Strychnine-sensitive Glycine Receptors Research Articles

Functional modulation of strychnine-sensitive glycine receptors in rat hippocampal pyramidal neurons by amyloid-β protein (1-42)

Amyloid-β peptide (Aβ) is considered a key protein in the pathogenesis of Alzheimer’s disease because of its neurotoxicity, resulting in impaired synaptic function and memory. On the other hand, it was demonstrated that low (picomolar) concentrations of Aβ enhance synaptic plasticity and memory, suggesting that in the healthy brain, physiological Aβ concentrations are necessary for normal cognitive functions. In the present study, we found that Aβ (1−42) in concentrations of 10 pМ – 100nМ enhanced desensitization of the glycine-activated current in isolated CA3 pyramidal neurons and also reversibly suppressed its peak amplitude during short (600ms) co-application with agonist. The effect was most prominent at low glycine concentrations. When glycine receptors were activated by other receptor agonists – taurine and β-alanine, the changes of current kinetics and amplitudes induced by Aβ had a similar character. When Aβ (100 pM) was added to the bath solution, it caused, besides acceleration of desensitization, more pronounced reduction of peak current amplitude. This effect developed slowly, during a few minutes, and was more prominent at saturating concentrations of agonists. The results suggest that Aβ interacts with glycine receptors through three different mechanisms - by enhancing receptor desensitization, by rapid inhibition of the receptor, and also by means of a slowly developing inhibition of the amplitude of the current, possibly through intracellular mechanisms. The observed changes in the activity of glycine receptors induced by Aβ can lead to suppression of the tonic inhibition of hippocampal neurons mediated by extrasynaptic glycine receptors.

Anti-anxiety effect of methanol extract of <i>Pericarpium zanthoxyli</i> using a strychnine-sensitive glycine receptor model

Purpose: To determine if the methanol extract of Pericarpium zanthoxyli exerts anti-anxiety effects and also to explore any probable anti-anxiety mechanism in vivo . Methods: The staircase test, elevated plus maze test, rota-rod treadmill test and convulsions induced by strychnine and picrotoxin on mice were tested to identify potential mechanism of anti-anxiety activity of the plant extract. Results: The plant extract (10 mg/kg, p.o.) significantly reduced rearing numbers in the staircase test while it increased the time spent in the open arms as well as the number of entries to the open arms in the elevated plus maze test, suggesting that it has significant anti-anxiety activity. Furthermore, the extract inhibited strychnine-induced convulsion. However, it had little effect on picrotoxin-induced convulsion, suggesting that its anti-anxiety activity may be linked to strychnine-sensitive glycine receptor and not GABA receptor. Conclusion: These results suggest that the Pericarpium zanthoxyli extract may be beneficial for the control of anxiety. Keywords: Anti-anxiety, Pericarpium zanthoxyli , Glycine Receptor, GABA Receptor

Insular balance of glutamatergic and GABAergic signaling modulates pain processing.

Neuroimaging studies of patients with chronic pain have shown that neurotransmitter abnormalities, including increases in glutamate and decreases in GABA, could be responsible for the cortical hyperactivity and hyperalgesia/allodynia observed in some pain conditions. These finding are particularly evident in the insula, a brain region known to play a role in both the sensory-discriminative and the affective-motivational aspects of pain processing. However, clinical studies are not entirely able to determine the directionality of these findings, nor whether they are causal or epiphenomenon. Thus, a set of animal studies was performed to determine whether alterations in glutamate and GABA are the result of injury, the cause of augmented pain processing, or both. Compared with controls, the excitatory neurotransmitters glutamate and aspartate are significantly higher in the rat insula after chronic constriction injury of the sciatic nerve (CCI). The CCI also produced significant increases in allodynia (mechanical and cold), thermal hyperalgesia, and nociceptive aversiveness. Unilateral microinjection of ionotropic glutamate receptor antagonists restored these nociceptive behaviors to preinjury values. Increasing endogenous levels of GABA or enhancing signaling at inhibitory glycinergic receptors had similar effects as the glutamate receptor antagonists. In naive rats, increasing endogenous levels of glutamate, decreasing endogenous levels of GABA, or blocking strychnine-sensitive glycine receptors in the insula significantly increased thermal hyperalgesia and mechanical allodynia. These data support the hypothesis that an altered balance of excitatory and inhibitory neurotransmitters in brain regions such as the insula occurs in chronic pain states and leads to augmented central pain processing and increased pain sensitivity.

