M. Capiscol C. Fernandez-Capitán C. Madero R. Montiel C. Clin. Exp. Immunol. 2002; 127: 331-336Crossref PubMed Scopus (104) Google Scholar). Concentrations of soluble TNF receptor type I + II (TNFR I + II) and IL-1 receptor antagonist (IL-1ra) were measured using a quantitative sandwich enzyme immunoassay (Quantikine; R & D Systems, Minneapolis, MN).Primary Neuronal Culture—Primary cultures of hippocampal neurons (for immunocytochemistry experiments) or mixed cortical plus hippocampal neurons (for immunoprecipitation experiments) were prepared from the hippocampi or from the cerebral cortices of Sprague-Dawley rat fetuses at embryonic day 18. Brains were removed and freed from the meninges, and the tissues were then dissected under a binocular microscope. Neurons were mechanically dispersed and plated on poly-l-lysin and laminin-treated 12-mm glass coverslips (at a density of 30,000/cm2) in B-27-supplemented Neurobasal media. The neurons were kept in an incubator at 5% CO2 without medium change for 14 days; experimental treatments were begun after this period.Immunocytochemistry and Confocal Imaging of Rat Hippocampal Neurons—After treatment with the cytokine, neurons were either fixed with 3% paraformaldehyde in phosphate buffer (PB) for 10 min at room temperature and permeabilized for 5 min in 0.25% Triton X-100 (in PBS, 10% normal goat serum) or fixed without permeabilization. Nonspecific immunolabeling sites were blocked by incubation with 10% normal goat serum in PBS. The overnight incubation with either the mouse monoclonal anti-GABAAR β2/β3 subunit antibody (bd-17, 20 μg/ml) or the rabbit polyclonal antibody raised against the type I receptor for IL-1β, IL-1RI (1:200), at 4 °C, was followed by incubation with Oregon Green 488 goat anti-mouse IgG (1:200) or the AlexaFluor 546 goat anti-rabbit IgG (1:300), respectively. Neurons treated as above but not incubated with the primary antibodies were used as negative controls. The stained neurons were imaged using a Leica TCS SP2 spectral confocal laser scanning microscope. All images were captured with the Leica confocal software as reported previously (25Solís-Garrido L.M. Pintado A.J. Andrés-Mateos E. Figueroa M. Matute C. Montiel C. J. Biol. Chem. 2004; 279: 52414-52424Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), using the same adjustments of laser intensity and photomultiplier sensitivity and then processed with Adobe Photoshop 6.0 software using identical contrast and brightness values for both control and IL-1β-treated neurons. Fluorescence intensity, expressed as arbitrary units (a.u.), was measured in a region of interest of 10 μm2 on the cell body membrane or within the soma. In other cases, fluorescence intensity was measured along the x axis of the entire cell body.For quantification of the number of GABAAR clusters in proximal neurites, hippocampal neurons were plated on coverslips and maintained in culture in the same conditions as above. After IL-1β treatment for 1 h, “live neurons” were incubated for 1 h at 37 °C with the primary anti-GABAAR β-chain antibody, at the same dilution as above. After rinsing, neurons were fixed with 3% paraformaldehyde in PB (5 min) followed by incubation with the secondary antibody for 45 min at room temperature. Coverslips were washed twice with PBS and mounted in glycerol/buffer (1:1). Images were collected with a DXM1200F digital camera incorporated to a personal computer, using the ×40 objective of a Nikon TE2000-S microscope. Image files were processed using Scion Image software (Scion Corp., Frederick, MD). The number of clusters was determined along dendritic segments of an area of 10 μm2, starting >5 μm from the soma; a single mean value was obtained for each neuron after analyzing three to four different fields per cell. At least five different neurons from each culture of three independent cultures were used in this analysis.Immunoprecipitation Assay—Cortical cultures, containing a mixed population of cortical plus hippocampal neurons, were prepared and maintained in culture for 14 days as described previously. After this period, neurons were homogenized in RIPA buffer (10 mm Na2HPO4, pH 7.2, 150 mm NaCl, 1% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS) containing protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 0.2 mm, 1,10-phenanthroline, 10 μg/ml pepstatin A, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 10 mm benzamide). The neuronal lysates (300 μg of protein) were then immunoprecipitated using the monoclonal anti-GABAAR β2/β3-chain antibody (2 μg) for 4 h at 4 °C, followed by the addition of 20 μl of a mixture of γ-bind protein G-Sepharose (Amersham Biosciences) and γ-bind protein A-agarose (Sigma) for 16 h at 4 °C. After three washings in lysis buffer, the immunoprecipitates were boiled in