Rapid Elevation of Calcium Concentration in Cultured Dorsal Spinal Cord Astrocytes by Corticosterone

In addition to the classic genomic effects, increasing evidence suggests that GC can generate multiple rapid effects on many tissues and cells through nongenomic pathway. In the present study, the effects of corticosterone (CORT) on the intracellular calcium concentration ([Ca²⁺]i) in cultured dorsal spinal cord astrocytes were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator that could monitor real-time alterations of [Ca²⁺]i. CORT (0.01–10 μM) caused a rapid increase in [Ca²⁺]i with a dose-dependent manner in cultured dorsal spinal cord astrocytes. The action of CORT on astrocytic [Ca²⁺]i was blocked by pertussis toxin (a blocker of G protein activation, 100 ng/ml), but was unaffected by RU38486 (glucocorticoid receptor antagonist, 10 μM). In addition, cycloheximide (protein-synthesis inhibitor, 10 μg/ml) pretreatment could not impair the CORT-evoked [Ca²⁺]i elevation. Furthermore, Ca²⁺ mobilization induced by CORT was abolished by chelerythrine chloride (protein kinase C inhibitor, 10 μM), but was not impaired by H89 (protein kinase A inhibitor, 10 μM). These observations suggest that a nongenomic pathways might be involved in the effect of CORT on [Ca²⁺]i in cultured dorsal spinal cord astrocytes. In addition, our results also raise a possibility that a putative pertussis toxin-sensitive mGCR (G-protein-coupled membrane-bound glucocorticoid receptor) and the downstream activation of protein kinase C may be responsible for CORT-induced Ca²⁺ mobilization in cultured dorsal spinal cord astrocytes.     

Purinergic P2Y1 Receptors Control Rapid Expression of Plasma Membrane Processes in Hippocampal Astrocytes

Astrocytes regulate neuronal activity and blood brain barrier through tiny plasma membrane branches or astrocytic processes (APs) making contact with synapses and brain vessels. Several transmitters released by astrocytes and exerting their action on several receptor classes expressed by astrocytes themselves influence their physiology. Here we found that APs are dynamically modulated by purines. In live imaging experiments carried out in rat hippocampal astrocytes, Gq-coupled P2Y1 receptor blockade with the selective antagonist MRS2179 (1 μM) or inhibition of its effector phospholipase C using U73122 (3 μM) produced APs retraction, while stimulation of the same receptor with the selective agonist 2MeSADP (100 μM) increased their number. Since astrocytes, among other transmitters, release ATP by several mechanisms including connexin hemichannels, we used the connexin hemichannel inhibitor carbenoxolone (100 μM) and APs retraction was observed. In our system we then measured expression or function of channels important for modulation of volume transmission and K+ buffering, aquaporin-4, and K+ inward rectifying (Kir) channels, respectively. Aquaporin-4 expression level did not change whereas, in whole-cell patch-clamp recordings performed to measure Kir current, we observed an increase in K+ current in all conditions where APs number was reduced. These data are supporting the idea of a dynamic modulation of astrocytic processes by purinergic signal, strengthening the role of purines in brain homeostasis.     

Corticosterone induces rapid spinogenesis via synaptic glucocorticoid receptors and kinase networks in hippocampus

Background

Modulation of dendritic spines under acute stress is attracting much attention. Exposure to acute stress induces corticosterone (CORT) secretion from the adrenal cortex, resulting in rapid increase of CORT levels in plasma and the hippocampus.

Methodology/Principal Findings

Here we demonstrated the mechanisms of rapid effect (∼1 h) of CORT on the density and morphology of spines by imaging neurons in adult male rat hippocampal slices. The application of CORT at 100–1000 nM induced a rapid increase in the density of spines of CA1 pyramidal neurons. The density of small-head spines (0.2–0.4 µm) was increased even at low CORT levels (100–200 nM). The density of middle-head spines (0.4–0.5 µm) was increased at high CORT levels between 400–1000 nM. The density of large-head spines (0.5–1.0 µm) was increased only at 1000 nM CORT. Co-administration of RU486, an antagonist of glucocorticoid receptor (GR), abolished the effect of CORT. Blocking a single kinase, such as MAPK, PKA, PKC or PI3K, suppressed CORT-induced enhancement of spinogenesis. Blocking NMDA receptors suppressed the CORT effect.

