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Subtype-Specific Effects of Lithium on Glutamate Receptor Function NIKOLAS B. KARKANIAS1 AND ROGER L. PAPKE1,21Department of Neuroscience and 2Department of Pharmacology and Therapeutics, University of Florida Medical College,J. H. Miller Health Center, Gainesville, Florida 32610-0267 Karkanias, Nikolas B. and Roger L. Papke. Subtype-specific ef-
how synaptic activity and neuronal plasticity may be coupled fects of lithium on glutamate receptor function. J. Neurophysiol. 81: 1506 –1512, 1999. We report that substitution of sodium with lithium Several subtypes of GluRs contribute to fast excitatory trans- (Liϩ) in the extracellular solution causes subtype-specific changes in mission, and they can be pharmacologically distinguished into the inward and outward currents of glutamate receptors (GluRs),without a shift in reversal potential. Liϩ produces an increase of two major classes, non–N-methyl-D-aspartate (NMDA) and inward and outward currents of ␣-amino-3-hydroxy-5-methyl-4-isox- NMDA sensitive. The non-NMDA–sensitive channels contain azole propionate receptors and decreases in the currents of kainate the receptor subunits GluR1–GluR7. The channels composed (KA) and N-methyl-D-aspartate receptors. The greatest effect of Liϩ of subunits GluR5–GluR7 can assemble with accessory sub- was observed with GluR3. A concentration-response curve for GluR3 units KA1 or KA2 to form receptors that are activated by reveals that the potentiation caused by Liϩ is greatest at saturating kainate (KA). Channels composed of GluR1–GluR4 are acti- agonist concentrations. GluR1, which shows no potentiation by Liϩ at vated by ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionate 100 ␮M KA, shows a small but significant potentiation at saturatingKA and glutamate concentrations. The effects of Liϩ on outward (AMPA) as well as KA. The NMDA-sensitive channels are current, where Liϩ is not the primary charge carrier, and the lack of composed of NMDAR1 and NMDAR2a–NMDAR2d. Func- reversal potential shift argue for a mechanism of potentiation not tional properties of these GluRs, such as their permeability to associated with Liϩ permeation. This potentiation of current is spe- sodium, potassium, and calcium and their kinetics, are influ- cific for Liϩ because rubidium, although causing an increase of enced by the specific subunit composition of the channel (Holl- inward current, shifted the reversal potential and did not increase mann et al. 1991; Monyer et al. 1992) .
outward current. The effects of Liϩ are different for KA, a weak Receptor desensitization is another property of GluRs that desensitizing agonist, and glutamate, a strong desensitizing agonist,suggesting that Liϩ might interact with a mechanism of desensitiza- may regulate synaptic function. When the glutamate transient tion. By using cyclothiazide (CTZ) to reduce desensitization of time course is slow, because of the nature of the synaptic GluR3, we find that for low concentrations of KA and glutamate morphology, the duration of synaptic current may be deter- potentiation of the response by a combination of CTZ and Liϩ is no mined primarily by desensitization kinetics (Barbour et al.
greater than by CTZ or Liϩ alone. However, at high concentrations of 1994). Desensitization is promoted by agonist exposure, and agonist, the potentiation of the response by a combination of CTZ and experimental agonists can vary in their relative desensitizing Liϩ is significantly greater than by CTZ or Liϩ alone. This potenti- effect. For example, KA produces less desensitization in ation was additive for glutamate but not for KA. At high agonist AMPA-selective receptors than either AMPA or glutamate.
concentration in the presence of CTZ, the intrinsically lower desen- The desensitization kinetics vary among the specific AMPA sitization produced with KA-evoked responses may preclude Liϩ receptor subtypes as a result of RNA editing and alternative from potentiating the current to the same degree as it can potentiateglutamate-evoked responses. The additive effects of CTZ and Liϩ splicing. The flip/flop domain is a 38-amino acid cassette were unique to the flop variant of GluR3.
located extracellularly and N-terminal to the final transmem-brane domain of AMPA receptors. Alternative splicing of thiscassette yields mature flip or flop AMPA receptors that vary intheir desensitization kinetics (Mosbacher et al. 1994; Sommer et al. 1990). Immediately before the flip/flop domain is the R/G Ionotropic glutamate receptors (GluRs) are responsible for site (Lomeli et al. 1994). RNA editing at the R/G site can also most of the fast excitatory neurotransmission in the mamma- influence AMPA receptor desensitization kinetics with edited lian brain, which includes activity-dependent synaptic modifi- channels (G) recovering from desensitization faster.
