Multidisciplinary characterization of U(VI) sequestration by Acidovorax facilis for bioremediation purposes.

The contamination of the environment by U may affect plant life and consequently may have an impact on animal and human health. The present work describes U(VI) sequestration by Acidovorax facilis using a multidisciplinary approach combining wet chemistry, transmission electron microscopy, and spectroscopy methods (e.g. cryo-time resolved laser-induced fluorescence spectroscopy, extended X-ray absorption fine structure spectroscopy, and in-situ attenuated total reflection Fourier transform infrared spectroscopy). This bacterial strain is widely distributed in nature including U-contaminated sites. In kinetic batch experiments cells of A. facilis were contacted for 5 min to 48 h with 0.1 mM U(VI). The results show that the local coordination of U species associated with the cells depends upon time contact. U is bound mainly to phosphate groups of lipopolysaccharide (LPS) at the outer membrane within the first hour. And, that both, phosphoryl and carboxyl functionality groups of LPS and peptidoglycan of A. facilis cells may effectuate the removal of high U amounts from solution at 24-48 h of incubation. It is clearly demonstrated that A. facilis may play an important role in predicting the transport behaviour of U in the environment and that the results will contribute to the improvement of bioremediation methods of U-contaminated sites.

Brain (Hyper)Excitability Revealed by Optimal Electrical Stimulation of GABAergic Interneurons.

BACKGROUND: Neurological disorders are often characterized by an excessive and prolonged imbalance between neural excitatory and inhibitory processes. An ubiquitous finding among these disorders is the disrupted function of inhibitory GABAergic interneurons. OBJECTIVE: The objective is to propose a novel stimulation procedure able to evaluate the efficacy of inhibition imposed by GABAergic interneurons onto pyramidal cells from evoked responses observed in local field potentials (LFPs). METHODS: Using a computational modeling approach combined with in vivo and in vitro electrophysiological recordings, we analyzed the impact of electrical extracellular, local, bipolar stimulation (ELBS) on brain tissue. We implemented the ELBS effects in a neuronal population model in which we can tune the excitation-inhibition ratio and we investigated stimulation-related parameters. Computer simulations led to sharp predictions regarding: i) the shape of evoked responses as observed in local field potentials, ii) the type of cells (pyramidal neurons and interneurons) contributing to these field responses and iii) the optimal tuning of stimulation parameters (intensity and frequency) to evoke meaningful responses. These predictions were tested in vivo (mouse). Neurobiological mechanisms were assessed in vitro (hippocampal slices). RESULTS: Appropriately-tuned ELBS allows for preferential activation of GABAergic interneurons. A quantitative neural network excitability index (NNEI) is proposed. It is computed from stimulation-induced responses as reflected in local field potentials. NNEI was used in four patients with focal epilepsy. Results show that it can readily reveal hyperexcitable brain regions. CONCLUSION: Well-tuned ELBS and NNEI can be used to locally probe brain regions and quantify the (hyper)excitability of the underlying brain tissue.

Combined use of flow cytometry and microscopy to study the interactions between the gram-negative betaproteobacterium Acidovorax facilis and uranium(VI).

The former uranium mine Königstein (Saxony, Germany) is currently in the process of remediation by means of controlled underground flooding. Nevertheless, the flooding water has to be cleaned up by a conventional wastewater treatment plant. In this study, the uranium(VI) removal and tolerance mechanisms of the gram-negative betaproteobacterium Acidovorax facilis were investigated by a multidisciplinary approach combining wet chemistry, flow cytometry, and microscopy. The kinetics of uranium removal and the corresponding mechanisms were investigated. The results showed a biphasic process of uranium removal characterized by a first phase where 95% of uranium was removed within the first 8h followed by a second phase that reached equilibrium after 24h. The bacterial cells displayed a total uranium removal capacity of 130mgU/g dry biomass. The removal of uranium was also temperature-dependent, indicating that metabolic activity heavily influenced bacterial interactions with uranium. TEM analyses showed biosorption on the cell surface and intracellular accumulation of uranium. Uranium tolerance tests showed that A. facilis was able to withstand concentrations up to 0.1mM. This work demonstrates that A. facilis is a suitable candidate for in situ bioremediation of flooding water in Königstein as well as for other contaminated waste waters.