D-Serine and Glycine Differentially Control Neurotransmission during Visual Cortex Critical Period.

N-methyl-D-aspartate receptors (NMDARs) play a central role in synaptic plasticity. Their activation requires the binding of both glutamate and d-serine or glycine as co-agonist. The prevalence of either co-agonist on NMDA-receptor function differs between brain regions and remains undetermined in the visual cortex (VC) at the critical period of postnatal development. Here, we therefore investigated the regulatory role that d-serine and/or glycine may exert on NMDARs function and on synaptic plasticity in the rat VC layer 5 pyramidal neurons of young rats. Using selective enzymatic depletion of d-serine or glycine, we demonstrate that d-serine and not glycine is the endogenous co-agonist of synaptic NMDARs required for the induction and expression of Long Term Potentiation (LTP) at both excitatory and inhibitory synapses. Glycine on the other hand is not involved in synaptic efficacy per se but regulates excitatory and inhibitory neurotransmission by activating strychnine-sensitive glycine receptors, then producing a shunting inhibition that controls neuronal gain and results in a depression of synaptic inputs at the somatic level after dendritic integration. In conclusion, we describe for the first time that in the VC both D-serine and glycine differentially regulate somatic depolarization through the activation of distinct synaptic and extrasynaptic receptors.

Activation of Strychnine-Sensitive Glycine Receptors by Shilajit on Preoptic Hypothalamic Neurons of Juvenile Mice.

Shilajit, a mineral pitch, has been used in Ayurveda and Siddha system of medicine to treat many human ailments, and is reported to contain at least 85 minerals in ionic form. This study examined the possible mechanism of Shilajit action on preoptic hypothalamic neurons using juvenile mice. The hypothalamic neurons are the key regulator of many hormonal systems. In voltage clamp mode at a holding potential of -60 mV, and under a high chloride pipette solution, Shilajit induced dose-dependent inward current. Shilajit-induced inward currents were reproducible and persisted in the presence of 0.5 μM tetrodotoxin (TTX) suggesting a postsynaptic action of Shilajit on hypothalamic neurons. The currents induced by Shilajit were almost completely blocked by 2 μM strychnine (Stry), a glycine receptor antagonist. In addition, Shilajit-induced inward currents were partially blocked by bicuculline. Under a gramicidin-perforated patch clamp mode, Shilajit induced membrane depolarization on juvenile neurons. These results show that Shilajit affects hypothalamic neuronal activities by activating the Stry-sensitive glycine receptor with α₂/α₂β subunit. Taken together, these results suggest that Shilajit contains some ingredients with possible glycine mimetic activities and might influence hypothalamic neurophysiology through activation of Stry-sensitive glycine receptor-mediated responses on hypothalamic neurons postsynaptically.

Glycine receptor mechanism elucidated by electron cryo-microscopy.

The strychnine-sensitive glycine receptor (GlyR) mediates inhibitory synaptic transmission in the spinal cord and brainstem and is linked to neurological disorders, including autism and hyperekplexia. Understanding of molecular mechanisms and pharmacology of glycine receptors has been hindered by a lack of high-resolution structures. Here we report electron cryo-microscopy structures of the zebrafish α1 GlyR with strychnine, glycine, or glycine and ivermectin (glycine/ivermectin). Strychnine arrests the receptor in an antagonist-bound closed ion channel state, glycine stabilizes the receptor in an agonist-bound open channel state, and the glycine/ivermectin complex adopts a potentially desensitized or partially open state. Relative to the glycine-bound state, strychnine expands the agonist-binding pocket via outward movement of the C loop, promotes rearrangement of the extracellular and transmembrane domain 'wrist' interface, and leads to rotation of the transmembrane domain towards the pore axis, occluding the ion conduction pathway. These structures illuminate the GlyR mechanism and define a rubric to interpret structures of Cys-loop receptors.