cations such as long-term potentiation and long-term depres- Although pharmacological modulators such as cyclothiazide sion (Bliss and Gardner-Medwin 1973; Bliss and Lynch 1988; (CTZ) are thought to reduce desensitization of AMPA recep- Collingridge and Bliss 1987). A differential expression of tors thus providing pharmacological tools with which to study GluR subunit genes gives rise to the functional diversity of this property (Partin et al. 1993; Vyklicky et al. 1991; Wong GluRs among brain regions with unique permeability and and Mayer 1993), desensitization was largely assumed to be kinetic properties for specific receptor subtypes. Characteris- independent of the charge-carrying ion. In a previous study we tics of GluRs such as ionic selectivity and kinetics are vital to reported the preliminary observation that Liϩ produced sub- the understanding of fast excitatory synaptic transmission and type-specific alterations of macroscopic current (Karkanias etal. 1998). Further investigation of Liϩ effects on GluR leads usto propose that modulation of receptor desensitization is a The costs of publication of this article were defrayed in part by the payment mechanism that causes the flop variant of GluR3 to display a of page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
modified conductance in the presence of Liϩ.
0022-3077/99 $5.00 Copyright 1999 The American Physiological Society LITHIUM AFFECTS GLUTAMATE RECEPTOR FUNCTION and then diluted in Ringer to Ͻ1% DMSO. No effect on controlresponse was observed when the agonist was dissolved in DMSO. In most experiments, barium was used instead of calcium in the Ringer Female Xenopus laevis frogs were purchased from Nasco (Fort to minimize contributions of endogenous calcium-activated chloride Atkinson, WI) and kept in tanks at 17°C. Frogs were anesthetized for current. However, similar results were obtained in the presence of 30 min on ice in 2.2 g/1.5 l of 3-aminobenzoic acid ethyl ester calcium. For experiments with GluR6(Q/R), a 2.0-ml, 10-s pulse of purchased from Sigma (St. Louis, MO). Oocytes were obtained in concanavalin A (Sigma; St. Louis, MO, type IV; 1.2 mg/ml) was lobes through a small abdominal incision made just above the leg and applied 5 min before applying agonist.
near the midline on the ventral surface of the frog. Two to three lobes Current-voltage relationships were performed by delivering a volt- were pulled from the frog, cut, and placed in collagenase from age ramp, Ϫ50.0 mV to ϩ50.0 mV, during the plateau phase of the Worthington Biochemical (Freehold, NJ) (1 mg/ml in calcium-free response to agonist (pClamp 5.5, Axon Instruments; Foster City, CA).
Barth’s solution containing 88 mM NaCl, 1 mM KCl, 15 mM HEPES, The passive current-voltage response of the cell membrane in the 0.33 mM MgSO , and 0.1 mg/ml gentamicin sulfate, pH 7.6) for 2 h absence of agonist was subtracted from the current-voltage response to enzymatically remove the native follicular cell layer. After the in the presence of agonist. Permeability ratios for Liϩ and rubidium follicular cell layer was removed, oocytes were washed several times with respect to sodium were calculated with Eq. 1 with calcium-free Barth’s and then washed several times with Barth’s solution containing 88 mM NaCl, 1 mM KCl, 15 mM HEPES, 0.33 mM CaNO , 0.41 mM CaCl , 0.33 mM MgSO , and 0.1 mg/ml E is the reversal potential in the presence of X (Liϩ or Rbϩ) or Naϩ, gentamicin sulfate (pH 7.6) and stored at 17°C. Mature oocytes were ⌬E is the difference between the reversal potentials in X and Na, [X ] injected the same or following day with the appropriate RNA tran- is the concentration of X outside of the cell (115 mM), [Na ] is the concentration of Naϩ outside of the cell (115 mM), and P is thepermeability of the ion.
cDNA clones containing the appropriate gene and T3/T7 bacterial Concentration-response relationships promoters were isolated from bacteria and purified with a kit from The responses of GluR3 expressing oocytes to various test concen- Qiagen (Santa Clarita, CA). Purified cDNA clones were linearized trations of KA were normalized by the response to the EC with the necessary restriction enzyme and then purified to serve as concentration (100 ␮M) immediately preceding the test concentration.