Conclusions/Significance

These results imply that stress levels of CORT (100–1000 nM) drive the spinogenesis via synaptic GR and multiple kinase pathways.     

Local perfusion of corticosterone in the rat medial hypothalamus potentiates d-fenfluramine-induced elevations of extracellular 5-HT concentrations

The dorsomedial hypothalamus (DMH) plays an important role in coordinating physiological and behavioral responses to stress-related stimuli. In vertebrates, DMH serotonin (5-HT) concentrations increase rapidly in response to acute stressors or corticosterone (CORT). Recent studies suggest that CORT inhibits postsynaptic clearance of 5-HT from the extracellular fluid in the DMH by blocking organic cation transporter 3 (OCT3), a polyspecific CORT-sensitive transport protein. Because OCTs are low-affinity, high-capacity transporters, we hypothesized that CORT effects on extracellular 5-HT are most pronounced in the presence of elevated 5-HT release. We predicted that local application of CORT into the DMH would potentiate the effects of d-fenfluramine, a 5-HT-releasing agent, on extracellular 5-HT. These experiments were conducted using in vivo microdialysis in freely-moving male Sprague-Dawley rats implanted with a microdialysis probe into the medial hypothalamus (MH), which includes the DMH. In Experiment 1, rats simultaneously received intraperitoneal (i.p.) injections of 1 mg/kg d-fenfluramine or saline and either 200 ng/mL CORT or dilute ethanol (EtOH) vehicle delivered to the MH by reverse-dialysis for 40 min. In Experiment 2, 5 μM d-fenfluramine and either 200 ng/mL CORT or EtOH vehicle were concurrently delivered to the MH for 40 min using reverse-dialysis. CORT potentiated the increases in extracellular 5-HT concentrations induced by either i.p. or intra-MH administration of d-fenfluramine. Furthermore, CORT and d-fenfluramine interacted to alter home cage behaviors. Our results support the hypothesis that CORT inhibition of OCT3-mediated 5-HT clearance from the extracellular fluid contributes to stress-induced increases in extracellular 5-HT and 5-HT signaling.     

Different GABAB-Mediated Effects on Protein Kinase C Activity and Immunoreactivity in Neonatal and Adult Rat Hippocampal Slices

Abstract: The effects of GABA on protein kinase C (PKC) were investigated in rat hippocampal slices at various postnatal ages [postnatal day (P) 1-P60]. At P4, GABA (300 µ M ) induced a rapid (in 1–2 min) 40–50% increase of PKC activity in the membrane fraction and a decrease in the cytosol. These effects were mediated by GABA_B receptors because (a) they were neither blocked by 10 µ M bicuculline nor reproduced by 10 µ M isoguvacine and (b) they were mimicked by the GABA_B agonist baclofen (3–30 µ M ), an effect fully antagonized by the GABA_B antagonist 2-hydroxysaclofen (10 µ M ). A baclofen-induced increased PKC activity in the membrane fraction was only present during the early postnatal period (P1–P14); it was associated with a translocation from the cytosol to the membrane of the immunoreactivity of some PKC isoforms (α-, β-, and ε-PKCs). In contrast, after P21, PKC activity and α-, β-, ε-, and γ-PKC immunoreactivities were decreased by baclofen in the membrane fraction and increased in the cytosol. These results suggest that the stimulation of GABA_B receptors differentially modulates PKC activity via distinct second messenger pathways in developing and mature hippocampi.     

Enhancement of acid-sensing ion channel activity by metabotropic P2Y UTP receptors in primary sensory neurons

Peripheral purinergic signaling plays an important role in nociception. Increasing evidence suggests that metabotropic P2Y receptors are also involved, but little is known about the underlying mechanism. Herein, we report that selective P2Y receptor agonist uridine 5′-triphosphate (UTP) can exert an enhancing effect on the functional activity of acid-sensing ion channels (ASICs), key sensors for extracellular protons, in rat dorsal root ganglia (DRG) neurons. First, UTP dose-dependently increased the amplitude of ASIC currents. UTP also shifted the concentration–response curve for proton upwards, with a 56.6?±?6.4 % increase of the maximal current response to proton. Second, UTP potentiation of proton-gated currents can be mimicked by adenosine 5′-triphosphate (ATP), but not by P2Y1 receptor agonist ADP. Potentiation of UTP was blocked by P2Y receptor antagonist suramin and by inhibition of intracellular G protein, phospholipase C (PLC), protein kinase C (PKC), or protein interacting with C-kinase 1 (PICK1) signaling. Third, UTP altered acidosis-evoked membrane excitability of DRG neurons and caused a significant increase in the amplitude of the depolarization and the number of spikes induced by acid stimuli. Finally, UTP dose-dependently exacerbated nociceptive responses to injection of acetic acid in rats. These results suggest that UTP enhanced ASIC-mediated currents and nociceptive responses, which reveal a novel peripheral mechanism underlying UTP-sensitive P2Y₂ receptor involvement in hyperalgesia by sensitizing ASICs in primary sensory neurons.     