Glycine transporter 1 is a target for the treatment of epilepsy

Glycine is the major inhibitory neurotransmitter in brainstem and spinal cord, whereas in hippocampus glycine exerts dual modulatory roles on strychnine-sensitive glycine receptors and on the strychnine-insensitive glycineB site of the N-methyl-d-aspartate receptor (NMDAR). In hippocampus, the synaptic availability of glycine is largely under control of glycine transporter 1 (GlyT1). Since epilepsy is a disorder of disrupted network homeostasis affecting the equilibrium of various neurotransmitters and neuromodulators, we hypothesized that changes in hippocampal GlyT1 expression and resulting disruption of glycine homeostasis might be implicated in the pathophysiology of epilepsy. Using two different rodent models of temporal lobe epilepsy (TLE) – the intrahippocampal kainic acid model of TLE in mice, and the rat model of tetanic stimulation-induced TLE – we first demonstrated robust overexpression of GlyT1 in the hippocampal formation, suggesting dysfunctional glycine signaling in epilepsy. Overexpression of GlyT1 in the hippocampal formation was corroborated in human TLE samples by quantitative real time PCR. In support of a role of dysfunctional glycine signaling in the pathophysiology of epilepsy, both the genetic deletion of GlyT1 in hippocampus and the GlyT1 inhibitor LY2365109 increased seizure thresholds in mice. Importantly, chronic seizures in the mouse model of TLE were robustly suppressed by systemic administration of the GlyT1 inhibitor LY2365109. We conclude that GlyT1 overexpression in the epileptic brain constitutes a new target for therapeutic intervention, and that GlyT1 inhibitors constitute a new class of antiictogenic drugs. These findings are of translational value since GlyT1 inhibitors are already in clinical development to treat cognitive symptoms in schizophrenia

Identification of amino acids involved in histamine potentiation of GABA A receptors.

Histamine is a neurotransmitter involved in a number of physiological and neuronal functions. In mammals, such as humans, and rodents, the histaminergic neurons found in the tuberomamillary nucleus project widely throughout the central nervous system. Histamine acts as positive modulator of GABAA receptors (GABAARs) and, in high concentrations (10 mM), as negative modulator of the strychnine-sensitive glycine receptor. However, the exact molecular mechanisms by which histamine acts on GABAARs are unknown. In our study, we aimed to identify amino acids potentially involved in the modulatory effect of histamine on GABAARs. We expressed GABAARs with 12 different point mutations in Xenopus laevis oocytes and characterized the effect of histamine on GABA-induced currents using the two-electrode voltage clamp technique. Our data demonstrate that the amino acid residues β2(N265) and β2(M286), which are important for modulation by propofol, are not involved in the action of histamine. However, we found that histamine modulation is dependent on the amino acid residues α1(R120), β2(Y157), β2(D163), β3(V175), and β3(Q185). We showed that the amino acid residues β2(Y157) and β3(Q185) mediate the positive modulatory effect of histamine on GABA-induced currents, whereas α1(R120) and β2(D163) form a potential histamine interaction site in GABAARs.

Glycine plays a crucial role as a co-agonist of NMDA receptors in the neuronal circuit generating body movements in rat fetuses

Neuronal circuits generating fetal movements in mammals are localized in the brainstem and the spinal cord. It has been shown that glycine plays an important role through the strychnine-sensitive glycine receptors in these circuits. However, the role of glycine as the NMDA receptor co-agonist in fetal period is not fully understood. In this study, we examined the contribution of glycine to the perinatal rat spinal circuit generating forelimb movements utilizing isolated brainstem-cervical-spinal-cord preparations. In late embryonic-days-preparations, spontaneous motor bursts related to forelimb movements (forelimb-movement-related bursts; FMRBs) and respiration-related activity were observed. In neonatal preparations, spontaneous FMRBs were not observed but periodic motor bursts resembling the FMRBs could be induced after bath application of strychnine (strychnine-induced motor bursts; SIMBs). Both FMRBs and SIMBs were blocked by either the NMDA receptor antagonist APV or the antagonists of the glycine binding site of NMDA receptors [5,7-dichlorokynurenic acid (DCKA) or L-689560]. Furthermore, these motor bursts were facilitated by the glycine uptake blocker sarcosine. This effect of sarcosine was blocked by DCKA. The findings indicate that glycine plays a crucial role as a NMDA receptor co-agonist in generating spontaneous fetal motor activity before functioning as a classical inhibitory neurotransmitter in suppressing the fetal neuronal circuits.