template for in vitro transcription. Briefly, in vitro cRNA transcripts First, a response was recorded to 100 ␮M KA, and after a 5-min were prepared with the appropriate mMessage mMachine kit from washout a response was recorded to a test dose of KA. After 5 min, Ambion (Austin, TX). Transcription reactions were performed with 1 100 ␮M KA was applied again to determine any residual effects from g cDNA as template, an RNA polymerase (T3 or T7, depending on the test dose of KA. If the response to 100 ␮M KA after the test dose clone), DTT, RNase inhibitor, dNTPs, and 32P. Nucleotide incorpo- was Ն75% of the response to 100 ␮M KA before the test dose, the ration was evaluated by DEAE81 filter binding assays and a liquid oocyte was tested further at other concentrations of KA. The resulting scintillation counter. RNA was stored in DEPC water stocks at concentration-response relationship was fitted with Eq. 2 (Luetje and 80°C, and aliquots were used for injection into the oocytes. The accession numbers for the clones used in this study were GluR1(X17184), GluR2 (M85035), GluR3 (M85036), GluR6 (Z11548), NMDAR1–1a (L19708), NMDAR2a (AF001423), and NMDAR2b (U11419). Unless otherwise noted, we used the flop variants ofAMPA receptors in our experiments.
Effect of Liϩ on neuronal GluR function For the conventional two-electrode, voltage-clamp experiments, Specific GluR subtypes were evaluated for their potential oocytes were placed in a Warner Instruments (Hamden, CT) recording modulation by Liϩ. Effects on GluR function were noted in the chamber and perfused with frog Ringer solution [containing (in mM) range of 5–115 mM Liϩ. For our standard agonist applications 115 NaCl, 2.5 KCl, 1.8 BaCl , and 10 HEPES, pH 7.3]. Ringer we used 100 ␮M KA for AMPA receptors, 100 ␮M glutamate solutions containing lithium (Liϩ) or rubidium were made by substi- for KA receptors, and 100 ␮M glutamate ϩ 10 ␮M glycine for tuting the ion for sodium. Osmolarity of different Ringer solutions NMDA receptors. Under these conditions, KA/NMDA-recep- was checked with a Precision Systems (Natick, MA) Osmette Aosmometer. A Warner Instruments Oocyte Clamp OC-725B and Fre- tor currents appeared to be reduced in Liϩ solutions, whereas quency Devices model 902 filter were interfaced with National In- AMPA receptor currents were potentiated in Liϩ solutions.
struments (Austin, TX) LabVIEW software and a Macintosh com- Subtype-selective differences in potentiation were observed puter for data acquisition. Electrodes were fabricated from glass within the AMPA receptor class and were investigated further capillary tubes (KG-33) from Garner Glass (Claremont, CA) with a with ionic variation in extracellular solutions, different ago- DKI (Tujunga, CA) model 750 needle/pipette puller. Voltage elec- nists at multiple concentrations, and chemical modulators.
trodes were filled with 3 M KCl and had resistances on the order of1–5 M⍀, whereas current electrodes were filled with 0.25 M CsCl,0.25 M CsF, and 100 mM EGTA (pH 7.3) and had resistances of Permeability and conductance of neuronal GluRs to Liϩ 0.5–3 M⍀. Experiments were performed at room temperature, and the Compared with their respective sodium controls, the ampli- oocyte membrane was clamped at Ϫ50 mV. Currents were measured tude of the KA-evoked current in Liϩ varied across subtype of to the nearest nanoampere. At least three and usually four or moreoocytes were used for each measurement. Drugs were dissolved in GluR. However, when extracellular sodium was replaced with Ringer and applied by filling a 2.0-ml length of tubing at the end of Liϩ, no shift in reversal potential was detected for any of the the perfusion line. A discrete volume of agonist was thereby admin- subtypes that were tested. For the AMPA-selective flop vari- istered over a 10-s period. Some drug stocks were dissolved in DMSO ants of GluRs, both inward and outward currents in Liϩ Ringer Current-voltage relationships of various glutamate re- ceptor (GluR) subtypes. A: current-voltage relationship a Xenopusoocyte expressing the ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-selective ionotropic GluR3 ϩ GluR2 in re-sponse to 100 ␮M kainate (KA). B: bar graph of the currentrecorded in lithium (Liϩ) relative to Naϩ for GluR1, GluR1 ϩ R2,GluR3 ϩ GluR2, and GluR3 receptors with 100 ␮M KA at 2different membrane voltages. Error bars represent SE. C: bar graphof the current recorded in Liϩ relative to Naϩ for GluR6Q andGluR6R receptors with 100 ␮M glutamate at 2 different membranevoltages. D: bar graph of the current recorded in Liϩ relative toNaϩ for NMDAR1a ϩ NMDAR2a and NMDAR1a ϩ NMDAR2breceptors with 100 ␮M glutamate ϩ 10 ␮M glycine at 2 differentmembrane voltages.
