Glycine-induced Currents Research Articles

Identification of a Subtype-Selective Allosteric Inhibitor of GluN1/GluN3 NMDA Receptors.

N-methyl-D-aspartate receptors (NMDARs) are Ca2+-permeable ionotropic glutamate receptors (iGluRs) in the central nervous system and play important roles in neuronal development and synaptic plasticity. Conventional NMDARs, which typically comprise GluN1 and GluN2 subunits, have different biophysical properties than GluN3-containing NMDARs: GluN3-containing NMDARs have smaller unitary conductance, less Ca2+-permeability and lower Mg2+-sensitivity than those of conventional NMDARs. However, there are very few specific modulators for GluN3-containing NMDARs. Here, we developed a cell-based high-throughput calcium assay and identified 3-fluoro-1,2-phenylene bis (3-hydroxybenzoate) (WZB117) as a relatively selective inhibitor of GluN1/GluN3 receptors. The IC50 value of WZB117 on GluN1/GluN3A receptors expressed in HEK-293 cells was 1.15 ± 0.34 μM. Consistently, WZB117 exhibited strong inhibitory activity against glycine-induced currents in the presence of CGP-78608 but only slightly affected the NMDA-, KA- and AMPA-induced currents in the acutely isolated rat hippocampal neurons. Among the four types of endogenous currents, only the first one is primarily mediated by GluN1/GluN3 receptors. Mechanistic studies showed that WZB117 inhibited the GluN1/GluN3A receptors in a glycine-, voltage- and pH-independent manner, suggesting it is an allosteric modulator. Site-directed mutagenesis and chimera construction further revealed that WZB117 may act on the GluN3A pre-M1 region with key determinants different from those of previously identified modulators. Together, our study developed an efficient method to discover modulators of GluN3-containing NMDARs and characterized WZB117 as a novel allosteric inhibitor of GluN1/GluN3 receptors.

Activation of M3-AChR and IP3/Ca2+/PKC signaling pathways by pilocarpine increases glycine-induced currents in ventral horn neurons of the spinal cord

Our study aimed to determine the effects of pilocarpine and the mechanisms involving muscarinic acetylcholine receptors (mAChRs) on glycine receptors (GlyRs) in neurons of the spinal cord ventral horn. An enzymatic digestion combined with acute mechanical separation was applied to isolate neurons from the spinal cord ventral horn. Patch-clamp recording was then used to investigate the outcomes of pilocarpine. Our results indicate that pilocarpine increased the glycine currents in a concentration-dependent manner, which was blocked by the M3-AChR selective antagonists 4-DAMP and J104129. Pilocarpine also enhanced the glycine currents in nominally Ca2+-free extracellular solution. Conversely, the enhancement of glycine currents by pilocarpine disappeared when intracellular Ca2+ was chelated by BAPTA. Heparin and Xe-C, which are IP3 receptor antagonists, also totally abolished the pilocarpine effect. Furthermore, Bis-IV, a PKC inhibitor, eliminated the pilocarpine effect. Additionally, PMA, a PKC activator, mimicked the pilocarpine effect. These results indicate that pilocarpine may increase the glycine currents by activating the M3-AChRs and IP3/Ca2+/PKC pathways.

Modulation of recombinant human alpha 1 glycine receptor by flavonoids and gingerols.