Laemmli sample buffer containing N-ethylmaleimide instead of β-mercaptoethanol and resolved in a 12% SDS-PAGE. Proteins were transferred to Immobilon membranes (Millipore, Billerica, MA) and immunoblotted with the phospho-Ser detection antibody set (Biomol, Plymouth Meeting, PA). The efficiency of receptor immunoprecipitation was determined by reprobing the same membrane with the anti-GABAAR β2/β3-chain antibody.Preparation of mRNA—Techniques for poly(A)+ RNA isolation from tissues as well as in vitro mRNA synthesis have been described elsewhere (25Solís-Garrido L.M. Pintado A.J. Andrés-Mateos E. Figueroa M. Matute C. Montiel C. J. Biol. Chem. 2004; 279: 52414-52424Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 26Pintado A.J. Herrero C.J. García A.G. Montiel C. Br. J. Pharmacol. 2000; 130: 1893-1902Crossref PubMed Scopus (67) Google Scholar, 27Herrero C.J. Ales E. Pintado A.J. López M.G. García-Palomero E. Mahata S.K. O'Connor D.T. García A.G. Montiel C. J. Neurosci. 2002; 22: 377-388Crossref PubMed Google Scholar). Briefly, poly(A)+ RNA was purified from mixed cortex plus hippocampus regions of brain from 3-week-old Sprague-Dawley rats using the FastTrack mRNA 2.0 kit from Invitrogen. The plasmid containing the entire coding region of the myristoylated conditionally active form of Akt1 (pcDNA3.1-myr-Akt1.ER*) was linearized with NotI and transcribed with T7 polymerase using the mCAP RNA capping kit (Stratagene, La Jolla, CA). Messenger was dissolved in RNase-free water (1 mg/ml) and aliquots stored at –80 °C until use. The myr-Akt1.ER* was constructed by fusing the c-Src myristoylation targeting sequence to a constitutively active form of Akt1 lacking the pleckstrin homology domain. Conditional activity was conferred in a manner similar to that described for other protein kinases by fusing the myr-Akt1 to a modified form of the hormone binding domain of the mouse estrogen receptor that binds 4-hydroxytamoxifen (4-HT) but is refractory to estrogen. This fusion protein is expressed in an inactive form and becomes activated in the presence of 4-HT as reported in a previous paper (28Rojo A.I. Salinas M. Martín D. Perona R. Cuadrado A. J. Neurosci. 2004; 24: 7324-7334Crossref PubMed Scopus (167) Google Scholar). As a result, myr-Akt1.ER* is rapidly activated in response to 4-HT and elicits effects that have been attributed to endogenous cellular Akt.Electrophysiology in Oocytes—Techniques for mRNA injection and electrophysiological recordings of foreign receptors expressed in Xenopus oocytes are described in detail elsewhere (25Solís-Garrido L.M. Pintado A.J. Andrés-Mateos E. Figueroa M. Matute C. Montiel C. J. Biol. Chem. 2004; 279: 52414-52424Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 26Pintado A.J. Herrero C.J. García A.G. Montiel C. Br. J. Pharmacol. 2000; 130: 1893-1902Crossref PubMed Scopus (67) Google Scholar, 27Herrero C.J. Ales E. Pintado A.J. López M.G. García-Palomero E. Mahata S.K. O'Connor D.T. García A.G. Montiel C. J. Neurosci. 2002; 22: 377-388Crossref PubMed Google Scholar, 29Herrero C.J. García-Palomero E. Pintado A.J. García A.G. Montiel C. Br. J. Pharmacol. 1999; 127: 1375-1387Crossref PubMed Scopus (36) Google Scholar, 30Miledi R. Parker I. Sumikawa K. EMBO J. 1989; 1: 1307-1312Crossref Scopus (61) Google Scholar). Mature female Xenopus laevis frogs obtained from a commercial supplier (Xenopus Express, Haute-Loire, France) were anesthetized with tricaine solution (0.125%) and allowed to recover after surgical removal of small pieces of ovary. Currents were recorded 3–5 days after injection of mRNA (50 ng/oocyte). The oocytes were impaled by two microelectrodes filled with 3 m KCl and voltage-clamped at –60 mV using a two-electrode voltage clamp amplifier (OC-725-B Warner Instrument Corp., Hamden, CT). Pulses of GABA or IL-1β, in the absence or presence of different antagonists (IL-1ra, LY294002, SH-5, API-2), were applied using a set of 2-mm diameter glass tubes located close to the oocyte surface. Pulses and data acquisition were controlled using Digi-data 1200 Interface and CLAMPEX software (Axon Instruments, Foster City, CA).Immunolabeling of Brain Receptors Transplanted to Oocytes—Techniques for immunodetection of foreign proteins expressed in intact mRNA-injected oocytes have been described elsewhere (25Solís-Garrido L.M. Pintado A.J. Andrés-Mateos E. Figueroa M. Matute C. Montiel C. J. Biol. Chem. 2004; 279: 52414-52424Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Briefly, oocytes were fixed in 4% paraformaldehyde in PBS buffer (0.1 m, pH 7.4), extensively washed with 3% bovine serum albumin in the same buffer, and permeabilized and blocked for 1 h with 0.05% Nonidet P-40, 3% bovine serum albumin in PBS. The oocytes were then immunostained with the same primary and secondary antibodies against