Cyr61 is a target for heparin in reducing MV3 melanoma cell adhesion and migration via the integrin VLA-4.

The integrin VLA-4 is important for the metastatic dissemination of melanoma cells. We could recently show that heparin can block VLA-4 binding, which contributes, next to blocking P- and L-selectin, to the understanding of antimetastatic activities of heparin. The matricellular ligand Cyr61, secreted by numerous tumours, is responsible for increased tumourigenicity and metastasis. This has been attributed to Cyr61 binding to, and thus activating integrins. However, a VLA-4/Cyr61 axis has not yet been reported. Since Cyr61 possesses heparin binding capabilities, Cyr61 can be supposed as potential target for heparin to indirectly interfere with integrin functions. The present in vitro studies address (i) the existence of a Cyr61/VLA-4 axis and (ii) the functional relevance of heparin interference via Cyr61. The C-terminal module III of Cyr61 could be exposed as nanomolar affine binding site for VLA-4. A shRNA-based knockdown of Cyr61 in MV3 human melanoma cells reduced VLA-4-mediated cell binding to VCAM-1, migration on fibronectin, and integrin signalling functions significantly. Using a biosensor approach we provide insight into heparin interference with this process. The low-molecular-weight heparin tinzaparin, but not the pentasaccharide fondaparinux, binds module IV of Cyr61 with micromolar affinity. But tinzaparin cannot interfere with Cyr61 accumulation onto syndecan-4, indicating different Cyr61 binding sites for heparin and other GAGs. Nonetheless, tinzaparin affects the VLA-4 binding and signalling functions selectively via Cyr61 already at very low concentration most likely by blocking the cellular secreted free Cyr61. This study emphasises Cyr61 as promising, and hitherto not considered target for heparin to selectively influence integrin functions.

End-tidal CO2: an important parameter for a correct interpretation in functional brain studies using speech tasks.

The aim was to investigate the effect of different speech tasks, i.e. recitation of prose (PR), alliteration (AR) and hexameter (HR) verses and a control task (mental arithmetic (MA) with voicing of the result on end-tidal CO2 (PETCO2), cerebral hemodynamics and oxygenation. CO2 levels in the blood are known to strongly affect cerebral blood flow. Speech changes breathing pattern and may affect CO2 levels. Measurements were performed on 24 healthy adult volunteers during the performance of the 4 tasks. Tissue oxygen saturation (StO2) and absolute concentrations of oxyhemoglobin ([O2Hb]), deoxyhemoglobin ([HHb]) and total hemoglobin ([tHb]) were measured by functional near-infrared spectroscopy (fNIRS) and PETCO2 by a gas analyzer. Statistical analysis was applied to the difference between baseline before the task, 2 recitation and 5 baseline periods after the task. The 2 brain hemispheres and 4 tasks were tested separately. A significant decrease in PETCO2 was found during all 4 tasks with the smallest decrease during the MA task. During the recitation tasks (PR, AR and HR) a statistically significant (p<0.05) decrease occurred for StO2 during PR and AR in the right prefrontal cortex (PFC) and during AR and HR in the left PFC. [O2Hb] decreased significantly during PR, AR and HR in both hemispheres. [HHb] increased significantly during the AR task in the right PFC. [tHb] decreased significantly during HR in the right PFC and during PR, AR and HR in the left PFC. During the MA task, StO2 increased and [HHb] decreased significantly during the MA task. We conclude that changes in breathing (hyperventilation) during the tasks led to lower CO2 pressure in the blood (hypocapnia), predominantly responsible for the measured changes in cerebral hemodynamics and oxygenation. In conclusion, our findings demonstrate that PETCO2 should be monitored during functional brain studies investigating speech using neuroimaging modalities, such as fNIRS, fMRI to ensure a correct interpretation of changes in hemodynamics and oxygenation.

Prolactin induces a hyperpolarising current in rat paraventricular oxytocinergic neurones.