The inhibitory glycine receptor (GlyR) is a principal mediator of fast synaptic inhibition in mammalian spinal cord, brainstem, and higher brain centres. Flavonoids are secondary plant metabolites that exhibit many beneficial physiological effects, including modulatory action on neuronal receptors. Using whole-cell current recordings from recombinant human α1 GlyRs, expressed in HEK293cells, we compared the flavonols kaempferol and quercetin, the flavanone naringenin, the flavones apigenin and nobiletin, the isoflavone genistein, and two gingerols, 6-gingerol and 8-gingerol for their modulation of receptor currents. All compounds were inhibitors of the GlyR with IC50 values ranging between 9.3±2.6µM (kaempferol) and 46.7±6.5µM (genistein), following a mixed mode of inhibition. Co-application of two inhibitors revealed distinct binding sites for flavonoids and gingerols. Pore-lining mutants T258A and T258S were strongly inhibited by quercetin and naringenin, but not by 6-gingerol, confirming the existence of distinct binding sites for flavonoids and gingerols. Apigenin, kaempferol, nobiletin, naringenin and 6-gingerol showed biphasic action, potentiating glycine-induced currents at low concentration of both, modulator and glycine, and inhibiting at higher concentrations. Identification of distinct modulatory sites for flavonoids and related compounds may present pharmacological target sites and aid the discovery of novel glycinergic drugs.

Voltage-Dependent Inhibition of Glycine Receptor Channels by Niflumic Acid

Niflumic acid (NFA) is a member of the fenamate class of nonsteroidal anti-inflammatory drugs. This compound and its derivatives are used worldwide clinically for the relief of chronic and acute pain. NFA is also a commonly used blocker of voltage-gated chloride channels. Here we present evidence that NFA is an efficient blocker of chloride-permeable glycine receptors (GlyRs) with subunit heterogeneity of action. Using the whole-cell configuration of patch-clamp recordings and molecular modeling, we analyzed the action of NFA on homomeric α1ΔIns, α2B, α3L, and heteromeric α1β and α2β GlyRs expressed in CHO cells. NFA inhibited glycine-induced currents in a voltage-dependent manner and its blocking potency in α2 and α3 GlyRs was higher than that in α1 GlyR. The Woodhull analysis suggests that NFA blocks α1 and α2 GlyRs at the fractional electrical distances of 0.16 and 0.65 from the external membrane surface, respectively. Thus, NFA binding site in α1 GlyR is closer to the external part of the membrane, while in α2 GlyR it is significantly deeper in the pore. Mutation G254A at the cytoplasmic part of the α1 GlyR pore-lining TM2 helix (level 2′) increased the NFA blocking potency, while incorporation of the β subunit did not have a significant effect. The Hill plot analysis suggests that α1 and α2 GlyRs are preferably blocked by two and one NFA molecules, respectively. Molecular modeling using Monte Carlo energy minimizations provides the structural rationale for the experimental data and proposes more than one interaction site along the pore where NFA can suppress the ion permeation.

A Glycine-Insulin Autocrine Feedback Loop Enhances Insulin Secretion From Human β-Cells and Is Impaired in Type 2 Diabetes.

The secretion of insulin from pancreatic islet β-cells is critical for glucose homeostasis. Disrupted insulin secretion underlies almost all forms of diabetes, including the most common form, type 2 diabetes (T2D). The control of insulin secretion is complex and affected by circulating nutrients, neuronal inputs, and local signaling. In the current study, we examined the contribution of glycine, an amino acid and neurotransmitter that activates ligand-gated Cl(-) currents, to insulin secretion from islets of human donors with and without T2D. We find that human islet β-cells express glycine receptors (GlyR), notably the GlyRα1 subunit, and the glycine transporter (GlyT) isoforms GlyT1 and GlyT2. β-Cells exhibit significant glycine-induced Cl(-) currents that promote membrane depolarization, Ca(2+) entry, and insulin secretion from β-cells from donors without T2D. However, GlyRα1 expression and glycine-induced currents are reduced in β-cells from donors with T2D. Glycine is actively cleared by the GlyT expressed within β-cells, which store and release glycine that acts in an autocrine manner. Finally, a significant positive relationship exists between insulin and GlyR, because insulin enhances the glycine-activated current in a phosphoinositide 3-kinase-dependent manner, a positive feedback loop that we find is completely lost in β-cells from donors with T2D.