GABAAR or IL-1RI used above for neurons following the same protocol. Injected oocytes treated as above, but not incubated with primary antibodies, were simultaneously used as negative controls. Oocytes were mounted in glycerol/buffer (70:30), under a glass coverslip on a glass dual-well slide. The slide was fastened upside down on the stage of the Leica confocal microscope and visualized with a ×20 lens. All images were captured at the same adjustments of laser intensity and photomultiplier sensitivity using Leica confocal software and were later processed with ADOBE Photoshop 6.0 software, using identical contrast and brightness values, for both control and treated oocytes.Immunoblot in Oocytes—Three days after injecting brain mRNA, oocytes were distributed in groups of 10 oocytes. Each group was exposed to the indicated treatment and processed separately. Oocytes were homogenized by vigorous vortex mixing, followed by several passes through an 18-gauge needle. A group of noninjected oocytes from the same donor was simultaneously homogenized and served as the control. Cell lysates were spun five times, at 13,000 × g for 10 min, to remove the lipid-rich upper phase and the insoluble pellet. Total protein lysates (20 μg per group) were resolved in SDS-PAGE and immunoblotted according to Rojo et al. (28Rojo A.I. Salinas M. Martín D. Perona R. Cuadrado A. J. Neurosci. 2004; 24: 7324-7334Crossref PubMed Scopus (167) Google Scholar). Blots were analyzed with the polyclonal antibodies (anti-Akt1, 1:500; or anti-phospho-Akt-S473, 1:500).Experimental Sepsis and Brain Immunocytochemistry—Male Sprague-Dawley rats (300–350 g), 3 months old, were used. The rats received an intraperitoneal injection of lipopolysaccharide (LPS) of 3 mg/kg body weight. According to our experience and in agreement with Klein et al. (31Klein D. Einspanier R. Bolder U. Jeschke M.G. Shock. 2003; 20: 536-543Crossref PubMed Scopus (26) Google Scholar), this dose of LPS induces a severe inflammatory response with good survival. Control rats were injected intraperitoneally with vehicle (saline). The animal experiments were conducted in accordance with European Community Council Directives 86/609/EEC and 2003/65/EC guidelines and sacrificed 14–20 h postinjection following deep sodium pentobarbital anesthesia. The rats were perfused through the left ventricle with saline, 4% paraformaldehyde in 0.1 m PB, and a graded series of cold sucrose solutions in PB. The brains were blocked in the coronal stereotaxic plane, cryo-protected by immersion in 30% sucrose until they sank, and frozen sectioned at 40 μm. Adjacent series of sections were collected and processed for GABAAR immunocytochemistry, acetylcholinesterase histochemistry, and cresyl violet staining. GABAAR immunohistochemistry was performed in two series of the control and two series of the septic rats. In order to facilitate comparison of results between the control and septic rats, tissues from both were processed simultaneously using identical solutions and incubation times. Immunocytochemistry was performed following a protocol described previously for dopamine transporter (32Sánchez-González M.A. García-Cabezas M. Rico B. Cavada C. J. Neurosci. 2005; 25: 6076-6083Crossref PubMed Scopus (220) Google Scholar). In this study, the primary antibody was the monoclonal anti-GABAAR β-chain antibody, diluted 1:100–1:200. The sections from control and septic brains were analyzed and photographed with a Zeiss Axiophot microscope, using identical contrast and brightness values. Negative control sections that omitted primary antibody were run in parallel and showed no immunostaining.Chemicals—Unless otherwise indicated, all products not specified were purchased from Sigma. The anti-GABAAR and anti-IL-1RI antibodies were purchased from Chemicon (Temecula, CA) and from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), respectively. The secondary antibodies Oregon Green 488 goat anti-mouse IgG and AlexaFluor 546 goat anti-rabbit IgG were purchased from Molecular Probes (Eugene, OR). The anti-Akt polyclonal antibody was obtained from BD Biosciences, and the anti-phospho-Akt-S473 was from Cell Signaling Technology, Inc. (Beverly, Ma). We obtained Neurobasal media from Invitrogen, and paraformaldehyde was from Merck. The human recombinant IL-1β and IL-1ra were purchased from PeproTech (London, UK); LY294002 was from Cayman Chemical. The Akt inhibitors (SH-5, specific inhibitor of the Akt1 isoform; API-2, an inhibitor of the three Akt isoforms) and the PKC inhibitor (Gö6850) were pur" @default.
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W2099206674 title "Interleukin-1β Enhances GABAA Receptor Cell-surface Expression by a Phosphatidylinositol 3-Kinase/Akt Pathway" @default.
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