Prolactin and oxytocin are important reproductive hormones implicated in several common adaptive functions during pregnancy, pseudopregnancy and lactation. Recently, extracellular recordings of supraoptic neurones have shown that prolactin may modulate the electrical activity of oxytocinergic neurones. However, no study has been conducted aiming to establish whether prolactin directly influences this activity in oxytocinergic paraventricular neurones. In the present study, we addressed this question by studying the effects of prolactin on the electrical activity and voltage-current relationship of identified paraventricular neurones in rat brain slices. Whole-cell recordings were obtained and neurones were classified on the basis of their morphological and electrophysiological fingerprint (i.e. magnocellular or parvicellular) and neuropeptide phenotype (i.e. oxytocinergic or non-oxytocinergic). We report that prolactin elicited a hyperpolarising current in 37% of the neurones in this nucleus, of which the majority (67%) were identified as putative magnocellular oxytocin neurones and the reminder (33%) were regarded as oxytocin-negative, parvicellular neuroendocrine neurones. Our results suggest that, in addition to the well-established negative feedback loop between prolactin-secreting lactotrophs and dopaminergic neurones in the arcuate nucleus, an inhibitory feedback loop also exists between lactotrophs and oxytocinergic paraventricular neurones.

Energy deprivation transiently enhances rhythmic inhibitory events in the CA3 hippocampal network in vitro.

Oxygen glucose deprivation (OGD) leads to rapid suppression of synaptic transmission. Here we describe an emergence of rhythmic activity at 8 to 20 Hz in the CA3 subfield of hippocampal slice cultures occurring for a few minutes prior to the OGD-induced cessation of evoked responses. These oscillations, dominated by inhibitory events, represent network activity, as they were abolished by tetrodotoxin. They were also completely blocked by the GABAergic antagonist picrotoxin, and strongly reduced by the glutamatergic antagonist NBQX. Applying CPP to block NMDA receptors had no effect and neither did UBP302, an antagonist of GluK1-containing kainate receptors. The gap junction blocker mefloquine disrupted rhythmicity. Simultaneous whole-cell voltage-clamp recordings from neighboring or distant CA3 pyramidal cells revealed strong cross-correlation of the incoming rhythmic activity. Interneurons in the CA3 area received similar correlated activity. Interestingly, oscillations were much less frequently observed in the CA1 area. These data, together with the observation that the recorded activity consists primarily of inhibitory events, suggest that CA3 interneurons are important for generating these oscillations. This transient increase in inhibitory network activity during OGD may represent a mechanism contributing to the lower vulnerability to ischemic insults of the CA3 area as compared to the CA1 area.

Structures of pulmonary surfactant films adsorbed to an air-liquid interface in vitro.

Phospholipid films can be preserved in vitro when adsorbed to a solidifiable hypophase. Suspensions of natural surfactant, lipid extract surfactants, and artificial surfactants were added to a sodium alginate solution and filled into a captive bubble surfactometer (CBS). Surfactant film was formed by adsorption to the bubble of the CBS for functional tests. There were no discernible differences in adsorption, film compressibility or minimal surface tension on quasi-static or dynamic compression for films formed in the presence or absence of alginate in the subphase of the bubble. The hypophase-film complex was solidified by adding calcium ions to the suspension with the alginate. The preparations were stained with osmium tetroxide and uranyl acetate for transmission electron microscopy. The most noteworthy findings are: (1) Surfactants do adsorb to the surface of the bubble and form osmiophilic lining layers. Pure DPPC films could not be visualized. (2) A distinct structure of a particular surfactant film depends on the composition and the concentration of surfactant in the bulk phase, and on whether or not the films are compressed after their formation. The films appear heterogeneous, and frequent vesicular and multi-lamellar film segments are seen associated with the interfacial films. These features are seen already upon film formation by adsorption, but multi-lamellar segments are more frequent after film compression. (3) The rate of film formation, its compressibility, and the minimum surface tension achieved on film compression appear to be related to the film structure formed on adsorption, which in turn is related to the concentration of the surfactant suspension from which the film is formed. The osmiophilic surface associated surfactant material seen is likely important for the surface properties and the mechanical stability of the surfactant film at the air-fluid interface.

Effects of fixatives on function of pulmonary surfactant.