4‐bromopropofol decreases action potential generation in spinal neurons by inducing a glycine receptor‐mediated tonic conductance

Impaired function of spinal strychnine-sensitive glycine receptors gives rise to chronic pain states and movement disorders. Therefore, increased activity of glycine receptors should help to treat such disorders. Although compounds targeting glycine receptors with a high selectivity are lacking, halogenated analogues of propofol have recently been considered as potential candidates. Therefore we asked whether 4-bromopropofol attenuated the excitability of spinal neurons by promoting glycine receptor-dependent inhibition. The actions of sub-anaesthetic concentrations of propofol and 4-bromopropofol were investigated in spinal tissue cultures prepared from mice. Drug-induced alterations in action potential firing were monitored by extracellular multi-unit recordings. The effects on GABAA and glycine receptor-mediated inhibition were quantified by whole-cell voltage-clamp recordings. Low concentrations of 4-bromopropofol (50 nM) reduced action potential activity of ventral horn neurons by about 30%, compared with sham-treated slices. This effect was completely abolished by strychnine (1 μM). In voltage-clamped neurons, 4-bromopropofol activated glycine receptors, generating a tonic current of 65 ± 10 pA, while GABAA - and glycine receptor-mediated synaptic transmission remained unaffected. The highest glycine levels in the CNS are found in the ventral horn of the spinal cord, a region mediating pain-induced motor reflexes and participating in the control of muscle tone. 4-Bromopropofol may serve as a starting point for the development of non-sedative, non-addictive, muscle relaxants and analgesics to be used to treat low back pain.

Involvement of the strychnine-sensitive glycine receptor in the anxiolytic effects of GlyT1 inhibitors on maternal separation-induced ultrasonic vocalization in rat pups

Several studies have shown that glycine transporter 1 (GlyT1) inhibitors have anxiolytic actions. There are two types of glycine receptor: the strychnine-sensitive glycine receptor (GlyA) and the strychnine-insensitive glycine receptor (GlyB); however, which receptor is the main contributor to the anxiolytic actions of GlyT1 inhibitors is yet to be determined. Here, we clarified which glycine receptor is the main contributor to the anxiolytic effects of GlyT1 inhibitors by using maternal separation-induced ultrasonic vocalization (USV) by rat pups as an index of anxiety. We confirmed that administration of the benzodiazepine diazepam or the selective serotonin reuptake inhibitor escitaloplam, which are both clinically proven anxiolytics, or the GlyT1 inhibitor SSR504734 (2-chloro-N-[(S)-phenyl[(2S)-piperidin-2-yl] methyl]-3-trifluoromethyl benzamide), decreases USV in rat pups. In addition, we showed that another GlyT1 inhibitor, ALX5407 ((R)-N-[3-(4′-fluorophenyl)-3(4′-phenylphenoxy)propyl]sarcosine) also decreases USV in rat pups. SSR504734- or ALX5407-induced decreases in USV were dose-dependently reversed by administration of the GlyA antagonist strychnine, whereas the diazepam- or escitalopram-induced decreases in USV were not. Furthermore, GlyT1-induced decreases in USV were not reversed by administration of the GlyB antagonist L-687,414. Together, these results suggest that GlyA activation is the main contributor to the anxiolytic actions of GlyT1 inhibitors and that the anxiolytic actions of diazepam and escitalopram cannot be attributed to GlyA activation. Our findings provide new insights into the importance of the activation of GlyA in the anxiolytic effects of GlyT1 inhibitors.

Ionotropic GABA and glycine receptor subunit composition in human pluripotent stem cell-derived excitatory cortical neurones.