Protein kinase C stimulates the acid-sensing ion channel ASIC2a via the PDZ domain-containing protein PICK1

Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular protons. They are expressed in central and sensory neurons where they are involved in neuromodulation and in pain perception. Recently, the PDZ domain-containing protein PICK1 (protein interacting with C-kinase) has been shown to interact with ASIC1a and ASIC2a, raising the possibility that protein kinase C (PKC) could regulate ASICs. We now show that the amplitude of the ASIC2a current, which was only modestly increased ( approximately +30%) by the PKC activator 1-oleyl-2-acetyl-sn-glycerol (OAG, 50 microm) in the absence of PICK1, was strongly potentiated ( approximately +300%) in the presence of PICK1. This PICK1-dependent regulatory effect was inhibited in the presence of a PKC inhibitory peptide and required the PDZ domain of PICK1 as well as the PDZ-binding domain of ASIC2a. We have also shown the direct PICK1-dependent phosphorylation of ASIC2a by [(32)P]phosphate labeling and immunoprecipitation and identified a major phosphorylation site, (39)TIR, on the N terminus part of ASIC2a. The OAG-induced increase in ASIC2a current amplitude did not involve any change in the unitary conductance of the ASIC2a channel, whether co-expressed with PICK1 or not. These data provide the first demonstration of a regulation of ASICs by protein kinase phosphorylation and its potentiation by the partner protein PICK1.     

ASIC2b-dependent regulation of ASIC3, an essential acid-sensing ion channel subunit in sensory neurons via the partner protein PICK-1

ASIC3, an acid-sensing ion channel subunit expressed essentially in sensory neurons, has been proposed to be involved in pain. We show here for the first time that native ASIC3-like currents were increased in cultured dorsal root ganglion (DRG) neurons following protein kinase C (PKC) stimulation. This increase was induced by the phorbol ester PDBu and by pain mediators, such as serotonin, which are known to activate the PKC pathway through their binding to G protein-coupled receptors. We demonstrate that this regulation involves the silent ASIC2b subunit, an ASIC subunit also expressed in sensory neurons. Indeed, heteromultimeric ASIC3/ASIC2b channels, but not homomeric ASIC3 channels, are positively regulated by PKC. The increase of ASIC3/ASIC2b current is accompanied by a shift in its pH dependence toward more physiological pH values and may lead to an increase of sensory neuron excitability. This regulation by PKC requires PICK-1 (protein interacting with C kinase), a PDZ domain-containing protein, which interacts with the ASIC2b C terminus.     

Dopamine Receptor Stimulation Decreases Cytosolic γ Protein Kinase C Immunoreactivity in Rat Hippocampal Slices: Evidence for Increased Ca2+-Dependent Proteolysis

Abstract: The effect of dopamine (DA) receptor stimulation on the distribution of γ protein kinase C (γPKC) in hippocampal slices was assessed. Nanomolar concentrations of DA decreased cytosolic γPKC (56%) without altering membrane γPKC levels, resulting in decreased total γPKC immunoreactivity. The maximal decrease in cytosolic γPKC occurred at 20 min of incubation and was significantly blocked by the D₁ DA antagonist SCH 23390 (10⁻⁶ M ) but not by the D₂ antagonist sulpiride (10⁻⁵ M ). The D₁ agonists SKF 38393 and A 77636 mimicked the effect of DA with similar responses produced at 10 µ M and 1 n M , respectively. The D₂ agonist quinpirole had no effect on γPKC immunoreactivity, thus indicating that this dopaminergic response is mediated through a D₁-like receptor. DA had no effect on α, δ, or ζPKC isozyme immunoreactivity in the same hippocampal preparations. The DA-induced decrease in cytosolic γPKC immunoreactivity was blocked by the Ca²⁺-dependent protease inhibitor N -acetyl-Leu-Leu-norleucinal (100 µ M ) and by the inorganic Ca²⁺ channel blocker Co²⁺. The data suggest that DA stimulates a D₁-like DA receptor, which increases the influx of Ca²⁺ and activates the Ca²⁺-dependent proteolysis of γPKC.     