The structure of pulmonary surfactant films remains ill defined. Although plausible film fragments have been imaged by electron microscopy, questions about the significance of the findings and even about the true fixability of surfactant films by the usual fixatives glutaraldehyde (GA), osmium tetroxide (OsO(4)), and uranyl acetate (UA) have not been settled. We exposed functioning natural surfactant films to fixatives within a captive bubble surfactometer and analyzed the effect of fixatives on surfactant function. The capacity of surfactant to reach near-zero minimum surface tension on film compression was barely impaired after exposure to GA or OsO(4). Although neither GA nor OsO(4) prevented the surfactant from forming a surface active film, GA increased the equilibrium surface tension to above 30 mN/m, and both GA and OsO(4) decreased film stability as seen in the slowly rising minimum surface tension from 1 to ~5 mN/m in 10 min. In contrast, the effect of UA seriously impaired surface activity in that both adsorption and minimum surface tension were substantially increased. In conclusion, the fixatives tested in this study are not suitable to fix, i.e., to solidify, surfactant films. Evidently, however, OsO(4) and UA may serve as staining agents.

Fast synaptic transmission mediated by P2X receptors in CA3 pyramidal cells of rat hippocampal slice cultures.

1. A fast ATP-mediated synaptic current was identified in CA3 pyramidal cells in organotypic hippocampal slice cultures. In the presence of inhibitors for ionotropic glutamate and GABA receptors, extracellular stimulation in the pyramidal cell layer evoked fast synaptic currents that reversed near 0 mV, reflecting an increase in a non-selective cationic conductance. This response was mimicked by focal application of ATP. Antagonists of ionotropic P2X receptors reduced both synaptic and ATP-induced currents. 2. Using a pharmacological approach, the source of synaptically released ATP was determined. Synaptic ATP responses were insensitive to presynaptic blockade of GABAergic transmission between interneurons and CA3 pyramidal cells with the mu-opioid receptor agonist D-Ala(2),MePhe(4),Met(O)(5)-ol-enkephalin (FK33-824), but were blocked by adenosine, which inhibits glutamate release from synaptic terminals in the hippocampus. However, selective inhibition of mossy fibre glutamatergic transmission with the metabotropic glutamate receptor group II agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) did not affect the response. This result points to the associational fibres as the source of the ATP-mediated synaptic response. 3. These results suggest that ATP, coreleased with glutamate, induces a synaptic response in CA3 pyramidal cells that is observed mainly under conditions of synchronous discharge from multiple presynaptic inputs.

G-protein-independent signaling by G-protein-coupled receptors.

Two classes of receptors transduce neurotransmitter signals: ionotropic receptors and heptahelical metabotropic receptors. Whereas the ionotropic receptors are structurally associated with a membrane channel, a mediating mechanism is necessary to functionally link metabotropic receptors with their respective effectors. According to the accepted paradigm, the first step in the metabotropic transduction process requires the activation of heterotrimeric G-proteins. An increasing number of observations, however, point to a novel mechanism through which neurotransmitters can initiate biochemical signals and modulate neuronal excitability. According to this mechanism metabotropic receptors induce responses by activating transduction systems that do not involve G-proteins.

Acute decrease in net glutamate uptake during energy deprivation.

The extracellular glutamate concentration ([glu](o)) rises during cerebral ischemia, reaching levels capable of inducing delayed neuronal death. The mechanisms underlying this glutamate accumulation remain controversial. We used N-methyl-D-aspartate receptors on CA3 pyramidal neurons as a real-time, on-site, glutamate sensor to identify the source of glutamate release in an in vitro model of ischemia. Using glutamate and L-trans-pyrrolidine-2,4-dicarboxylic acid (tPDC) as substrates and DL-threo-beta-benzyloxyaspartate (TBOA) as an inhibitor of glutamate transporters, we demonstrate that energy deprivation decreases net glutamate uptake within 2-3 min and later promotes reverse glutamate transport. This process accounts for up to 50% of the glutamate accumulation during energy deprivation. Enhanced action potential-independent vesicular release also contributes to the increase in [glu](o), by approximately 50%, but only once glutamate uptake is inhibited. These results indicate that a significant rise in [glu](o) already occurs during the first minutes of energy deprivation and is the consequence of reduced uptake and increased vesicular and nonvesicular release of glutamate.

Apamin-sensitive conductance mediates the K(+) current response during chemical ischemia in CA3 pyramidal cells.