We have assessed, using whole-cell patch-clamp recording and RNA-sequencing (RNA-seq), the properties and composition of GABAA receptors (GABAARs) and strychnine-sensitive glycine receptors (GlyRs) expressed by excitatory cortical neurons derived from human embryonic stem cells (hECNs). The agonists GABA and muscimol gave EC50 values of 278 μm and 182 μm, respectively, and the presence of a GABAAR population displaying low agonist potencies is supported by strong RNA-seq signals for α2 and α3 subunits. GABAAR-mediated currents, evoked by EC50 concentrations of GABA, were blocked by bicuculline and picrotoxin with IC50 values of 2.7 and 5.1 μm, respectively. hECN GABAARs are predominantly γ subunit-containing as assessed by the sensitivity of GABA-evoked currents to diazepam and insensitivity to Zn2+, together with the weak direct agonist action of gaboxadol; RNA-seq indicated a predominant expression of the γ2 subunit. Potentiation of GABA-evoked currents by propofol and etomidate and the lack of inhibition of currents by salicylidine salycylhydrazide (SCS) indicate expression of the β2 or β3 subunit, with RNA-seq analysis indicating strong expression of β3 in hECN GABAARs. Taken together our data support the notion that hECN GABAARs have an α2/3β3γ2 subunit composition – a composition that also predominates in immature rodent cortex. GlyRs expressed by hECNs were activated by glycine with an EC50 of 167 μm. Glycine-evoked (500 μm) currents were blocked by strychnine (IC50 = 630 nm) and picrotoxin (IC50 = 197 μm), where the latter is suggestive of a population of heteromeric receptors. RNA-seq indicates GlyRs are likely to be composed of α2 and β subunits.

Strychnine-sensitive glycine receptors on pyramidal neurons in layers II/III of the mouse prefrontal cortex are tonically activated.

Processing of signals within the cerebral cortex requires integration of synaptic inputs and a coordination between excitatory and inhibitory neurotransmission. In addition to the classic form of synaptic inhibition, another important mechanism that can regulate neuronal excitability is tonic inhibition via sustained activation of receptors by ambient levels of inhibitory neurotransmitter, usually GABA. The purpose of this study was to determine whether this occurs in layer II/III pyramidal neurons (PNs) in the prelimbic region of the mouse medial prefrontal cortex (mPFC). We found that these neurons respond to exogenous GABA and to the α4δ-containing GABAA receptor (GABA(A)R)-selective agonist gaboxadol, consistent with the presence of extrasynaptic GABA(A)R populations. Spontaneous and miniature synaptic currents were blocked by the GABA(A)R antagonist gabazine and had fast decay kinetics, consistent with typical synaptic GABA(A)Rs. Very few layer II/III neurons showed a baseline current shift in response to gabazine, but almost all showed a current shift (15-25 pA) in response to picrotoxin. In addition to being a noncompetitive antagonist at GABA(A)Rs, picrotoxin also blocks homomeric glycine receptors (GlyRs). Application of the GlyR antagonist strychnine caused a modest but consistent shift (∼15 pA) in membrane current, without affecting spontaneous synaptic events, consistent with the tonic activation of GlyRs. Further investigation showed that these neurons respond in a concentration-dependent manner to glycine and taurine. Inhibition of glycine transporter 1 (GlyT1) with sarcosine resulted in an inward current and an increase of the strychnine-sensitive current. Our data demonstrate the existence of functional GlyRs in layer II/III of the mPFC and a role for these receptors in tonic inhibition that can have an important influence on mPFC excitability and signal processing.

Background synaptic activity in rat entorhinal cortex shows a progressively greater dominance of inhibition over excitation from deep to superficial layers.

The entorhinal cortex (EC) controls hippocampal input and output, playing major roles in memory and spatial navigation. Different layers of the EC subserve different functions and a number of studies have compared properties of neurones across layers. We have studied synaptic inhibition and excitation in EC neurones, and we have previously compared spontaneous synaptic release of glutamate and GABA using patch clamp recordings of synaptic currents in principal neurones of layers II (L2) and V (L5). Here, we add comparative studies in layer III (L3). Such studies essentially look at neuronal activity from a presynaptic viewpoint. To correlate this with the postsynaptic consequences of spontaneous transmitter release, we have determined global postsynaptic conductances mediated by the two transmitters, using a method to estimate conductances from membrane potential fluctuations. We have previously presented some of this data for L3 and now extend to L2 and L5. Inhibition dominates excitation in all layers but the ratio follows a clear rank order (highest to lowest) of L2>L3>L5. The variance of the background conductances was markedly higher for excitation and inhibition in L2 compared to L3 or L5. We also show that induction of synchronized network epileptiform activity by blockade of GABA inhibition reveals a relative reluctance of L2 to participate in such activity. This was associated with maintenance of a dominant background inhibition in L2, whereas in L3 and L5 the absolute level of inhibition fell below that of excitation, coincident with the appearance of synchronized discharges. Further experiments identified potential roles for competition for bicuculline by ambient GABA at the GABAA receptor, and strychnine-sensitive glycine receptors in residual inhibition in L2. We discuss our results in terms of control of excitability in neuronal subpopulations of EC neurones and what these may suggest for their functional roles.