The N-terminal domain of the GluN3A subunit determines the efficacy of glycine-activated NMDA receptors

N-methyl-d-aspartate (NMDA) receptors composed of glycine-binding GluN1 and GluN3 subunits function as excitatory glycine receptors that respond to agonist application only with a very low efficacy. Binding of glycine to the high-affinity GluN3 subunits triggers channel opening, whereas glycine binding to the low-affinity GluN1 subunits causes an auto-inhibition of the maximal glycine-inducible receptor current (Imax). Hence, competitive antagonists of the GluN1 subunit strongly potentiate glycine responses of wild type (wt) GluN1/GluN3 receptors. Here, we show that co-expression of N-terminal domain (NTD) deleted GluN1 (GluN1ΔNTD) and GluN3 (GluN3ΔNTD) subunits in Xenopus oocytes generates GluN1/GluN3 receptors with a large increase in the glycine-inducible Imax accompanied by a strongly impaired GluN1 antagonist-mediated potentiation. Affinity purification after metabolic or surface labeling revealed no differences in subunit stoichiometry and surface expression between wt GluN1/GluN3A and mutant GluN1ΔNTD/GluN3AΔNTD receptors, indicating a specific effect of NTD deletions on the efficacy of receptor opening. Notably, GluN1/GluN3AΔNTD receptors showed a similar increase in Imax and a greatly reduced GluN1 antagonist-mediated current potentiation as GluN1ΔNTD/GluN3AΔNTD receptors, whereas the glycine-induced currents of GluN1ΔNTD/GluN3A receptors resembled those of wt GluN1/GluN3A receptors. Furthermore, oxidative crosslinking of the homophilic GluN3A NTD intersubunit interface in mutant GluN1/GluN3AR319C receptors caused both a decrease in the glycine-induced Imax concomitantly with a marked increase in GluN1 antagonist-mediated current potentiation, whilst mutations within the intrasubunit region linking the GluN3A NTD to the ligand binding domain had opposite effects. Together these results show that the GluN3A NTD constitutes a crucial regulatory determinant of GluN1/GluN3A receptor function.

Ginkgolic acid specifically potentiates alpha 1 glycine receptors.

Ginkgo biloba extract is a neuroactive agent that is widely used for correction of age-associated impairment of memory, attention deficit and other cognitive functions. It has been shown that ginkgolides and bilobalides, Ginkgo biloba extract components, are potent blockers of glycine receptors (Kondratskaya et al., 2002, Hawthorne et al., 2006). However, the effect of ginkgolic acid, the other Ginkgo biloba extract constituent, on ligand-gated ion channels was not investigated. In the present study, using patch-clamp technique and transient transfection of different subunits in CHO cells we have shown that glycine receptors (GlyRs) are modulated by ginkgolic acid in a subunit-specific manner. After pre-application of ginkgolic acid (0.5-2 min), glycine-induced currents mediated by α1 GlyRs were strongly potentiated, while currents mediated by α2 GlyRs exhibited weak inhibition. There was no significant effect of ginkgolic acid on amplitudes of currents mediated by α3 GlyRs or on GABAARs composed of 1α/1β/2γ subunits. In order to further investigate subunit-specific effect of ginkgolic acid we have focused on possible interaction sites for this compound inside different GlyR domains. We found that mutation of three residues (T59A/A261G/A303S) in α2 subunit can convert the inhibitory action of ginkgolic acid into potentiation. Indeed, application of ginkgolic acid to cells expressing α2 T59A/A261G/A303S subunits resulted in an increase of responses to low concentrations of glycine and abolishment of the inhibitory effect, typical for wild type α2 GlyR. Our results suggest that (i) ginkgolic acid selectively enhances the function of α1 GlyRs and attenuates the function of α2 GlyRs, (ii) mutation of α2 subunit converts effect of ginkgolic acid from inhibition to potentiation.

Estradiol inhibits the activity of proton-coupled amino acid transporter PAT1 expressed in Xenopus oocytes

Estrogen has great potential as a therapeutic agent in focal ischemic brain injury. Amino acids as energy resources and neurotransmitters in the central nervous system are crucial for proper neuronal function and excitability. The proton-coupled amino acid transporter PAT1 has clear potential in drug absorption. In this paper, human brain PAT1 was cloned and expressed in Xenopus oocytes. The effects of estradiol on the activity of PAT1 were investigated. Glycine-induced membrane currents mediated by PAT1 were measured using the two-electrode voltage clamp technique. The amplitude of the glycine-elicited current was decreased progressively with increasing concentrations of β-estradiol. A concentration-dependent outwards current of PAT1 was also detected by the presence of β-estradiol. We conclude that estrogen attenuates the activity of PAT1 by directly closing PAT1 channel. Our results may provide an additional mechanism for estrogen on neurotransmission and neuronal metabolism during ischemic injury.

Lidocaine Metabolites Inhibit Glycine Transporter 1

Lidocaine exerts antinociceptive effects when applied systemically. The mechanisms are not fully understood but glycinergic mechanisms might be involved. The synaptic glycine concentration is controlled by glycine transporters. Whereas neurons express two types of glycine transporters, astrocytes specifically express glycine transporter 1 (GlyT1). This study focuses on effects of lidocaine and its major metabolites on GlyT1 function. The effects of lidocaine and its metabolites monoethylglycinexylidide (MEGX), glycinexylidide, and N-ethylglycine on GlyT1 function were investigated in uptake experiments with [¹⁴C]-labeled glycine in primary rat astrocytes. Furthermore, the effect of lidocaine and its metabolites on glycine-induced currents were investigated in GlyT1-expressing Xenopus laevis oocytes. Lidocaine reduced glycine uptake only at toxic concentrations. The metabolites MEGX, glycinexylidide, and N-ethylglycine, however, significantly reduced glycine uptake (P < 0.05). Inhibition of glycine uptake by a combination of lidocaine with its metabolites at a clinically relevant concentration was diminished with increasing extracellular glycine concentrations. Detailed analysis revealed that MEGX inhibits GlyT1 function (P < 0.05), whereas N-ethylglycine was identified as an alternative GlyT1 substrate (EC₅₀ = 55 μM). Although lidocaine does not function directly on GlyT1, its metabolites MEGX and N-ethylglycine [corrected] were shown to inhibit GlyT1-mediated glycine uptake by at least two different mechanisms. Whereas N-ethylglycine [corrected] was demonstrated to be an alternative GlyT1 substrate, MEGX was shown to inhibit GlyT1 activity in both primary astrocytes and in GlyT1-expressing Xenopuslaevis oocytes at clinically relevant concentrations. These findings provide new insights into the possible mechanisms for the antinociceptive effect of systemic lidocaine.

Glycine receptor internalization by protein kinases activation

Although glycine-induced currents in the central nervous system have been proven to be modulated by protein kinases A (PKA) and C (PKC), the mechanism is not well understood. In order to better comprehend the mechanism involved in this phenomenon, we tested the PKA and PKC activation effect on the specific [(3) H]glycine and [(3) H]strychnine binding to postsynaptic glycine receptor (GlyR) in intact rat retina. The specific binding constituted about 20% of the total radioligand binding. Kinetic analysis of the specific binding exhibited a sigmoidal behavior with three glycine and two strychnine binding sites and affinities of 212 nM for [(3) H]glycine and 50 nM for [(3) H]strychnine. Specific radioligand binding was decreased (60-85%) by PKA and PKC activation, an effect that was blocked by specific kinases inhibitors, as well as by cytochalasin D. GlyR expressed in the plasma membrane decreased about 50% in response to kinases activation, which was consistent with an increase of the receptor in the microsomal fraction when PKA was activated. Moreover, immunoprecipitation studies indicated that these kinases lead to a time-dependent receptor phosphorylation. Our results suggest that in retina, GlyR is cross-regulated by G protein-coupled receptors, activating PKA and PKC.

A Salt Bridge Crucial for Glycine Receptor Function

The glycine receptor (GlyR) chloride channel is a member of the pentameric ligand-gated ion channel (pLGIC) receptor family. These channels mediate fast inhibitory transmission in the nervous system. Recent structural data and a wide body of functional data have underlined the importance of the interface between the ligand-binding domain and the transmembrane domain in channel gating. A number of charged residues at this interface have been shown to form salt bridges critical for channel function in other pLGICs. However, no such interaction has been demonstrated in the GlyR. Mutations at D148 (Cys-loop) and R218 (pre-M1) in the homomeric alpha 1 GlyR can have drastic effects on GlyR function and the single mutants D148C and R218C failed to produce glycine-induced currents in Xenopus oocytes. However, the D148C, R218C double mutant resulted in receptors that produced glycine-gated currents with only a small decrease in glycine sensitivity compared to WT. Importantly, sub-saturating glycine-evoked currents were dramatically increased by the reducing agent dithiothreitol. This suggests that D148C and R218C can form a disulphide bridge, possibly indicating an electrostatic interaction between D148 and R218 that stabilizes the channel closed state. The possibilty of a state-dependent salt bridge between D148 and R218 raised the question how the charges are stabilised in the open state. Interestingly, structural studies have suggested that R218 is flanked by two highly conserved phenylalanine side chains. To test, if these aromatic amino acids interact with R218 electrostically via a cation-pi interaction, we incorporated a series of fluorinated Phe derivatives using the in vivo non-sense suppression method. However, the fluorination-induced changes in the glycine sensitivity were not consistent with a cation-pi interaction. Taken together, these results point towards a crucial salt bridge between D148 and R218 but no electrostatic contributions by the flanking phenylalanines.

Direct and indirect control of orexin/hypocretin neurons by glycine receptors.

Hypothalamic hypocretin/orexin (hcrt/orx) neurons promote arousal and reward seeking, while reduction in their activity has been linked to narcolepsy, obesity and depression. However, the mechanisms influencing the activity of hcrt/orx networks in situ are not fully understood. Here we show that glycine, a neurotransmitter best known for its actions in the brainstem and spinal cord, elicits dose dependent postsynaptic Cl⁻ currents in hcrt/orx cells in acute mouse brain slices. The effect was blocked by the glycine receptor (GLyR) antagonist strychnine and mimicked by the GlyR agonist alanine. Postsynaptic GlyRs on hcrt/orx cells remained functional during both early postnatal and adult periods, and gramicidin-perforated patch-clamp recordings revealed that they progressively switch from excitatory to inhibitory during the first two postnatal weeks. The pharmacological profile of the glycine response suggested that developed hcrt/orx neurons contain α/β-heteromeric GlyRs that lack α2-subunits, whereas α2-subunits, whereas α2-subunits are present in early postnatal hcrt/orx neurons. All postsynaptic currents (PSCs) in developed hcrt/orx cells were blocked by inhibitors of GABA and glutamate receptors, with no evidence of GlyR-mediated PSCs. However, the frequency but not amplitude of miniature PSCs was reduced by strychnine and increased by glycine in ~50% of hcrt/orx neurons. Together, these results provide the first evidence for functional GlyRs in identified hcrt/orx circuits and suggest that the activity of developed hcrt/orx cells is regulated by two GlyR pools: inhibitory extrasynaptic GlyRs located on all hcrt/orx cells and excitatory GlyRs located on presynaptic terminals contacting some hcrt/orx cells.