The involvement of AMPA–ERK1/2–BDNF pathway in the mechanism of new antidepressant action of prokinetic meranzin hydrate

It was recently discovered that ketamine can relieve depression in a matter of hours through an action on α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. This is much more rapid than the several weeks required for the available antidepressants to show therapeutic efficacy. However, ketamine has negative side effects. The aim of this study was to determine whether the natural prokinetic drug meranzin hydrate (MH) has a fast-acting antidepressant effect mediated by AMPA receptors. By means of in vivo and in vitro experiments, we found that (1) treatment of rats with MH at 9 mg/kg decreased immobility time in a forced swimming test (FST), as did the popular antidepressant fluoxetine and the AMPA receptor positive modulator aniracetam. Pretreatment of rats with NBQX (10 mg/kg), an antagonist of AMPA receptors, blocked this effect of MH. (2) MH increased number of crossings of forced swimming rats in the open field test. (3) FST enhanced hippocampal ERK1/2, p-ERK1/2 and BDNF expression levels. MH (9 mg/kg) treatment further up-regulated hippocampal p-ERK1/2 and BDNF expression levels, and this effect was prevented by NBQX. (4) MH-increased BDNF expression corresponded with MH-decreased immobility time in the FST. (5) In vitro experiments, we found that incubation of rats hippocampus slices with MH (10, 20 μM respectively) increased concentrations of BDNF and p-ERK1/2. This effect of MH (20 μM) were prevented by NBQX. In conclusion, in animals subjected to acute stress, the natural prokinetic drug MH produced a rapid effect mediated by AMPA receptors and involving BDNF modulation through the ERK1/2 pathway.     

Role of Calcineurin-Mediated Dephosphorylation in Modulation of an Inwardly Rectifying K+ Channel in Human Proximal Tubule Cells

Activity of an inwardly rectifying K⁺ channel with inward conductance of about 40 pS in cultured human renal proximal tubule epithelial cells (RPTECs) is regulated at least in part by protein phosphorylation and dephosphorylation. In this study, we examined involvement of calcineurin (CaN), a Ca²⁺/calmodulin (CaM)–dependent phosphatase, in modulating K⁺ channel activity. In cell-attached mode of the patch-clamp technique, application of a CaN inhibitor, cyclosporin A (CsA, 5 μM) or FK520 (5 μM), significantly suppressed channel activity. Intracellular Ca²⁺ concentration ([Ca²⁺] _i ) estimated by fura-2 imaging was elevated by these inhibitors. Since inhibition of CaN attenuates some dephosphorylation with increase in [Ca²⁺] _i , we speculated that inhibiting CaN enhances Ca²⁺-dependent phosphorylation, which might result in channel suppression. To verify this hypothesis, we examined effects of inhibitors of PKC and Ca²⁺/CaM-dependent protein kinase-II (CaMKII) on CsA-induced channel suppression. Although the PKC inhibitor GF109203X (500 nM) did not influence the CsA-induced channel suppression, the CaMKII inhibitor KN62 (20 μM) prevented channel suppression, suggesting that the channel suppression resulted from CaMKII-dependent processes. Indeed, Western blot analysis showed that CsA increased phospho-CaMKII (Thr286), an activated CaMKII in inside–out patches, application of CaM (0.6 μM) and CaMKII (0.15 U/ml) to the bath at 10^?6 M Ca²⁺ significantly suppressed channel activity, which was reactivated by subsequent application of CaN (800 U/ml). These results suggest that CaN plays an important role in supporting K⁺ channel activity in RPTECs by preventing CaMKII-dependent phosphorylation.     