Zinc-Dependent Modulation of α2- and α3-Glycine Receptor Subunits by Ethanol

Strychnine-sensitive glycine receptors (GlyRs) are expressed throughout the brain and spinal cord and are among the strongly supported protein targets of alcohol. This is based largely on studies of the α1-subunit; however, α2- and α3-GlyR subunits are as or more abundantly expressed than α1-GlyRs in multiple forebrain brain areas considered to be important for alcohol-related behaviors, and uniquely some α3-GlyRs undergo RNA editing. Nanomolar and low micromolar concentrations of zinc ions potentiate GlyR function, and in addition to zinc's effects on glycine-activated currents, we have recently shown that physiological concentrations of zinc also enhance the magnitude of ethanol (EtOH)'s effects on α1-GlyRs. Using 2-electrode voltage-clamp electrophysiology in oocytes expressing either α2- or α3-GlyRs, we first tested the hypothesis that the effects of EtOH on α2- and α3-GlyRs would be zinc dependent, as we have previously reported for α1-GlyRs. Next, we constructed an α3P185L-mutant GlyR to test whether RNA-edited and unedited GlyRs contain differences in EtOH sensitivity. Last, we built a homology model of the α3-GlyR subunit. The effects of EtOH (20 to 200 mM) on both subunits were greater in the presence than in the absence of 500 nM added zinc. The α3P185L-mutation that corresponds to RNA editing increased sensitivity to glycine and decreased sensitivity to EtOH. Our findings provide further evidence that zinc is important for determining the magnitude of EtOH's effects at GlyRs and suggest that by better understanding zinc/EtOH interactions at GlyRs, we may better understand the sites and mechanisms of EtOH action.

New insights in endogenous modulation of ligand-gated ion channels: Histamine is an inverse agonist at strychnine sensitive glycine receptors

Histamine is involved in many physiological functions in the periphery and is an important neurotransmitter in the brain. It acts on metabotropic H1–H4 receptors mediating vasodilatation, bronchoconstriction and stimulation of gastric acid secretion. In the brain histamine is produced by neurons in the tuberomamillary nucleus (TMN), which controls arousal. Histamine is also a positive modulator of the inhibitory Cys-loop ligand-gated ion channel GABAA. We investigated now its effect on the second member of inhibitory Cys-loop ligand-gated ion channels, the strychnine sensitive glycine receptor. We expressed different human and rat glycine receptor subunits in Xenopus laevis oocytes and characterized the effect of histamine using the two electrode voltage clamp technique. Furthermore we investigated native glycine receptors in hypothalamic neurons using the patch-clamp technique. Histamine inhibited α1β glycine receptors with an IC50 of 5.2±0.3mM. In presence of 10mM histamine the glycine dose–response curve was shifted, increasing the EC50 for glycine from 25.5±1.4μM to 42.4±2.3μM. In addition, histamine blocked the spontaneous activity of RNA-edited α3β glycine receptors. Histamine inhibited glycine receptors expressed in hypothalamic TMN neurons with an IC50 of 4.6±0.3mM. Our results give strong evidence that histamine is acting on the same binding site as glycine, being an inverse agonist that competitively antagonizes glycine receptors. Thus, we revealed histamine as an endogenous modulator of glycine receptors.

P.1.011 Identification of a significant role for the ventral hippocampus in neuropeptide S-elicited anxiolysis