were equal to or greater than the current in Naϩ Ringer. A creased 134 Ϯ 26% above control (n ϭ 3) in the presence of representative I-V relationship for GluR3 ϩ R2 is shown in Liϩ (Fig. 2B). These results indicate that GluR2 is intrinsically Fig. 1A. The increase of both inward and outward currents capable of Liϩ modulation. However, further experiments fo- without a shift in the reversal potential suggests an effect on the cused on the GluR3 subtype because it was most sensitive to rather than an increase in single channel conductance Liϩ potentiation and easily formed homomeric receptors.
with Liϩ. In general, the inward and outward currents ofvarious AMPA receptors were potentiated by Liϩ, rangingfrom 40 to 200% above control (Fig. 1B). However, the current Specificity of the effect of Liϩ on GluR currents evoked by 100 ␮M KA in Liϩ Ringer through GluR1 was notsignificantly increased. In contrast, under these conditions the To test the specificity of Liϩ effect on GluR currents, we current in Liϩ Ringer was decreased by 40 – 60% for KA and performed similar experiments in rubidium-based Ringer. Raw NMDA receptors (Fig. 1, C and D). Because GluR1 ϩ R2 waveforms recorded from a GluR3-expressing oocyte in Naϩ, receptor currents were potentiated but GluR1 currents were not Rbϩ, and Liϩ Ringer are shown in Fig. 3A. Rubidium produced (Fig. 1B), we investigated the role of the GluR2 subunit in Liϩ a mean increase in current of 56 Ϯ 4.6% (n ϭ 4) but corre- potentiation. Wild-type GluR2 alone does not function well in spondingly shifted the reversal potential 6.60 Ϯ 1.03 mV (n ϭ oocytes. Therefore for these experiments we expressed wild- 7) for GluR3 (Fig. 3A, inset) and 4.05 Ϯ 0.46 mV (n ϭ 10) for type GluR2 with a Q/R site mutant, GluR2(R586Q), as well as GluR3 ϩ R2 (Fig. 4, A and B). There is no potentiation of the mutant alone. The inward and outward currents of hetero- outward current by Rbϩ for GluR3 (Fig. 3A, inset) or for meric GluR2 ϩ GluR2(R586Q) receptors were increased in GluR3 ϩ R2 (Fig. 4B). By using the shift in reversal potentials Liϩ by 74 Ϯ 7.9% (n ϭ 6) and 72 Ϯ 10% (n ϭ 6), respectively and solving for the ratio P /P (Fig. 2A). The conductance of the mutant homomer was in- of 1.30 for GluR3 and 1.17 for GluR3 ϩ R2.
Q/R site mutant heteromer and homomer. A:current-voltage relationship from a Xenopusoocyte expressing the AMPA-selective iono-tropic GluR2 ϩ GluR2(R586Q) in response to100 ␮M KA. B: GluR2(R586Q).
LITHIUM AFFECTS GLUTAMATE RECEPTOR FUNCTION Effects of Rbϩ on GluR3 ϩ GluR2. A: mean data for the reversal potential shift of GluR3 and GluR3 ϩ R2 caused by Rbϩ. B: I-V relationshipfor GluR3 ϩ GluR2 in Naϩ and Rbϩ. Note the rightward shift in reversalpotential and that the outward current is not affected as in Fig. 1A. The agonistused was 100 ␮M KA.
Liϩ, 20 mM sucrose). The presence of 10 mM Naϩ in 115 mMLiϩ Ringer did not significantly reduce the response to 1 mMKA (data not shown). These results indicate that the extremepotentiation reported for 1 mM KA responses obtained in thepresence of 115 mM Liϩ was not due to a specific effectassociated with the removal of sodium. Note that when 1 mMGlu was used as the agonist the GluR3 current displayed anincreased threshold for potentiation by Liϩ that did not appearto saturate and reached a maximum potentiation of 357% with100% mole fraction of Liϩ (Fig. 7B).
It is interesting to note the receptor sensitivity to potentiation by concentrations of Liϩ that approach levels used for treat-ment of bipolar disorder. We observed an increase of 21 Ϯ 3%(n ϭ 10, P Ͻ 0.001) for GluR3 and 26 Ϯ 3% (n ϭ 6, P Ͻ 0.05)for GluR3 ϩ R2 in response to 100 ␮M KA at 5 mM extra-cellular Liϩ. If the potentiation was linearly dependent onextracellular Liϩ concentration, at 5 mM extracellular Liϩ(1/23 of 115 mM) one would expect that the potentiation wouldbe ϳ1/23 of the maximum potentiation observed with 100 ␮MKA and 115 mM Liϩ (or 7– 8%). This observation furthersupports the hypothesis that the effects of Liϩ are most likelydue to effects on P response waveforms recorded in 115 mM Naϩ, 115 mM Liϩ,and 5 mM Liϩ from GluR3-expressing oocytes are shown in Comparison of the effects of Rbϩ and Liϩ. A: response waveforms Fig. 6, AC. Rubidium has no effect on GluR3 current at 5 mM evoked with 100 ␮M KA from a GluR3 expressing oocyte in Naϩ, Rbϩ, andLiϩ Ringer solutions. The response of GluR3 in Naϩ Ringer is the smallest of extracellular concentration in contrast to Liϩ (Figs. 3B and 6).