Pyramidal cells typically respond to ischemia with initial transient hyperpolarization, which may represent a neuroprotective response. To identify the conductance underlying this hyperpolarization in CA3 pyramidal neurons of rat hippocampal organotypic slice cultures, recordings were obtained using the single-electrode voltage-clamp technique. Brief chemical ischemia (2 mM 2-deoxyglucose and 3 mM NaN(3), for 4 min) induced a response mediated by an increase in K(+) conductance. This current was blocked by intracellular application of the Ca(2+) chelator, bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid (BAPTA), reduced with low external [Ca(2+)], and inhibited by a selective L-type Ca(2+) channel inhibitor, isradipine, consistent with the activation of a Ca(2+)-dependent K(+) conductance. Experiments with charybdotoxin (10 nM) and tetraethylammonium (TEA; 1 mM), or with the protein kinase C activator, phorbol 12,13-diacetate (PDAc; 3 microM), ruled out an involvement of a large conductance-type or an apamin-insensitive small conductance, respectively. In the presence of apamin (1 microM), however, the outward current was significantly reduced. These results demonstrate that in rat hippocampal CA3 pyramidal neurons an apamin-sensitive Ca(2+)-dependent K(+) conductance is activated in response to brief ischemia generating a pronounced outward current.

G-protein-independent signaling mediated by metabotropic glutamate receptors.

Synaptically released glutamate activates ionotropic and metabotropic receptors at central synapses. Metabotropic glutamate receptors (mGluRs) are thought to modulate membrane conductances through transduction cascades involving G proteins. Here we show, in CA3 pyramidal cells from rat hippocampus, that synaptic activation of type 1 mGluRs by mossy fiber stimulation evokes an excitatory postsynaptic response independent of G-protein function, while inhibiting an afterhyperpolarization current through a G-protein-coupled mechanism. Experiments using peptide activators and specific inhibitors identified a Src-family protein tyrosine kinase as a component of the G-protein-independent transduction pathway. These results represent the first functional evidence for a dual signaling mechanism associated with a heptahelical receptor such as mGluR1, in which intracellular transduction involves activation of either G proteins or tyrosine kinases.

Involvement of the N-methyl-D-aspartate receptor in neuronal cell death induced by cytotoxic T cell-derived secretory granules.

The mechanisms underlying neurotoxicity mediated by cytotoxic T lymphocytes (CTL) and their secretory granule proteins perforin and granzymes remain unclear. We evaluated the possible role of the neurotransmitter glutamate in cell death observed in differentiated neurons exposed to CTL-derived granules. Excitotoxicity induced by excessive concentrations of extracellular glutamate is associated with a rise in intracellular calcium and can lead to generation of NO through the activation of glutamatergic N-methyl-D-aspartate (NMDA) receptors. Consistent with an involvement of glutamate, we found that cell death in mature cerebral granule cells was inhibited by 65-80% by two NMDA receptor blockers (MK-801 and D-2-amino-5-phosphonovaleric acid) or a NO synthase blocker (N(G)-nitro-L-arginine methylester). Furthermore, neurons treated with secretory granules responded with a biphasic rise in the intracellular calcium concentration ([Ca2+]i). Whereas MK-801 did not interfere with the immediate rise of [Ca2+]i, the second wave of calcium accumulation starting at 40 min was delayed by 20 min and reduced in amplitude in the presence of MK-801. In immature, NMDA receptor-negative neurons, MK-801 prevented neither the cytotoxicity nor the calcium influx observed 5 min after addition of cytotoxic granules. The demonstration that NMDA receptors and NO are involved in granule-mediated killing of mature neurons opens new avenues in the treatment of neuronal cell death in CTL-mediated diseases such as viral encephalitis.

Inhibition of uptake unmasks rapid extracellular turnover of glutamate of nonvesicular origin.

Maintaining glutamate at low extracellular concentrations in the central nervous system is necessary to protect neurons from excitotoxic injury and to ensure a high signal-to-noise ratio for glutamatergic synaptic transmission. We have used DL-threo-beta-benzyloxyaspartate (TBOA), an inhibitor of glutamate uptake, to determine the role of glutamate transporters in the regulation of extracellular glutamate concentration. By using the N-methyl-D-aspartate receptors of patched CA3 hippocampal neurons as "glutamate sensors," we observed that application of TBOA onto organotypic hippocampal slices led to a rapid increase in extracellular glutamate concentration. This increase was Ca(2+)-independent and was observed in the presence of tetrodotoxin. Moreover, prevention of vesicular glutamate release with clostridial toxins did not affect the accumulation of glutamate when uptake was inhibited. Inhibition of glutamine synthase, however, increased the rate of accumulation of extracellular glutamate, indicating that glial glutamate stores can serve as a source in this process. TBOA blocked synaptically evoked transporter currents in astrocytes without inducing a current mediated by the glutamate transporter. This indicates that this inhibitor is not transportable and does not release glutamate by heteroexchange. These results show that under basal conditions, the activity of glutamate transporters compensates for the continuous, nonvesicular release of glutamate from the intracellular compartment. As a consequence, acute disruption of transporter activity immediately results in significant accumulation of extracellular glutamate.

L-Type Ca2+ channels mediate the slow Ca2+-dependent afterhyperpolarization current in rat CA3 pyramidal cells in vitro.

Single-electrode voltage-clamp recordings were obtained from CA3 pyramidal cells in rat hippocampal organotypic slice cultures, and the slow Ca2+-dependent K+ current or afterhyperpolarization current (IAHP) was elicited with brief depolarizing voltage jumps. The slow IAHP was suppressed by the selective L-type Ca2+ channel antagonists isradipine (2 microM) or nifedipine (10 microM). In contrast, neither omega-conotoxin MVIIA (1 microM) nor omega-agatoxin IVA (200 nM), N-type and P/Q-type Ca2+ channel antagonists, respectively, attenuated this slow outward current. The slow IAHP was significantly reduced by thapsigargin (10 microM), a Ca2+ ATPase inhibitor that depletes intracellular Ca2+ stores, and by ryanodine (10-100 microM), which blocks Ca2+-induced Ca2+ release from intracellular compartments. At this concentration thapsigargin did not modify high-threshold Ca2+ current, which was, however, blocked by isradipine. Thus, in hippocampal CA3 pyramidal cells, Ca2+ influx through L-type Ca2+ channels is necessary to trigger the slow IAHP. Furthermore, intracellular Ca2+-activated Ca2+ stores represent a critical component in the transduction pathway leading to the generation of the slow IAHP.

Effects of transient oxygen-glucose deprivation on G-proteins and G-protein-coupled receptors in rat CA3 pyramidal cells in vitro.

The role of guanosine triphosphate-binding proteins (G-proteins) in the generation of the outward current during transient oxygen-glucose deprivation (OGD) was investigated in CA3 pyramidal cells in rat hippocampal organotypic slice cultures using the single-electrode voltage-clamp technique with KMeSO4-filled microelectrodes. To simulate ischaemia, brief chemical OGD (2 mM 2-deoxyglucose and 3 mM NaN3 for 4-9 min) was used, which induced an outward K+ current associated with an increase in input conductance. OGD failed to induce the outward current under conditions where G-protein function was disrupted by loading cells with guanosine 5'-O-(2-thiodiphosphate) [GDPbetaS] or after prolonged injection of guanosine 5'-O(3-thiotdphosphate) [GTPgammaS]. However, in slices treated with pertussis toxin (PTX), OGD still elicited the outward current, indicating that PTX-insensitive G-proteins are involved. Consistent with this insensitivity to PTX, neither adenosine receptors nor GABA(B) (gamma-aminobutyric acid) receptors, which operate via PTX-sensitive G-proteins, mediate the OGD-induced outward current. When adenosine receptors or GABA(B) receptors were blocked with 1,3-dipropyl-8-psulphophenylxanthine (DPSPX, 5 microM) or CGP 52 432 (10 microM), respectively, the OGD-induced response was not modified. The response also persisted following pretreatment of slice cultures with tetanus toxin to prevent vesicular release of neurotransmitters and neuromodulators from presynaptic terminals. Both PTX-sensitive and PTX-insensitive G-protein-mediated responses were suppressed during OGD. The inward current induced by the metabotropic glutamate receptor agonist 1 S, 3R-1-aminocyclopentane-1,3-dicarboxylate (1S,3R-ACPD) and the outward current elicited by adenosine or baclofen were strongly or completely attenuated. In contrast, the ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) response was not affected. These findings suggest that during OGD there is a functional uncoupling of receptors from G-proteins, and a direct receptor-independent activation of PTX-insensitive G-proteins leading to an increase in membrane K+ conductance.