Glycine-induced currents are insensitive to the glycine receptor α 1 subunit-specific blocker, cyanotriphenylborate, in older circling mice

The pharmacologic characteristics of glycine receptors (GlyRs) in the lateral superior olive (LSO) of circling mice, animal model for inherited deafness, were investigated using a GlyR α 1 subunit-specific receptor blocker (cyanotriphenylborate [CTB]). There was a statistically significant age-dependent increase in the antagonistic effect of CTB in heterozygous (+/ cir) mice. In postnatal (P)0–P3 heterozygous (+/ cir) mice, glycine currents evoked by glycine puffs were reduced to 20.4 ± 2.6, 37.1 ± 3.1, and 63.9 ± 2.5% at 0.1, 1, and 10 μM CTB ( n = 13) compared to controls, while the glycine currents were reduced to 22.3 ± 3.5, 52.9 ± 4.1, and 78.3 ± 3.5% at 0.1, 1, and 10 μM CTB ( n = 7) in P8–P12 heterozygous (+/ cir) mice. In contrast, the antagonistic effect of CTB was not strong and even less than that of younger animals in older homozygous ( cir/ cir) mice. In P0–P3 homozygous ( cir/ cir) mice, the extent of inhibition was 20.2 ± 3.7, 37.8 ± 4.3, and 66.8 ± 4.2% at 0.1, 1, and 10 μM CTB ( n = 6) compared to controls, while the extent of inhibition was 18.7 ± 2.4, 28.1 ± 3.9, and 39.1 ± 8.2% ( n = 6) in P8–P12 homozygous ( cir/ cir) mice. The age-dependent decrease in the antagonistic effect of CTB indicates the abnormal development of the α 1 subunit-containing GlyRs in homozygous ( cir/ cir) mice.

Decreased Immunoreactivities and Functions of the Chloride Transporters, KCC2 and NKCC1, in the Lateral Superior Olive Neurons of Circling Mice

ObjectivesWe tested the possibility of differential expression and function of the potassium-chloride (KCC2) and sodium-potassium-2 chloride (NKCC1) co-transporters in the lateral superior olive (LSO) of heterozygous (+/cir) or homozygous (cir/cir) mice.MethodsMice pups aged from postnatal (P) day 9 to 16 were used. Tails from mice were cut for DNA typing. For Immunohistochemical analysis, rabbit polyclonal anti-KCC2 or rabbit polyclonal anti-NKCC1 was used and the density of immunolabelings was evaluated using the NIH image program. For functional analysis, whole cell voltage clamp technique was used in brain stem slices and the changes of reversal potentials were evaluated at various membrane potentials.ResultsImmunohistochemical analysis revealed both KCC2 and NKCC1 immunoreactivities were more prominent in heterozygous (+/cir) than homozygous (cir/cir) mice on P day 16. In P9-P12 heterozygous (+/cir) mice, the reversal potential (Egly) of glycine-induced currents was shifted to a more negative potential by 50 µM bumetanide, a known NKCC1 blocker, and the negatively shifted Egly was restored by additional application of 1 mM furosemide, a KCC2 blocker (-58.9±2.6 mV to -66.0±1.5 mV [bumetanide], -66.0±1.5 mV to -59.8±2.8 mV [furosemide+bumetanide], n=11). However, only bumetanide was weakly, but significantly effective (-60.1±2.9 mV to -62.7±2.6 mV [bumetanide], -62.7±2.6 mV to -62.1±2.5 mV [furosemide+bumetanide], n=7) in P9-P12 homozygous (cir/cir) mice.ConclusionThe less prominent immunoreactivities and weak or absent responses to bumetanide or furosemide suggest impaired function or delayed development of both transporters in homozygous (cir/cir) mice.

Frequency-Dependent Cannabinoid Receptor-Independent Modulation of Glycine Receptors by Endocannabinoid 2-AG