The negative symptoms of schizophrenia are reverted by treatment with glycine or other agonists of the glycine-B site which facilitate NMDA receptor function. On the other hand, there are experimental observations showing that exogenous application of glycine (0.5–10 mM) results in a long-lasting potentiation of glutamatergic synaptic transmission (LTP-GLY). The characterization of the mechanisms underlying LTP-GLY could be useful to develop new therapies for schizophrenia. The main goal of this work is to deepen the understanding of this potentiation phenomenon. The present study demonstrates in rat hippocampal slices that superfusion of glycine 1 mM during 30 min produces a potentiation of excitatory postsynaptic potentials in CA3-CA1 pathway lasting at least 1 h. Glycine application does not modify neither presynaptic fiber volley nor paired-pulse facilitation of synaptic potentials. This LTP-GLY is independent of both strychnine-sensitive glycine receptors and nifedipine-sensitive calcium channels. Interestingly, LTP-GLY is not inhibited but strengthened by NMDA receptors antagonists such as AP-5 or MK-801. In contrast, LTP-GLY is partially or totally blocked with the antagonists of glycine transporter GLYT1, sarcosine or ALX-5407, respectively. These results indicate that LTP-GLY requires the activation of GLYT1, a glycine transporter co-localized and associated to NMDA receptors. In addition, the fact that NMDA receptor inhibition increases LTP-GLY magnitude, opens the possibility that these receptors could have a negative control on GLYT1 activity.

4-Chloropropofol enhances chloride currents in human hyperekplexic and artificial mutated glycine receptors

BackgroundThe mammalian neurological disorder hereditary hyperekplexia can be attributed to various mutations of strychnine sensitive glycine receptors. The clinical symptoms of “startle disease” predominantly occur in the newborn leading to convulsive hypertonia and an exaggerated startle response to unexpected mild stimuli. Amongst others, point mutations R271Q and R271L in the α1-subunit of strychnine sensitive glycine receptors show reduced glycine sensitivity and cause the clinical symptoms of hyperekplexia.Halogenation has been shown to be a crucial structural determinant for the potency of a phenolic compound to positively modulate glycine receptor function.The aim of this in vitro study was to characterize the effects of 4-chloropropofol (4-chloro-2,6-dimethylphenol) at four glycine receptor mutations.MethodsGlycine receptor subunits were expressed in HEK 293 cells and experiments were performed using the whole-cell patch-clamp technique.Results4-chloropropofol exerted a positive allosteric modulatory effect in a low sub-nanomolar concentration range at the wild type receptor (EC50 value of 0.08 ± 0.02 nM) and in a micromolar concentration range at the mutations (1.3 ± 0.6 μM, 0.1 ± 0.2 μM, 6.0 ± 2.3 μM and 55 ± 28 μM for R271Q, L, K and S267I, respectively).Conclusions4-chloropropofol might be an effective compound for the activation of mutated glycine receptors in experimental models of startle disease.

Hydroxylated biphenyl derivatives are positive modulators of human GABAA receptors

A series of 7 hydroxylated biphenyl derivatives (1–7) were prepared to evaluate their ability to modulate the function of several ligand gated ion channel (LGIC) recombinant receptors expressed in Xenopus laevis oocytes. Compounds 1, 3, 4, 6 and 7 are natural occurring compounds whereas the synthesis of compounds 2 and 5 was previously reported (Delogu et al., 2004; Fabbri et al., 2007). None of the compounds tested were able to modify, the activity of the strychnine-sensitive glycine receptor, or the activity of nicotinic receptor. The function of the 5HT3A receptor was partially inhibited by all compounds tested, however this inhibition occurred at relatively high concentrations (100μM). All compounds, with the exception of compound 6, potentiate the action of gamma-aminobutyric acid (GABA)-evoked Cl− currents in Xenopus laevis oocytes expressing recombinant human α1β2γ2L GABAA receptors. Compounds 1, 2, 5 and 7 enhance the function of the GABAA receptor at concentrations higher than 3–10μM. Compound 4 was the most efficacious. However, compound 3 was the most potent (EC50 0.8μM). The potency of compound 3 in modulating the function of the GABAA receptor was comparable to that of diazepam, propofol or allopregnanolone. The enhancement of the GABA evoked Cl− currents by compound 3 was not affected by flumazenil. Compound 3 did not induce loss of the righting reflex in rats suggesting that it is not an anesthetic agent, however, its ability in protecting the animals from seizures induced by picrotoxin confirm that its action occurs through the GABAA receptor.

Differentiated human midbrain-derived neural progenitor cells express excitatory strychnine-sensitive glycine receptors containing α2β subunits.

BackgroundHuman fetal midbrain-derived neural progenitor cells (NPCs) may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson’s disease. While glutamate and GABAA receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown.Methodology/Principal FindingsHere we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na+-K+-Cl− co-transporter 1 (NKCC1)-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. Conclusions/SignificanceThese data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro.