Silent synapses in developing cerebellar granule neurons. (49/267)

Silent synapses are excitatory synapses endowed exclusively with N-methyl-D-aspartate (NMDA) responses that have been proposed to acquire alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) responses during development and after long-term potentiation (LTP). These synapses are functionally silent because of the Mg(2+) block of NMDA receptors at resting potentials. Here we provide evidence for the presence of silent synapses in developing cerebellar granule cells. Using the patch-clamp technique in the whole-cell configuration, we recorded the spontaneous excitatory postsynaptic currents (sEPSCs) from rat cerebellar granule cells in culture and in slices at physiological concentration of Mg(2+) (1 mM). A holding potential of +60 mV removes Mg(2+) block of NMDA channels, allowing us to record NMDA-sEPSCs. We thus compared the frequency of AMPA-sEPSCs, recorded at -60 mV, with that of NMDA-sEPSCs, recorded at +60 mV. NMDA-sEPSCs occurred at higher frequency than the AMPA-sEPSCs in most cells recorded in slices from rats at postnatal day (P) <13 and in culture at 6-8 days after plating (DIV6-8). In a few cells from young rats (P6-9) and in most neurons in culture at DIV6 we recorded exclusively NMDA-sEPSCs, supporting the hypothesis of existence of functional synapses with NMDA and without AMPA receptors. Increasing glutamate release in the slice with cyclothiazide and temperature increased AMPA and NMDA-sEPSCs frequencies but failed to alter the relative ratio of frequency of occurrence. Frequency ratio of NMDA versus AMPA-sEPSCs in slices was correlated with the weighted time constant of decay (tau(w)) of NMDA-sEPSCs and decreased with development along the reported decrease of tau(w). We suggest that the prevalence of synaptic NR2A subunits that confer faster kinetics is paralleled by the disappearance of silent synapses early in cerebellar development.  (+info)

Contributions of receptor desensitization and saturation to plasticity at the retinogeniculate synapse. (50/267)

The retinogeniculate synapse conveys visual information from the retina to thalamic relay neurons. Here, we examine the mechanisms of short-term plasticity that can influence transmission at this connection in mouse brain slices. Our studies show that synaptic strength is modified by physiological activity patterns due to marked depression at high frequencies. Postsynaptic mechanisms of plasticity make prominent contributions to this synaptic depression. During trains of retinal input stimulation, receptor desensitization attenuates the AMPA EPSC while the NMDA EPSC saturates. This differential plasticity may help explain the distinct roles of these receptors in shaping the relay neuron response to visual stimulation with the AMPA component being important for transient responses, while sustained high frequency responses rely more on the NMDA component.  (+info)

Functional differences between flounder and rat thiazide-sensitive Na-Cl cotransporter. (51/267)

The purpose of the present study was to determine the major functional, pharmacological, and regulatory properties of the flounder thiazide-sensitive Na-Cl cotransporter (flTSC) to make a direct comparison with our recent characterization of the rat TSC (rTSC; Monroy A, Plata C, Hebert SC, and Gamba G. Am J Physiol Renal Physiol 279: F161-F169, 2000). When expressed in Xenopus laevis oocytes, flTSC exhibits lower affinity for Na(+) than for Cl(-), with apparent Michaelis-Menten constant (K(m)) values of 58.2 +/- 7.1 and 22.1 +/- 4.2 mM, respectively. These K(m) values are significantly higher than those observed in rTSC. The Na(+) and Cl(-) affinities decreased when the concentration of the counterion was lowered, suggesting that the binding of one ion increases the affinity of the transporter for the other. The effect of several thiazides on flTSC function was biphasic. Low concentrations of thiazides (10(-9) to 10(-7) M) resulted in activation of the cotransporter, whereas higher concentrations (10(-6) to 10(-4) M) were inhibitory. In rTSC, this biphasic effect was observed only with chlorthalidone. The affinity for thiazides in flTSC was lower than in rTSC, but the affinity in flTSC was not affected by the Na(+) or the Cl(-) concentration in the uptake medium. In addition to thiazides, flTSC and rTSC were inhibited by Hg(2+), with an apparent higher affinity for rTSC. Finally, flTSC function was decreased by activation of protein kinase C with phorbol esters and by hypertonicity. In summary, we have found significant regulatory, kinetic, and pharmacological differences between flTSC and rTSC orthologues.  (+info)

Activity-dependent change in AMPA receptor properties in cerebellar stellate cells. (52/267)

