Receptor-mediated Whole-cell Currents Research Articles

Xenon modulates the GABA and glutamate responses at genuine synaptic levels in rat spinal neurons

Effects of xenon (Xe) on whole-cell currents induced by glutamate (Glu), its three ionotropic subtypes, and GABA, as well as on the fast synaptic glutamatergic and GABAergic transmissions, were studied in the mechanically dissociated “synapse bouton preparation” of rat spinal sacral dorsal commissural nucleus (SDCN) neurons. This technique evaluates pure single or multi-synapse responses from native functional nerve endings and enables us to quantify how Xe influences pre- and postsynaptic transmissions accurately. Effects of Xe on glutamate (Glu)-, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-, kainate (KA)- and N-methyl-d-aspartate (NMDA)- and GABAA receptor-mediated whole-cell currents were investigated by the conventional whole-cell patch configuration. Excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) were measured as spontaneous (s) and evoked (e) EPSCs and IPSCs. Evoked synaptic currents were elicited by paired-pulse focal electric stimulation. Xe decreased Glu, AMPA, KA, and NMDA receptor-mediated whole-cell currents but did not change GABAA receptor-mediated whole-cell currents. Xe decreased the frequency and amplitude but did not affect the 1/e decay time of the glutamatergic sEPSCs. Xe decreased the frequency without affecting the amplitude and 1/e decay time of GABAergic sIPSCs. Xe decreased the amplitude and increased the failure rate (Rf) and paired-pulse ratio (PPR) without altering the 1/e decay time of both eEPSC and eIPSC, suggesting that Xe acts on the presynaptic side of the synapse. The presynaptic inhibition was greater in eEPSCs than in eIPSCs. We conclude that Xe decreases glutamatergic and GABAergic spontaneous and evoked transmissions at the presynaptic level. The glutamatergic presynaptic responses are the main target of anesthesia-induced neuronal responses. In contrast, GABAergic responses minimally contribute to Xe anesthesia.

Effects of propofol on glycinergic neurotransmission in a single spinal nerve synapse preparation

The effects of the intravenous anesthetic, propofol, on glycinergic transmission and on glycine receptor-mediated whole-cell currents (IGly) were examined in the substantia gelatinosa (SG) neuronal cell body, mechanically dissociated from the rat spinal cord. This “synaptic bouton” preparation, which retains functional native nerve endings, allowed us to evaluate glycinergic inhibitory postsynaptic currents (IPSCs) and whole-cell currents in a preparation in which experimental solution could rapidly access synaptic terminals. Synaptic IPSCs were measured as spontaneous (s) and evoked (e) IPSCs. The eIPSCs were elicited by applying paired-pulse focal electrical stimulation, while IGly was evoked by a bath application of glycine. A concentration-dependent enhancement of IGly was observed for ≥10µM propofol. Propofol (≥3µM) significantly increased the frequency of sIPSCs and prolonged the decay time without altering the current amplitude. However, propofol (≥3µM) also significantly increased the mean amplitude of eIPSCs and decreased the failure rate (Rf). A decrease in the paired-pulse ratio (PPR) was noted at higher concentrations (≥10µM). The decay time of eIPSCs was prolonged only at the maximum concentration tested (30µM). Propofol thus acts at both presynaptic glycine release machinery and postsynaptic glycine receptors. At clinically relevant concentrations (<1μM) there was no effect on IGly, sIPSCs or eIPSCs suggesting that at anesthetic doses propofol does not affect inhibitory glycinergic synapses in the spinal cord.

Neuroprotection of GluR5-containing Kainate Receptor Activation against Ischemic Brain Injury through Decreasing Tyrosine Phosphorylation of N-Methyl-d-aspartate Receptors Mediated by Src Kinase

Previous studies indicate that cerebral ischemia breaks the dynamic balance between excitatory and inhibitory inputs. The neural excitotoxicity induced by ionotropic glutamate receptors gain the upper hand during ischemia-reperfusion. In this paper, we investigate whether GluR5 (glutamate receptor 5)-containing kainate receptor activation could lead to a neuroprotective effect against ischemic brain injury and the related mechanism. The results showed that (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA), a selective GluR5 agonist, could suppress Src tyrosine phosphorylation and interactions among N-methyl-D-aspartate (NMDA) receptor subunit 2A (NR2A), postsynaptic density protein 95 (PSD-95), and Src and then decrease NMDA receptor activation through attenuating tyrosine phosphorylation of NR2A and NR2B. More importantly, ATPA had a neuroprotective effect against ischemia-reperfusion-induced neuronal cell death in vivo. However, four separate drugs were found to abolish the effects of ATPA. These were selective GluR5 antagonist NS3763; GluR5 antisense oligodeoxynucleotides; CdCl(2), a broad spectrum blocker of voltage-gated calcium channels; and bicuculline, an antagonist of gamma-aminobutyric acid A (GABA(A)) receptor. GABA(A) receptor agonist muscimol could attenuate Src activation and interactions among NR2A, PSD-95 and Src, resulting the suppression of NMDA receptor tyrosine phosphorylation. Moreover, patch clamp recording proved that the activated GABA(A) receptor could inhibit NMDA receptor-mediated whole-cell currents. Taken together, the results suggest that during ischemia-reperfusion, activated GluR5 may facilitate Ca(2+)-dependent GABA release from interneurons. The released GABA can activate postsynaptic GABA(A) receptors, which then attenuates NMDA receptor tyrosine phosphorylation through inhibiting Src activation and disassembling the signaling module NR2A-PSD-95-Src. The final result of this process is that the pyramidal neurons are rescued from hyperexcitability.

Deletion of the NR2A subunit prevents developmental changes of NMDA‐mEPSCs in cultured mouse cerebellar granule neurones

We investigated the role N-methyl-d-aspartate (NMDA) receptor subunits play in shaping excitatory synaptic currents in cultures of cerebellar granule cells (CGCs) from NR2A knockout (NR2A-/-) and wild-type (+/+) mice. Cultures were maintained in a condition that facilitates the occurrence of functional synapses, allowing us to record NMDA-miniature excitatory postsynaptic currents (mEPSCs) in addition to NMDA receptor-mediated whole-cell currents at three ages in vitro. Whole-cell NMDA current density decreased with development in both strains though currents from NR2A-/- neurones demonstrated greater sensitivity to CP101 606, an NR2B subunit specific blocker. Sensitivity to Mg(2+) blockade decreased with age in vitro in +/+ but not in NR2A-/- CGCs. Immunocytochemistry revealed that dendrites and somas displayed distinct NR1 and NR2A subunit clusters which became increasingly colocalized in +/+ neurones. Qualitatively the overall NR2B subunit staining pattern was similar in +/+ and NR2A-/- neurones throughout development, suggesting that the NR2B subunit distribution is not mediated by the NR2A subunit. In addition, staining with markers for excitatory synapses showed that expression of NR2A subunit (but not NR2B) increases at both synaptic and extrasynaptic sites in +/+ neurones during development. In parallel, NMDA-mEPSCs were faster in +/+ compared with NR2A-/- neurones at all time points studied, suggesting that the NR2A subunit begins to replace NR2B-rich NMDA receptors even at early stages of development. Many NR2A-/- neurones were devoid of NMDA-mEPSCs at the later time point, and transfection of the NR2A subunit in these neurones restored fast decay and the occurrence of NMDA-mEPSCs. Taken together, our results indicate that the NR2A subunit is mainly responsible for the developmental changes observed in the maturation of excitatory synapses.

Regulation of single NMDA receptor channel activity by alpha-actinin and calmodulin in rat hippocampal granule cells.

The NMDA receptor is modulated by changes in the intracellular calcium concentration, through activation of various intracellular calcium-dependent proteins. We have investigated regulation of single NMDA receptor channel activity by the calcium-sensing proteins alpha-actinin and calmodulin. Both of these proteins bind to the NMDA receptor NR1 subunit C-terminus at the C0 region where they compete for occupation of the C0 site and contribute to calcium-dependent inactivation of NMDA receptor-mediated whole-cell currents. Calmodulin has also been shown to bind to the neighbouring C1 region where it has been shown to reduce single channel open time. To investigate regulation of single NMDA channel activity by alpha-actinin and calmodulin, we selected concentrations of these two proteins that would result in maximal binding to the C0 region and/or the C1 region in the case of calmodulin. Alpha-actinin binding was found to predominantly decrease single channel shut time, resulting in an increased open probability (P(open)), whereas calmodulin binding reduced single channel mean open time, resulting in an overall reduction in P(open). The physiological implications of these findings are discussed.

Kainate Receptor Subunits Underlying Presynaptic Regulation of Transmitter Release in the Dorsal Horn

Presynaptic kainate (KA) receptors regulate synaptic transmission at both excitatory and inhibitory synapses in the spinal cord dorsal horn. Previous work has demonstrated pharmacological differences between the KA receptors expressed by rat dorsal horn neurons and those expressed by the primary afferent sensory neurons that innervate the dorsal horn. Here, neurons isolated from KA receptor subunit-deficient mice were used to evaluate the contribution of glutamate receptor subunit 5 (GluR5) and GluR6 to the presynaptic control of transmitter release and to KA receptor-mediated whole-cell currents in these two cell populations [corrected]. Deletion of GluR6 produced a significant reduction in KA receptor-mediated current density in dorsal horn neurons, whereas GluR5 deletion caused no change in current density but removed sensitivity to GluR5-selective antagonists. Presynaptic modulation of inhibitory transmission between dorsal horn neurons was preserved in cells from either GluR5- or GluR6-deficient mice. In DRG neurons, in contrast, GluR5 deletion abolished KA receptor function, whereas deletion of GluR6 had little effect on peak current density but increased the rate and extent of desensitization. These results highlight fundamental differences in KA receptor physiology between the two cell types and suggest possible strategies for the pharmacological modulation of nociception.

Modulation of GABA A receptor-mediated currents by benzophenone derivatives in isolated rat Purkinje neurones

We investigated modulation of GABA A receptor-mediated whole-cell currents in cerebellar Purkinje neurones by several derivatives of benzophenone. A metabolite of phenazepam, 5-bromo-2’-chloro-2-aminobenzophenone ( I), caused dual modification of peak amplitudes of GABA-gated currents that depended upon the concentration of applied GABA and incubation time. Following short 10 s pre-incubations, 1–30 μM I facilitated activation and delayed deactivation of currents evoked by 500 ms pulses of 20 μM GABA. In addition, 10 μM I prominently enhanced desensitisation of currents during applications of 500 μM GABA mainly by decreasing the value of the fast time constant of the desensitisation. Continuous 6 min incubation with 10 μM I during GABA stimulation or its administration between but not during 1 s pulses of 500 μM GABA led to a gradual, partly reversible attenuation of GABA-activated currents. This inhibition was not observed when I was applied only during pulses of GABA, indicating that the blockade was not use-dependent. One of the possible mechanisms of this down-modulation could be an intracellular effect of I, because when applied intracellularly it caused slow inhibition of responses to consecutive GABA pulses. When 3–30 μM I was applied on the background of small ‘plateau’-like current 5–7 s after application of 500 μM GABA, it was able to block open channels with on and off rates similar to those observed with 30 μM picrotoxin but much slower than in the case of 500 μM benzylpenicillin. At a concentration of 10 μM, 5-substituted benzophenones, but not 2-aminobenzophenone or benzophenone itself, exhibited modulatory properties similar to I and distinct from those of picrotoxin and benzylpenicillin. Therefore, we conclude that derivatives of benzophenone are a novel class of GABA A receptor modulators with a unique pharmacological profile.

Deletion of the C-terminal domain of the NR2B subunit alters channel properties and synaptic targeting of N-methyl-D-aspartate receptors in nascent neocortical synapses.

Channel properties and synaptic targeting of N-methyl-D-aspartate (NMDA) receptors determine their importance in synaptic transmission, long-term synaptic plasticity, and developmental reorganization of synaptic circuits. To investigate the involvement of the C-terminal domain of the NR2B subunit in regulating channel properties and synaptic localization, we analyzed gene-targeted mice expressing C-terminally truncated NR2B subunits (NR2B(DeltaC/DeltaC) mice; Sprengel et al. [1998] Cell 92:279-89). Because homozygous NR2B(DeltaC/DeltaC) mice die perinatally, we studied embryonic neocortical neurons differentiating in culture. At early stages in vitro, neurons predominantly expressed NR1/NR2B receptors, as shown by the NR2B subunit-specific antagonist ifenprodil. At these nascent synapses, NMDA excitatory postsynaptic currents (EPSCs) in neurons from NR2B(DeltaC/DeltaC) mice showed a strong-amplitude reduction to 20% of control, but AMPA EPSCs were unaltered. Analysis of the MK-801 block of NMDA receptor-mediated whole-cell currents revealed a decreased peak open probability of NMDA receptor channels (to about 60%) in neurons from NR2B(DeltaC/DeltaC) mice, although their single channel conductance was unchanged. To study effects on synaptic targeting, we determined the fraction of synaptically localized NMDA receptors relative to the whole-cell NMDA receptor population. In neurons from NR2B(DeltaC/DeltaC) mice, the synaptic NMDA receptor fraction was drastically reduced, suggesting that the C-terminal domain of the NR2B subunit plays a major role in synaptic targeting of NMDA receptors at nascent synapses. With increasing time in culture, the reduction in NMDA EPSCs in neurons from NR2B(DeltaC/DeltaC) mice diminished. This is explained by the expression of additional NMDA receptor subtypes containing NR2A subunits at more mature synapses.

Synaptic activity-dependent developmental regulation of NMDA receptor subunit expression in cultured neocortical neurons.

The biophysical properties of NMDA receptors are thought to be critical determinants involved in the regulation of long-term synaptic plasticity during neocortical development. NMDA receptor channel properties are strongly dependent on the subunit composition of heteromeric NMDA receptors. During neocortical development in vivo, the expression of the NMDA receptor 2A (NR2A) subunit is up-regulated at the mRNA and protein level correlating with changes in the kinetic and pharmacological properties of functional NMDA receptors. To investigate the developmental regulation of NMDA receptor subunit expression, we studied NR2 mRNA expression in cultured neocortical neurons. With increasing time in culture, they showed a similar up-regulation of NR2A mRNA expression as described in vivo. As demonstrated by chronic blockade of postsynaptic glutamate receptors in vitro, the regulation of NR2A mRNA was strongly dependent on synaptic activity. In contrast, NR2B mRNA expression was not influenced by activity blockade. Moreover, as shown pharmacologically, the regulation of NR2A mRNA expression was mediated by postsynaptic Ca(2+) influx through both NMDA receptors and L-type Ca(2+) channels. It is interesting that even relatively weak expression of NR2A mRNA was correlated with clearly reduced sensitivity of NMDA receptor-mediated whole-cell currents against the NR2B subunit-specific antagonist ifenprodil. Developmental changes in the expression of NR1 mRNA splice variants were also strongly dependent on synaptic activity and thus might, in addition to regulation of NR2 subunit expression, contribute to developmental changes in the properties of functional NMDA receptors. In summary, our results demonstrate that synaptic activity is a key factor in the regulation of NMDA receptor subunit expression during neocortical development.

Synthesis and characterization of photolabile derivatives of serotonin for chemical kinetic investigations of the serotonin 5-HT(3) receptor.

A series of photolabile o-nitrobenzyl derivatives of serotonin (caged serotonin) were synthesized: the amine-linked serotonin derivatives N-(2-nitrobenzyl) serotonin (Bz-5HT) and N-(alpha-carboxy-2-nitrobenzyl) serotonin (N-CNB-5HT), and O-alpha-carboxy-2-nitrobenzyl) serotonin (O-CNB-5HT), which has the caging group attached to the phenolic OH group. All the derivatives released free serotonin when excited by 308-nm or 337-nm laser pulses. The time constant of serotonin release from N-CNB-5HT was 1. 2 ms, with a quantum yield of 0.08. This is too slow for rapid chemical kinetic measurements. O-CNB-5HT is suitable for transient kinetic investigations of the serotonin 5-HT(3) receptor. It released serotonin with a time constant of 16 micros and a quantum yield of 0.03. The biological properties of O-CNB-5HT were evaluated, and the applicability of the compound for kinetic studies of the 5-HT(3) receptor was demonstrated. O-CNB-5HT does not activate the 5-HT(3) receptor by itself, nor does it modulate the response of a cell when co-applied with serotonin. When irradiated with a 337-nm laser pulse, O-CNB-5HT released free serotonin that evoked 5-HT(3) receptor-mediated whole-cell currents in NIE-115 mouse neuroblastoma cells.

Pregnenolone sulfate antagonizes GABA A receptor-mediated currents via a reduction of channel opening frequency

Our previous study showed antagonism of GABA A receptor-mediated whole-cell currents by pregnenolone sulfate (PS). Here, the effects of PS, picrotoxin (PTX) and pentobarbital (PB) were tested on GABA-activated single Cl − channels recorded from membrane patches of rat cortical neurons in primary cultures. PS and PTX selectively decreased the opening frequency of the channels, while PB increased mean open time and burst duration without affecting opening frequency. It is suggested that PS and PTX may antagonize GABA A receptor function through the same mechanism and/or the same binding site.