Heavy metal-induced oxidative damage is reduced by β-estradiol application in lentil seedlings

The protective effect of β-estradiol (E) application against heavy metal (HM) toxicity in lentil (Lens culinaris) seedlings was investigated. Seeds were treated with distilled water (control) or aqueous solutions of 100 μM CdCl₂, 200 μM CuCl₂ and 1 μM E singly or in combinations (1 μM E+100 μM CdCl₂ and 1 μM E+200 μM CuCl₂). HM treatments resulted in increase in the activities of antioxidative enzymes, including superoxide dismutase (SOD), catalase (CAT), guaicol peroxidase and ascorbate peroxidase. In a similar manner, Cd and Cu affected significantly oxidative injury indicators measured as electrolyte leakage (electrical conductivity of germination medium), lipoxygenase (LOX) activity and contents of malondialdehyde (MDA; lipoperoxidation marker), carbonyl groups (protein oxidation marker) and hydrogen peroxide (a reactive oxygen species). However, E was effective in reducing HM-induced toxicity. The steroid (1) alleviated HM-induced increase in the electrolyte leakage, LOX activity and contents of MDA, carbonyl and H₂O₂ and (2) improved the activities of SOD and CAT, but not the peroxidase ones, as compared to treatments with HM singly. In addition, E application prevented HM-induced decrease in dry weight production, but did not reduce the accumulation of Cd and Cu in tissues. Results of the present study suggest that E is able to protect lentil from HM-induced oxidative damage most likely by avoidance of H₂O₂ generation and improving antioxidative enzyme activities and, thereby, decreasing oxidative stress injury, but not by reducing Cd and Cu uptake.     

Effect of Systemic Administration of N-Methyl-d-Aspartic Acid on Extracellular Taurine Level Measured by Microdialysis in the Hippocampal CA1 Field and Striatum of Rats

Abstract: The extracellular concentrations of amino acids in the hippocampal CA1 field and striatum of conscious freely moving rats were monitored simultaneously by in vivo brain microdialysis using HPLC with electrochemical detection. Under basal conditions, aspartate, glutamate, glutamine, glycine, taurine, and alanine were detected, but γ-aminobutyric acid was undetectable in both regions. In-traperitoneal injection of N -methyl- d -aspartic acid (NMDA; 10 mg/kg) caused a significant increase (three-to fivefold) in the taurine concentration in the dialysate obtained from both the hippocampal CA1 and striatum, whereas other amino acids (aspartate, glutamate, and alanine) did not show significant changes. Local application of NMDA (300 γ) to both regions via the dialysis probes also caused a similar increase (three-to fivefold) in both regions. Under infusion of hypertonic Ringer's solution containing 150 m M sucrose, the effect of NMDA on the level of taurine in both the regional dialysates was not affected. The effect of NMDA was totally reduced by intraperitoneal administration of MK-801 (0.3–1.0 mg/kg), a noncompetitive antagonist of NMDA receptors. Continuous infusion of dl -2-amino-5-phosphonovaleric acid (1.0 mM), a competitive antagonist of NMDA receptors, via the dialysis probes completely inhibited the effect of NMDA. These findings suggest that systemic administration of NMDA is effective as well as local administration into the brain and that NMDA receptors might be involved in the regulation of the extracellular taurine level in the brain without dependence on cell swelling.     

Effect of GABA Receptor Agonists or Antagonists Injected Spinally on the Blood Glucose Level in Mice

The possible roles of gamma-amino butyric acid (GABA) receptors located in the spinal cord for the regulation of the blood glucose level were studied in ICR mice. We found in the present study that intrathecal (i.t.) injection with baclofen (a GABA_B receptor agonist; 1–10 μg/5 μl) or bicuculline (a GABA_A receptor antagonist; 1–10 μg/5 μl) caused an elevation of the blood glucose level in a dose-dependent manner. The hyperglycemic effect induced by baclofen was more pronounced than that induced by bicuculline. However, muscimol (a GABA_A receptor agonist; 1–5 μg/5 μl) or phaclofen (a GABA_B receptor antagonist; 5–10 μg/5 μl) administered i.t. did not affect the blood glucose level. Baclofen–induced elevation of the blood glucose was dose-dependently attenuated by phaclofen. Furthermore, i.t. pretreatment with pertussis toxin (PTX; 0.05 or 0.1 μg/5 μl) for 6 days dose-dependently reduced the hyperglycemic effect induced by baclofen. Our results suggest that GABA_B receptors located in the spinal cord play important roles for the elevation of the blood glucose level. Spinally located PTX-sensitive G-proteins appear to be involved in hyperglycemic effect induced by baclofen. Furthermore, inactivation of GABA_A receptors located in the spinal cord appears to be responsible for tonic up-regulation of the blood glucose level.     

Radiosensitization by Thymidine Phosphorylase Inhibitor in Thymidine Phosphorylase Negative and Overexpressing Bladder Cancer Cell Lines

TAS-102 (trifluorothymidine [TFT] and thymidine phosphorylase inhibitor [TPI] in a molar ratio of 1:0.5) has activity in 5-fluorouracil resistant colon cancer. TPI is added to increase TFT's bioavailability. TFT has a dual mechanism of action by inhibiting thymidylate synthase and by its incorporation into DNA. Interesting radiosensitizing effects of TPI were recently reported. The aim of our study was to determine whether TP expression would affect radiosensitivity and to characterize the effect of TPI. Two bladder cancer cell lines RT112 (TP negative) and RT112/TP (TP overexpression) were tested for drug sensitivity and radiosensitivity (clonogenic assay), with and without TFT and/or TPI. Expression of γ H2AX was used as marker for DNA damage. RT112 cells were not more sensitive to TFT then RT112/TP cells. TPI alone did not inhibit cell growth of RT112 even at 100 μM, but inhibited that of RT112/TP by 27%. In both RT112 and RT112/TP cells 10 μM TPI did not or slightly affect radiosensitivity, but 100 μM TPI alone enhanced the radiation response (p <.05). TFT alone at 1 μM and in combination with 10 μM TPI did not affect the radiation response of both cell lines. TPI alone induced expression of ?H2AX, which was increased in combination with radiation. In conclusion, TPI enhanced radiosensitivity at high concentrations, independent of TP expression, while TFT and TPI at a low concentration did not affect the radiosensitivity of RT112 and RT112/TP cell lines.     

Cellular Localization of Acid-Sensing Ion Channel 1 in Rat Nucleus Tractus Solitarii

By determining its cellular localization in the nucleus tractus solitarii (NTS), we sought anatomical support for a putative physiological role for acid-sensing ion channel Type 1 (ASIC1) in chemosensitivity. Further, we sought to determine the effect of a lesion that produces gliosis in the area. In rats, we studied ASIC1 expression in control tissue with that in tissue with gliosis, which is associated with acidosis, after saporin lesions. We hypothesized that saporin would increase ASIC1 expression in areas of gliosis. Using fluorescent immunohistochemistry and confocal microscopy, we found that cells and processes containing ASIC1-immunoreactivity (IR) were present in the NTS, the dorsal motor nucleus of vagus, and the area postrema. In control tissue, ASIC1-IR predominantly colocalized with IR for the astrocyte marker, glial fibrillary acidic protein (GFAP), or the microglial marker, integrin αM (OX42). The subpostremal NTS was the only NTS region where neurons, identified by protein gene product 9.5 (PGP9.5), contained ASIC1-IR. ASIC1-IR increased significantly (157 ± 8.6% of control, p < 0.001) in the NTS seven days after microinjection of saporin. As we reported previously, GFAP-IR was decreased in the center of the saporin injection site, but GFAP-IR was increased in the surrounding areas where OX42-IR, indicative of activated microglia, was also increased. The over-expressed ASIC1-IR colocalized with GFAP-IR and OX42-IR in those reactive astrocytes and microglia. Our results support the hypothesis that ASIC1 would be increased in activated microglia and in reactive astrocytes after injection of saporin into the NTS.     

ACh受体在皮质酮引起的大鼠延髓腹外侧区前交感神经元反应中的作用

目的：研究乙酰胆碱（ACh)受体在皮质酮（CORT）对大鼠头端延髓腹外侧区（RVLM）前交感神经元快速效应中的作用,探讨糖皮质激素在交感心血管活动调节中的非基因组机制。方法：本研究采用细胞外记录和微电泳等方法观察CORT对氨基甲酸乙酯麻醉大鼠RVLM前交感神经元的作用,观察分别给予ACh受体拮抗剂阿托品（ATR）、筒箭毒（d-TC)或六烃季铵（C6）后CORT对RVLM前交感神经元的影响。结果：在RVLM共记录到33个前交感神经元,CORT能导致25（76％）个前交感神经元快速兴奋,且具有剂量依赖性,余8个前交感神经元没有反应;其中被CORT兴奋的10个单位微电泳ART后神经元的放电明显下降,但对CORT导致的兴奋作用没有明显的影响。分别向7和6个被CORT兴奋的前交感神经元微电泳d-TC和C6后,单位放电没有变化,同时对CORT导致的兴奋作用无影响。结论：CORT对RVLM前交感神经元具有快速的兴奋作用,这种作用可能并不通过ACh受体介导。     

Biphasic increase of gap junction coupling induced by dipyridamole in the rat aortic A-10 vascular smooth muscle cell line

The rat aortic smooth muscle cell line A-10 was used to investigate the effect of dipyridamole on the gap junction coupling of smooth muscle cells. The scrape loading/dye transfer (SL/DT) technique revealed that dipyridamole concentrations between 5 μM and 100 μM significantly increased gap junction coupling. The adenosine receptor antagonist MRS 1754, as well as the PKA inhibitors Rp-cAMPS and H-89 were able to inhibit the dipyridamole-related increase in coupling, while forskolin and Br-cAMP also induced an enhancement of the gap junction coupling. Regarding the time-dependent behaviour of dipyridamole, a short-term effect characterised by an oscillatory reaction was observed for application times of less than 5 h, while applications times of at least 6 h resulted in a long-term effect, characterised by a constant increase of gap junction coupling to its maximum levels. This increase was not altered by prolonged presence of dipyridamole. In parallel, a short application of dipyridamole for at least 15 min was found to be sufficient to evoke the long-term effect measured 6 h after drug washout. We propose that in both the short-term and long-term effect, cAMP-related pathways are activated. The short-term phase could be related to an oscillatory cAMP effect, which might directly affect connexin trafficking, assembly and/or gap junction gating. The long-term effect is most likely related to the new expression and synthesis of connexins. With previous data from a bovine aortic endothelial cell line, the present results show that gap junction coupling of vascular cells is a target for dipyridamole.     

Electrophysiological approach of the antiepileptic effect of dexamethasome on hippocampal slice culture usihg a multirecording system: The physiocard®

The in vitro antiepileptic activity of the synthetic glucocorticoid dexamethasone (DEX) was tested in rat hippocampal organotypic cultures on the field potential epileptiform activity induced by picrotoxin (PTX). Spontaneous as well as evoked electrophysiological activities have been studied through the extracellular multirecording Physiocard® system. PTX typically elicited seizure-like discharges (epileptiform bursts) in the hippocampus neurons. Those epileptiform bursts can be divided in two groups, one rhythmic which lasted 43 ± 24s (mean ± sd) at a frequency of 4.6 ± 1.9Hz and the other arhythmic composed of population spikes, which occurred during 14.3 ± 6.9min. In the presence of DEX at different concentrations, results obtained were: 1) DEX 1μM decreased the occurrence of the two different groups of spontaneous epileptiform bursts, most of the time to zero. 2) DEX 50μM prevented totally the occurrence of epileptiform bursts. 3) DEX 50μM contrarily to DEX 1μM avoided the decrease of evoked field potentials' amplitude induced by PTX 3μM on all simultaneous recorded points. Those results suggest that synthetic glucocorticoid DEX presents an acute antiepileptic effect in a dose dependent manner on the hippocampus tissue.     

An evaluation of feather corticosterone as a biomarker of fitness and an ecologically relevant stressor during breeding in the wild

Feather corticosterone (CORT) levels are increasingly employed as biomarkers of environmental stress. However, it is unclear if feather CORT levels reflect stress and/or workload in the wild. We investigated whether feather CORT represents a biomarker of environmental stress and reproductive effort in tree swallows (Tachycineta bicolor). Specifically, we examined whether individual state and investment during reproduction could predict feather CORT levels in subsequently moulted feathers and whether those levels could predict future survival and reproductive success. Through a manipulation of flight cost during breeding, we also investigated whether an increase in stress level would be reflected in subsequently grown feathers, and whether those levels could predict future success. We found that CORT levels of feathers grown during moult did not (1) reflect past breeding experience (n = 29), (2) predict reproductive output (n = 18), or (3) respond to a manipulation of flight effort during reproduction (10 experimental, 14 control females). While higher feather CORT levels predicted higher return rate (a proxy for survival), they did so only in the manipulated group (n = 36), and this relationship was opposite to expected. Overall, our results add to the mixed literature reporting that feather CORT levels can be positively, negatively, or not related to proxies of within-season and longer-term fitness (i.e., carryover effects). In addition, our results indicate that CORT levels or disturbances experienced during one time (e.g., breeding) may not carry over to subsequent stages (e.g., moult). We, therefore, petition for directed research investigating whether feather CORT represents exposure to chronic stress in feathers grown during moult.