High-frequency synaptic stimulation is thought to cause a rapid and lasting change in the expression of GluR2 subunit-containing AMPA receptors (AMPARs) at synapses in cerebellar stellate cells. We examined whether spontaneous synaptic activity affects the expression of GluR2-containing synaptic AMPARs and whether the change in AMPAR subtypes alters their Ca(2+) permeability and kinetic properties. We used intracellular spermine, which blocks GluR2-lacking receptors at depolarized potentials, to distinguish the presence of GluR2. In most cells, the spontaneous EPSC frequency was low, and evoked EPSCs displayed inwardly rectifying I-V relationships, indicative of the presence of GluR2-lacking AMPARs. However, in cells that displayed a higher rate of spontaneous synaptic activity, EPSCs gave linear I-V plots, suggesting the presence of GluR2-containingAMPARs. This is consistent with the idea that spontaneous synaptic activity increased the expression of GluR2-containing AMPARs at synapses. The Ca(2+) permeability of AMPARs that gave inwardly rectifying currents in outside-out patches from TTX-treated cells was six times higher than in control cells that gave linear or outwardly rectifying I-V plots. However, increased spontaneous synaptic activity did not significantly alter the EPSC decay time. Furthermore, the decay time course ofEPSCs mediated by GluR2-containing receptors was not different from that mediated by a mixed population of receptors at the same synapse. Our results suggest that the level of spontaneous synaptic activity can determine the subunit composition of postsynaptic receptors at this synapse. The activity-induced expression of GluR2-containing receptors significantly reduced the Ca(2+) permeability of AMPARs in stellate cells but did not slow the decay time course of synaptic currents.  (+info)

The novel diazoxide analog 3-isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine 1,1-dioxide is a selective Kir6.2/SUR1 channel opener. (53/267)

ATP-sensitive K(+) (K(ATP)) channels are activated by a diverse group of compounds known as potassium channel openers (PCOs). Here, we report functional studies of the Kir6.2/SUR1 Selective PCO 3-isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine 1,1-dioxide (NNC 55-9216). We recorded cloned K(ATP) channel currents from inside-out patches excised from Xenopus laevis oocytes heterologously expressing Kir6.2/SUR1, Kir6.2/SUR2A, or Kir6.2/SUR2B, corresponding to the beta-cell, cardiac, and smooth muscle types of the K(ATP) channel. NNC 55-9216 reversibly activated Kir6.2/SUR1 currents (EC(50) = 16 micromol/l). This activation was dependent on intracellular MgATP and was abolished by mutation of a single residue in the Walker A motifs of either nucleotide-binding domain of SUR1. The drug had no effect on Kir6.2/SUR2A or Kir6.2/SUR2B currents. We therefore used chimeras of SUR1 and SUR2A to identify regions of SUR1 involved in the response to NNC 55-9216. Activation was completely abolished and significantly reduced by swapping transmembrane domains 8-11. The reverse chimera consisting of SUR2A with transmembrane domains 8-11 and NBD2 consisting SUR1 was activated by NNC 55-9216, indicating that these SUR1 regions are important for drug activation. [(3)H]glibenclamide binding to membranes from HEK293 cells transfected with SUR1 was displaced by NNC 55-9216 (IC(50) = 105 micromol/l), and this effect was impaired when NBD2 of SUR1 was replaced by that of SUR2A. These results suggest NNC 55-9216 is a SUR1-selective PCO that requires structural determinants, which differ from those needed for activation of the K(ATP) channel by pinacidil and cromakalim. The high selectivity of NNC 55-9216 may prove to be useful for studies of the molecular mechanism of PCO action.  (+info)

Long-term potentiation alters the modulator pharmacology of AMPA-type glutamate receptors. (54/267)

Changes in the biophysical properties of AMPA-type glutamate receptors have been proposed to mediate the expression of long-term potentiation (LTP). The present study tested if, as predicted from this hypothesis, AMPA receptor modulators differentially affect potentiated versus control synaptic currents. Whole cell recordings were collected from CA1 pyramidal neurons in hippocampal slices from adult rats. Within-neuron comparisons were made of the excitatory postsynaptic currents (EPSCs) elicited by two separate groups of Schaffer-collateral/commissural synapses. LTP was induced by theta burst stimulation in one set of inputs; cyclothiazide (CTZ), a drug that acts on the desensitization kinetics of AMPA receptors, was infused 30 min later. The decay time constants of the potentiated EPSCs prior to drug infusion were slightly, but significantly, shorter than those of control EPSCs. CTZ slowed the decay of the EPSCs, as reported in prior studies, and did so to a significantly greater degree in the potentiated synapses. Additionally, infusion of CTZ resulted in significantly greater effects on amplitude in potentiated pathways as compared with control pathways. The interaction between LTP and CTZ was also obtained in a separate set of experiments in which GABA receptor antagonists were used to block inhibitory postsynaptic currents. Additionally, there was no significant change in paired-pulse facilitation in the presence of CTZ, indicating that presynaptic effects of the drug were negligible. These findings provide new evidence that LTP modifies AMPA receptor kinetics. Candidates for the changes responsible for the observed effects of LTP were evaluated using a model of AMPA receptor kinetics; a simple increase in the channel opening rate provided the most satisfactory match with the LTP data.  (+info)

AMPA and NMDA currents show different short-term depression in the dorsal lateral geniculate nucleus of the rat. (55/267)

Paired-pulse depression was studied at the glutamatergic synapse between retinal afferents and thalamocortical cells in the rat dorsal lateral geniculate nucleus. The main objective of this study was to examine the contributions of the pre- and postsynaptic sites to this depression by comparing AMPA- and NMDA-receptor-mediated responses. Equal depression of the two receptor components would indicate involvement of presynaptic mechanisms, while differences in depression would indicate involvement of postsynaptic mechanisms. Pharmacologically isolated AMPA- and NMDA-receptor-mediated currents were recorded using the whole-cell patch-clamp technique in acute thalamic slices. Both the AMPA and the NMDA components showed pronounced depression when retinal afferents were activated by paired pulses. The depression decayed within 5 s. The AMPA component was more strongly depressed than the NMDA component at paired-pulse intervals ranging from 20 to 200 ms, suggesting the involvement of postsynaptic mechanisms. For intervals of 500 ms and longer, the depression of the two components was identical, suggesting the involvement of purely presynaptic mechanisms. The degree of depression measured without the use of pharmacological tools produced similar results, thus excluding the involvement of presynaptic ionotropic glutamate receptors. Cyclothiazide, a blocker of AMPA-receptor desensitisation, reduced the difference in depression between the two components, suggesting that desensitisation of the AMPA receptors is a postsynaptic mechanism that contributes to the difference in depression between the AMPA and the NMDA components.  (+info)

Functional characteristics of non-NMDA-type ionotropic glutamate receptor channels in AII amacrine cells in rat retina. (56/267)

The properties of non-NMDA glutamate receptor channels in AII amacrine cells were studied by patch-clamp recording from rat retinal slices. Application of AMPA or kainate to intact cells evoked currents with no apparent desensitization (EC50 of 118 microM for AMPA and 169 microM for kainate). Application of AMPA to patches evoked desensitizing responses with an EC50 of 217 and 88 microM for the peak and steady-state responses, respectively. Kainate-evoked responses of patches displayed no desensitization (EC50 = 162 microM). Cyclothiazide strongly potentiated AMPA-evoked responses and the AMPA-receptor antagonist GYKI 53655 inhibited both AMPA- and kainate-evoked responses (IC50 = 0.5-1.7 microM). Pre-equilibration with GYKI 53655 completely blocked the response to kainate and pretreatment with concanavalin A did not unmask a response mediated by kainate receptors. AMPA- and kainate-evoked currents reversed close to 0 mV. AMPA-evoked peak and steady-state response components in patches displayed linear and outwardly rectifying I-V relations with an RI (ratio of the slope conductances at +40 mV and -60 mV) of 0.96 +/- 0.11 and 5.6 +/- 1.3, respectively. AMPA-evoked currents displayed a voltage-dependent relaxation after steps to positive or negative membrane potentials, indicating that the outward rectification of the steady-state response is caused by a voltage-dependent kinetic parameter of channel gating. Under bi-ionic conditions ([Ca2+](out) = 30 mM, [Cs+)(in) = 171 mM), the reversal potentials of AMPA- and kainate-evoked currents indicated channels with significant Ca2+ permeability (P(Ca)/P(Cs) = 1.9-2.1). Stationary noise analysis indicated that kainate activated channels with an apparent chord conductance of approximately 9 pS. Non-stationary noise analysis indicated that AMPA and glutamate activated channels with apparent chord conductances of approximately 9, approximately 15, approximately 23 and approximately 38 pS. Discrete single-channel gating corresponding to chord conductances of approximately 23 pS could be directly observed in some responses. Thus, our results indicate expression of high-affinity, voltage-sensitive AMPA receptors with significant Ca2+ permeability and relatively large single-channel chord conductances in AII amacrine cells.  (+info)