the 3 solutions tested. The response of GluR3 in Rbϩ Ringer is ϳ56% larger Potentiation by Liϩ is readily reversible at both high and low than in Naϩ and is approximately 170% larger in Liϩ Ringer than in Naϩ. The concentrations on washout of the Liϩ Ringer. In Fig. 6D, an inset shows a representative I-V for GluR3 in Naϩ and Rbϩ. The reversalpotential is shifted 6.60 Ϯ 1.03 mV in the positive direction. B: comparison ofthe potentiation caused by 2 different concentrations of Liϩ (open bar) andRbϩ (shaded bar), relative to Naϩ (solid line; SE, dashed line). Asterisksindicate significant differences compared with Naϩ. * P Ͻ 0.001, ** P Ͻ0.0001.
Concentration dependence of extracellular Liϩ We wished to determine if the magnitude of Liϩ potentiation of GluR3 response varies linearly with external Liϩ concen-tration. We noted that when 100 ␮M KA is used as the agonistan increase of the extracellular Liϩ concentration causes acurrent potentiation that could be fit by Eq. 2 with an EC 14.85 Ϯ 1.71 mM (n Ն 4) (Fig. 5). However, when saturatingconcentrations (1 mM) of KA were used we saw a furtheranomalous increase in potentiation at the highest extracellularLiϩ concentration. To test if this peculiar increase with 1 mMKA in 100% Liϩ (115 mM Liϩ, 0 mM Naϩ) could be asso- Effect of extracellular [Liϩ] titration on GluR3 response potentia- ciated with the total absence of sodium we performed experi- tion. The percent increase in response caused by Liϩ relative to Naϩ Ringer isplotted vs. extracellular [Liϩ] for GluR3 expressing oocytes. Two different ments at the same Liϩ concentration but with added Naϩ (115 agonist concentrations were used; 100 ␮M KA (Ⅺ) and 1 mM KA (E). Each mM Liϩ, 10 mM Naϩ) as well as an osmotic control (115 mM point represents the means Ϯ SE of 4 –11 oocytes.
therapeutic window. A: depicted here are re-sponses to 100 ␮M KA in Naϩ Ringer fromoocytes expressing GluR3 channels. B: first 2responses are recorded in Naϩ Ringer, the next2 in Ringer with 115 mM Liϩ, and the finalresponse is a washout back to Naϩ Ringer. C:paradigm is the same as in B except the thirdand fourth responses are recorded in 110 mMNaϩ/5 mM Liϩ. D: I-V relationship for a Xe-nopus oocyte expressing GluR3 ϩ GluR2 re-corded in 110 mM Naϩ/5 mM Liϩ showingpotentiation of both inward and outward cur-rent.
I-V relationship of a GluR3 ϩ R2-expressing oocyte recorded Because flip/flop variants were reported to vary in their in 5 mM Liϩ showed an increase of both inward and outward intrinsic desensitization, we set out to determine the interaction currents analogous to that shown in Fig. 1A.
of the flip/flop domain with Liϩ potentiation. We investigatedGluR3 flip currents for their capacity to be modulated by Liϩ Liϩ effects on GluR agonist potency at saturating agonist concentrations in the presence and ab-sence of CTZ and Liϩ (Fig. 8, E and F). We found that Liϩ We examined the potency and efficacy of KA in the pres- potentiated the current evoked from GluR3 flip channels less ence and absence of Liϩ. The concentration-response relation-ship determined for GluR3 in Naϩ Ringer yielded a Hill slopeof 1.14 Ϯ 0.07 and an EC Liϩ Ringer, the concentration-response relationship yielded aHill slope of 2.19 Ϯ 0.56 and an EC 7A, Œ). Liϩ increased the maximal attainable response by Ͼ500% and increased the EC by 60% compared with so- dium. The total percent increase caused by Liϩ for glutamateand KA at low and high concentrations relative to the sameagonist and concentration in Naϩ is presented in Fig. 7B.
GluR1 does not show the same magnitude of increase com-pared with GluR3 with either KA or glutamate at saturatingconcentrations.
Liϩ effects and desensitization We examined the effect of Liϩ when desensitization of GluR3 channels was reduced with the compound CTZ (Fig. 8).
With 100 ␮M KA or glutamate as the agonist, both Liϩ and100 ␮M CTZ potentiate GluR3 responses to the same extent.
There was no further increase when the treatments were com-bined (Fig. 8, A and B). With 1 mM KA as the agonist, thecombination of 100 ␮M CTZ and Liϩ substitution produced acurrent increase that was 30% larger than the increase CTZproduced in Naϩ Ringer (Fig. 8C, P Ͻ 0.05, unpaired t-test).
Concentration-response relationship. A: concentration-response re- At a high glutamate concentration (1 mM), the combination of lationship of KA for GluR3 in Naϩ and Liϩ Ringer solution. All responses 100 ␮M CTZ and Liϩ substitution produced a current increase were normalized to the response of 100 ␮M KA in Naϩ Ringer. B: percent that was 353 Ϯ 54% larger than the increase CTZ produced in increase in response caused by Liϩ with different agonists and agonist con- Naϩ Ringer (Fig. 8D, P Ͻ 0.001, unpaired t-test). Although centrations. For 100 ␮M KA the increase was 220 Ϯ 14% (n ϭ 19), for 1 mM CTZ potentiated GluR1 currents in sodium, there was no KA the increase was 518 Ϯ 30% (n ϭ 19), for 100 ␮M glutamate the increasewas 237 Ϯ 29% (n ϭ 5), and for 1 mM glutamate the increase was 357 Ϯ 54% apparent interaction between the combination of CTZ with Liϩ (n ϭ 11). The increase is presented normalized to the response to the respective at either agonist concentration (data not shown).
LITHIUM AFFECTS GLUTAMATE RECEPTOR FUNCTION than from GluR3 flop. Potentiation of responses to 1 mM KA (Fig. 8F, P Ͻ 0.05, unpaired t-test). This reduction in GluR3 and 1 mM Glu was only 19 and 67% of the potentiation flip current when CTZ and Liϩ are combined was also seen obtained with GluR3 flop. We also examined the effect of Liϩ when lower agonist concentrations were used (100 ␮M Glu), when desensitization of GluR3 flip channels was reduced with indicating that this effect is not specific to high agonist con- the compound CTZ (Fig. 8, E and F). In contrast to the results centrations (n ϭ 3, P Ͻ 0.05, data not shown).
obtained with GluR3 flop, Liϩ actually decreased 1 mM KA ϩ100 ␮M CTZ-evoked GluR3 flip currents by 19% (n ϭ 6) compared with the 1 mM KA ϩ 100 ␮M CTZ-evoked currentsin Naϩ Ringer (n ϭ 6) (Fig. 8E, P Ͻ 0.05, unpaired t-test).
We characterized the subtype-selective potentiation of Similarly, Liϩ decreased 1 mM Glu ϩ 100 ␮M CTZ-evoked GluR3 by Liϩ through an evaluation of Liϩ effects with GluR3 flip currents by 35% (n ϭ 11) compared with the 1 mM agonist concentration, mole fraction of extracellular Liϩ, and Glu ϩ 100 ␮M CTZ-evoked currents in Naϩ Ringer (n ϭ 11) compounds affecting receptor desensitization. Our results per-mit us to propose that the effect of Liϩ is to modify desensi-tization in a manner that depends on the flop domain.
Liϩ, rubidium, and sodium are all monovalent cations in group I of the periodic table, but only Liϩ is used for thetreatment of the mental illness bipolar disorder. Studies involv-ing Liϩ inhibition of second-messenger systems often userubidium to confirm the specificity of the inhibition by Liϩ(Ebstein et al. 1980). We therefore compared GluR3 currents inLiϩ-, Naϩ-, and Rbϩ-based Ringer. Although we observed apotentiation of the responses in both Rbϩ and Liϩ Ringercompared with Naϩ, the potentiation in Rbϩ was qualitativelydifferent than in Liϩ because it coincided with a shift of thereversal potential in the positive direction and was not ob-served on outward currents. These data suggest that the effectsof Rbϩ were largely due to an increased permeability of Rbϩthrough the channel.
The magnitude of Liϩ potentiation increased with the con- centration of extracellular Liϩ independently of agonist con-centration except at saturating agonist concentration in thepresence of 115 mM Liϩ. One potential explanation for thisobservation is that there are two processes that contribute toLiϩ potentiation of GluR current. One process, which is inde-pendent of agonist concentration, may predominate at lowerLiϩ concentrations. At very high Liϩ concentrations, a secondform of potentiation may manifest that selectively enhancesresponses to high, potentially desensitizing concentrations ofagonists.