Endocannabinoids are known as retrograde messengers, being released from the postsynaptic neuron and acting on specific presynaptic G-protein-coupled cannabinoid (CB) receptors to decrease neurotransmitter release. Also, at physiologically relevant concentrations cannabinoids can directly modulate the function of voltage-gated and receptor-operated ion channels. Using patch-clamp recording we analyzed the consequences of the direct action of an endocannabinoid, 2-arachidonoylglycerol (2-AG), on the functional properties of glycine receptor channels (GlyRs) and ionic currents in glycinergic synapses. At physiologically relevant concentrations (0.1–1 μM), 2-AG directly affected the functions of recombinant homomeric α1H GlyR: it inhibited peak amplitude and dramatically enhanced desensitization. The action of 2-AG on GlyR-mediated currents developed rapidly, within ∼300 ms. Addition of 1 μM 2-AG strongly facilitated the depression of glycine-induced currents during repetitive (4–10 Hz) application of short (2 ms duration) pulses of glycine to outside-out patches. In brainstem slices from CB1 receptor knockout mice, 2-AG significantly decreased the extent of facilitation of synaptic currents in hypoglossal motoneurons during repetitive (10–20 Hz) stimulation. These observations suggest that endocannabinoids can modulate postsynaptic metaplasticity of glycinergic synaptic currents in a CB1 receptor-independent manner.

Brain derived neurotrophic factor and neurotrophic factor 3 modulate neurotransmitter receptor expressions on developing spiral ganglion neurons

Cochlear spiral ganglion neurons (SGN) provide the only pathway for transmitting sound evoked activity from the hair cells to the central auditory system. Neurotrophic factor 3 (NT-3) and brain derived neurotrophic factor (BDNF) released from hair cells and supporting cells exert a profound effect on SGN survival and neural firing patterns; however, it is unclear what the effects NT-3 and BDNF have on the type of neurotransmitter receptors expressed on SGN. To address this question, the whole-cell patch clamp recording technique was used to determine what effect NT-3 and BDNF had on the function and expression of glutamate, GABA and glycine receptors (GlyR) on SGN of cochlea from postnatal C57 mouse. Receptor currents induced by the agonist of each receptor were recorded from SGN cultured with or without BDNF or NT-3. NT-3 and BDNF exerted different effects. NT-3, and to a lesser extent BDNF, enhanced the expression of GABA receptors and had comparatively little effect on glutamate receptors. Absence of BDNF and NT-3 resulted in the emergence of glycine-induced currents; however, GlyR currents were absent from the short term cultured SGN. In contrast, NT-3 and BDNF suppressed GlyR expression on SGN. These results indicate that NT-3 and BDNF exert a profound effect on the types of neurotransmitter receptors expressed on postnatal SGN, results that may have important implications for neural development and plasticity.

Nitrous oxide and the inhibitory synaptic transmission in rat dorsal horn neurons

The analgesic effect of nitrous oxide (N 2O) is thought to depend on noradrenaline release in the spinal cord following activation of descending inhibitory neurons. In addition to this indirect facilitation of inhibition in the spinal cord, we previously showed direct inhibition of glutamate receptors in dorsal horn neurons by N 2O. Since general anesthetics could possibly affect excitatory and/or inhibitory components of synaptic transmission, we sought to evaluate the direct effect of N 2O on inhibitory transmission in spinal cord neurons. Using whole-cell patch-clamp recording from rat transversal spinal cord slices, we investigated the actions of 50% N 2O and 0.5% isoflurane (both 0.3 rat MAC; minimum alveolar concentration) on exogenously applied γ-aminobutyric acid (GABA)- and glycine-induced currents in rat dorsal horn lamina II neurons. The amplitudes and integrated areas of GABA- and glycine-induced currents were not significantly affected by N 2O, but were increased in the presence of isoflurane. N 2O did not affect the amplitude, frequency or decay time probability distribution of either GABA or glycine receptor-mediated miniature postsynaptic currents. We further sought to determine the effect of N 2O on focal stimulation-evoked synaptic currents mediated by GABA and glycine receptors, and found no effect in the majority of neurons. These and other findings suggest that N 2O has a discrete action in the spinal cord, distinct from the effects of the volatile anesthetics, consisting of inhibition of excitation in SG neurons through an action on ionotropic glutamatergic receptors and potentiation of inhibition through the descending noradrenergic system.

Functional expression of the glycine transporter 1 on bullfrog retinal cones

Using patch clamp techniques, we characterized glycine-induced currents from cones in bullfrog retinal slices. Application of glycine to cone terminals induced an inward current, which was in part suppressed by strychnine. The remaining strychnine-resistant current component, which did not show polarity reversion in a range of -120 mV to +40 mV, was blocked by N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl] sarcosine, an antagonist of glycine transporter 1 (GlyT1), but not affected by amoxapine, an inhibitor of glycine transporter 2. Application of sarcosine, an agonist of GlyT1, to cone terminals induced an inward current that was completely suppressed by N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl] sarcosine or when external Na in Ringer's was replaced by choline. All these results show for the first time the functional expression of GlyT1 on bullfrog cones.

Fluoxetine inhibition of glycine receptor activity in rat hippocampal neurons

Fluoxetine is a selective serotonin reuptake inhibitor widely used for treating depression. However, fluoxetine treatment may lead to seizures at higher doses, which underlying mechanism remains largely unknown. In this study, we examined the effects of fluoxetine on glycine receptor (GlyR) activity. Using the whole-cell patch-clamp recording method, we found that fluoxetine and its metabolite norfluoxetine inhibited glycine-induced currents in cultured rat hippocampal neurons. This inhibition was dose-dependent, and voltage-independent. Fluoxetine shifted the glycine concentration–response curve to the right without altering the maximal current. Both Lineweaver–Burk and Schild plots suggest competitive inhibition. The amount of fluoxetine inhibition significantly increased when homomeric GlyRs were selectively inhibited with picrotoxin. Moreover, fluoxetine inhibited the current mediated by heteromeric α2β- but not homomeric α2-GlyRs transiently expressed in HEK293T cells. These results suggest that fluoxetine is a competitive and subtype-selective GlyR inhibitor, which may explain its capacity to induce seizures.

Cytokine Mechanisms of Central Sensitization: Distinct and Overlapping Role of Interleukin-1β, Interleukin-6, and Tumor Necrosis Factor-α in Regulating Synaptic and Neuronal Activity in the Superficial Spinal Cord

Central sensitization, increased sensitivity in spinal cord dorsal horn neurons after injuries, plays an essential role in the induction and maintenance of chronic pain. However, synaptic mechanisms underlying central sensitization are incompletely known. Growing evidence suggests that proinflammatory cytokines (PICs), such as interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNFalpha), are induced in the spinal cord under various injury conditions and contribute to pain hypersensitivity. Using patch-clamp recordings in lamina II neurons of isolated spinal cord slices, we compared the effects of IL-1beta, IL-6, and TNFalpha on excitatory and inhibitory synaptic transmission. Whereas TNFalpha enhanced the frequency of spontaneous EPSCs (sEPSCs), IL-6 reduced the frequency of spontaneous IPSCs (sIPSCs). Notably, IL-1beta both enhanced the frequency and amplitude of sEPSCs and reduced the frequency and amplitude of sIPSCs. Consistently, TNFalpha and IL-1beta enhanced AMPA- or NMDA-induced currents, and IL-1beta and IL-6 suppressed GABA- and glycine-induced currents. Furthermore, all the PICs increased cAMP response element-binding protein (CREB) phosphorylation in superficial dorsal horn neurons and produced heat hyperalgesia after spinal injection. Surprisingly, soluble IL-6 receptor (sIL-6R) produced initial decrease of sEPSCs, followed by increase of sEPSCs and CREB phosphorylation. Spinal injection of sIL-6R also induced heat hyperalgesia that was potentiated by coadministration with IL-6. Together, our data have demonstrated that PICs induce central sensitization and hyperalgesia via distinct and overlapping synaptic mechanisms in superficial dorsal horn neurons either by increasing excitatory synaptic transmission or by decreasing inhibitory synaptic transmission. PICs may further induce long-term synaptic plasticity through CREB-mediated gene transcription. Blockade of PIC signaling could be an effective way to suppress central sensitization and alleviate chronic pain.