G-protein-mediated desensitization of metabotropic glutamatergic and muscarinic responses in CA3 cells in rat hippocampus.

1. Desensitization of a metabotropic response was investigated in CA3 pyramidal neurons in hippocampal slice cultures using the patch-clamp technique. 2. 1S,3R-1-aminocyclopentane-1,3-dicarboxylate (1S,3R-ACPD), an agonist at metabotropic glutamate receptors (mGluRs), and metacholine (MCh), an agonist at muscarinic receptors, induced a cationic current that appears to be activated through a G-protein-independent transduction process, as previously shown. Prolonged or repetitive bath application of agonists led to rapid desensitization of the cationic current with a time constant of approximately 20 s. 3. Complete recovery from desensitization was observed within 6 min. 4. These responses mediated by mGluRs and muscarinic receptors cross-desensitized. 5. Preventing the activation of G-proteins by loading cells with GDP beta S strongly reduced or suppressed desensitization, and resulted in a sustained inward cationic current. When cells were filled with GTP gamma S to irreversibly activate G-proteins, the desensitization process was enhanced such that a first application of agonist caused a markedly reduced response. 6. These results show that a cationic current induced by metabotropic agonists in hippocampal pyramidal cells undergoes apparent desensitization and suggests that this process occurs through a G-protein-mediated inhibition of the underlying membrane conductance.

1S, 3R-ACPD induces a region of negative slope conductance in the steady-state current-voltage relationship of hippocampal pyramidal cells.

Synaptic responses mediated by metabotropic glutamate receptors (mGluRs) display a marked voltage-dependent increase in amplitude when neurons are moderately depolarized beyond membrane potential. We have investigated the basis for this apparent nonlinear behavior by activating mGluRs with 1S, 3R-1-aminocyclopentane-1, 3-dicarboxylate (1S, 3R-ACPD; 10 microM) in CA3 pyramidal cells from rat hippocampal slice cultures with the use of the single-electrode voltage-clamp technique. Under control conditions, cells depolarized from resting potential by 10-20 mV responded with delayed outwardly rectifying currents due to activation of voltage- and Ca(2+)-dependent K+ conductances. In contrast, in the continuous presence of 1S, 3R-ACPD, small depolarizations (10-20 mV) induced a delayed inward current. The steady-state current-voltage relationship for this response displayed a region of negative slope conductance at potentials between -55 and -40 mV. The reversal potential of the corresponding 1S,3R-ACPD-sensitive tail currents (-93.0 +/- 2.2 mV, mean +/- SE) was close to the potassium reversal potential, consistent with an mGluR-mediated suppression of K+ current. When external K+ concentration was increased to 8 mM, there was a positive shift in reversal potential to -76.9 +/- 5.1 mV. The depolarization-induced inward current in the presence of 1S,3R-ACPD was blocked by Ba2+ (1 mM). The response was not dependent on changes in intracellular Ca2+ concentration and was insensitive to bath-applied Cs+ (1 mM), ruling out a contribution of Ca(2+)-dependent currents or the inward rectifier lQ. Furthermore, the effect of 1S,3R-ACPD was not mimicked by inhibiting afterhyperpolarizing current and M current with low-Ca2+ saline (0.5 mM Ca2+, 10 mM Mg2+) containing 10 mM tetraethylammonium chloride. A comparison of the responses induced by 1S,3R-ACPD and N-methyl-D-aspartate showed that both induce an inward current with small depolarizations from resting potential but with different kinetics and Mg2+ sensitivity. These results indicate that the suppression of K+ currents in response to activation of mGluRs is markedly voltage dependent, increasing at depolarized potentials and decreasing at hyperpolarized potentials. The negative slope conductance at membrane voltages positive to resting potential may underlie the amplification of mGluR-mediated responses when the membrane potential approaches action potential threshold.