The results of our concentration-response experiments fur- ther suggest that Liϩ caused a dramatic change in the apparentefficacy of the agonist. A raised maximal response suggests anincrease in the probability of a channel being open (P ) or an increase in the single-channel conductance (␥). However, theeffects of Liϩ on outward current, where it is not the primarycharge carrier, favors the interpretation that there is an alter- Interactions among cyclothiazide (CTZ), agonist, and Liϩ. Shaded ation in the percentage of time that channels are open.
bars represent mean current potentiation in Liϩ relative to Naϩ (dashed line).
CTZ was reported to potentiate AMPA receptor currents by Open bars represent mean current potentiation by 100 ␮M CTZ relative to Naϩ reducing receptor desensitization (Partin et al. 1996). Because (dashed line). Striped bars represent mean current potentiation in Liϩ ϩ100 Liϩ effects were greatest under desensitizing conditions, we ␮M CTZ relative to Naϩ (dashed line). Error bars represent SE. A: GluR3 flop hypothesized that the effect of Liϩ might also be to reduce responses to 100 ␮M KA in 115 mM Liϩ (n ϭ 13), 115 mM Naϩ ϩ 100 ␮MCTZ (n ϭ 13), and 115 mM Liϩ ϩ 100 ␮M CTZ (n ϭ 14). B: GluR3 flop desensitization, and we sought to determine if CTZ and Liϩ responses to 100 ␮M glutamate in 115 mM Liϩ (n ϭ 7), 115 mM Naϩ ϩ 100 acted through similar or different mechanisms. If Liϩ and CTZ ␮M CTZ (n ϭ 7), and 115 mM Liϩ ϩ 100 ␮M CTZ (n ϭ 8). C: GluR3 flop work through distinct mechanisms, the potentiation that each responses to 1 mM KA in 115 mM Liϩ (n ϭ 9), 115 mM Naϩ ϩ 100 ␮M CTZ causes individually might be additive when they are applied in (n ϭ 9), and 115 mM Liϩ ϩ 100 ␮M CTZ (n ϭ 7). D: GluR3 flop responsesto 1 mM glutamate in 115 mM Liϩ (n ϭ 4), 115 mM Naϩ ϩ 100 ␮M CTZ combination. Our results indeed suggest that, at high concen- (n ϭ 4), and 115 mM Liϩ ϩ 100 ␮M CTZ (n ϭ 4). E: GluR3 flip responses trations of KA, Liϩ and CTZ do not act entirely through the to 1 mM KA in 115 mM Liϩ (n ϭ 6), 115 mM Naϩ ϩ 100 ␮M CTZ (n ϭ 6), same mechanism. Moreover, when a high concentration of and 115 mM Liϩ ϩ 100 ␮M CTZ (n ϭ 6). F: GluR3 flip responses to 1 mM glutamate (1 mM) was used, the effects of Liϩ and CTZ glutamate in 115 mM Liϩ (n ϭ 11), 115 mM Naϩ ϩ 100 ␮M CTZ (n ϭ 11), appeared to be completely additive for GluR3. These observa- and 115 mM Liϩ ϩ 100 ␮M CTZ (n ϭ 11). Asterisks indicate significantdifferences compared with Naϩ ϩ CTZ. * P Ͻ 0.05, ** P Ͻ 0.001.
tions are consistent with the idea that Liϩ produces potentia- tion of current in GluR3 by modulating the amount of receptor BLISS, T.V.P. AND LYNCH, M. A. Long-term potentiation of synaptic transmis- desensitization and that desensitization of the channel can be sion in the hippocampus: properties and mechanisms. In: Long-term Poten- influenced by specific agonists. Desensitization is also con- tiation: From Biophysics to Behavior. New York: Liss, 1988, p. 3–72.
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alternative splice variants, flip and flop, in AMPA receptors.
EBSTEIN, R. P., HERMONI, M., AND BELMAKER, R. H. The effect of lithium on Flop receptors are thought to desensitize more than flip vari- noradrenaline-induced cyclic AMP accumulation in rat brain: inhibition ants. Indeed, it appears that flop receptor currents are also after chronic treatment and absence of supersensitivity. J Pharmacol. Exp. potentiated to a greater degree by Liϩ than are flip receptor currents. The specificity of the Liϩ effect for the flop domain HOLLMANN, M., HARTLEY, M., AND HEINEMANN, S. Ca2ϩ permeability of KA-AMPA-gated glutamate receptor channels depends on subunit compo- is further supported by the observation that, under conditions sition. Science 252: 851– 853, 1991.
of the additive CTZ effect for flop variants, there was an KARKANIAS, N. B., KABAKOV, A. Y., LENOX, R. H., AND PAPKE, R. L. Synapse- antagonism of the CTZ effect in flip variants.
specific accumulation of lithium in intracellular microdomains: a model for The inconsistent interaction of Liϩ with CTZ suggests a uncoupling coincidence detection in the brain. Synapse 28: 271–279, 1998.
novel mechanism for this effect, although with a similar re- LOMELI, H., MOSBACHER, J., MELCHER, T., HOGER, T., GEIGER, JR., KUNER, T., quirement for the flop domain. The modulation of desensitiza- MONYER, H., HIGUCHI, M., BACH, A., AND SEEBURG, P. H. Control of kineticproperties of AMPA receptor channels by nuclear RNA editing. Science tion could involve alterations of rate constants into or out of the desensitized state as well as alterations of rate constants lead- LUETJE, C. W. AND PATRICK, J. Both ␣- and ␤-subunits contribute to the agonist ing toward the desensitized state. Our data suggest that further sensitivity of neuronal nicotinic acetylcholine receptors. J. Neurosci. 11: detailed studies of desensitization may exploit the use of Liϩ as a tool to dissect the mechanisms of desensitization.
MONYER, H., SPRENGEL, R., SCHOEPFER, R., HERB, A., HIGUCHI, M., LOMELI, H., BURNASHEV, N., SAKMANN, B., AND SEEBURG, P. H. Heteromeric NMDA The authors thank Drs. Michael Hollmann, Jane Sullivan, Steve Heinemann, receptors: molecular and functional distinction of subtypes. Science 256: and Jim Boulter for the glutamate receptor (GluR) subunit genes and Dr. R.
Dingledine for providing the mutant GluR2 channel cDNA. We thank Dr.
MOSBACHER, J., SCHOEPFER, R., MONYER, H., BURNASHEV, N., SEEBURG, P., Robert Lenox for helpful discussion.
AND RUPPERSBERG, J. A molecular determinant for submillisecond desensi- This work was supported by predoctoral fellowships to N. B. Karkanias tization in glutamate receptors. Science 266: 1059 –1062, 1994.
from the University of Florida Center for Neurobiology and Behavior and the PARTIN, K. M., FLECK, M. W., AND MAYER, M. L. AMPA receptor flip/flop University of Florida Division of Sponsored Research. Technical assistance mutants affecting deactivation, desensitization, and modulation by cy- clothiazide, aniracetam, and thiocyanate. J. Neurosci. 16: 6634 – 6647, 1996.
Address for reprint requests: R. L. Papke, Depts. Of Pharmacology & PARTIN, K. M., PATNEAU, D. K., WINTERS, C. A., MAYER, M. L., AND BUO- Therapeutics and Neuroscience, University of Florida Medical College, J. H.
NANNO, A. Selective modulation of desensitization at AMPA versus kainate Miller Health Center, Box 100267, Gainesville, FL 32610-0267.
receptors by cyclothiazide and concanavalin A. Neuron 11: 1069 –1082,1993.
Received 14 September 1998; accepted in final form 15 December 1998.
SOMMER, B., KEIN¨ANEN, K., VERDOORN, T. A., WISDEN, W., BURNASHEV, N., HERB, A., KOHLER, M., TAKAGI, T., SAKMANN, B., AND SEEBURG, P. H. Flip and flop: a cell-specific functional switch in glutamate-operated channels ofthe CNS. Science 249: 1580 –1585, 1990.
BARBOUR, B., KELLER, B. U., LLANO, I., AND MARTY, A. I. Prolonged presence VYKLICKY, J. L., PATNEAU, D. K., AND MAYER, M. L. Modulation of excitatory of glutamate during excitatory synaptic transmission to cerebellar Purkinje synaptic transmission by drugs that reduce desensitization at AMPA/kainate cells. Neuron 12: 1331–1343, 1994.
receptors. Neuron 7: 971–984, 1991.
BLISS, T.V.P. AND GARDNER-MEDWIN, A. R. Long-lasting potentiation of WONG, L. A. AND MAYER, M. L. Differential modulation by cyclothiazide and synaptic transmission in the dentate area of the unanaesthetized rabbit concanavalin A of desensitization at native alpha-amino-3-hydroxy-5- following stimulation of the perforant path. J. Physiol. (Lond.) 232: 357– methyl-4-isoxazolepropionic acid- and kainate-preferring glutamate recep- tors. Mol. Pharmacol. 44: 504 –510, 1993.

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