Glycinergic Synaptic Transmission Research Articles

Neurexins control the strength and precise timing of glycinergic inhibition in the auditory brainstem.

Neurexins play diverse functions as presynaptic organizers in various glutamatergic and GABAergic synapses. However, it remains unknown whether and how neurexins are involved in shaping functional properties of the glycinergic synapses, which mediate prominent inhibition in the brainstem and spinal cord. To address these issues, we examined the role of neurexins in a model glycinergic synapse between the principal neuron in the medial nucleus of the trapezoid body (MNTB) and the principal neuron in the lateral superior olive (LSO) in the auditory brainstem. Combining RNAscope with stereotactic injection of AAV-Cre in the MNTB of neurexin1/2/3 conditional triple knockout mice, we showed that MNTB neurons highly express all isoforms of neurexins although their expression levels vary remarkably. Selective ablation of all neurexins in MNTB neurons not only reduced the amplitude but also altered the kinetics of the glycinergic synaptic transmission at LSO neurons. The synaptic dysfunctions primarily resulted from an impaired Ca2+ sensitivity of release and a loosened coupling between voltage-gated Ca2+ channels and synaptic vesicles. Together, our current findings demonstrate that neurexins are essential in controlling the strength and temporal precision of the glycinergic synapse, which therefore corroborates the role of neurexins as key presynaptic organizers in all major types of fast chemical synapses.

GABAergic and Glycinergic Synaptic Transmission in Respiratory‐Controlling Kolliker‐Fuse Neurons in Female and Male Rett Syndrome Mice

Rett Syndrome (RTT) is a neurodevelopmental disorder characterized by severe motor behavior disturbances including breathing problems. RTT is caused by silencing mutations in the methyl‐CpG binding protein 2 (MeCP2) gene located on the X chromosome. Accordingly, girls and women account for the majority of RTT patients. The genetic nature of RTT lends itself to study in mice with loss of function mutations in the MeCP2 gene. Importantly, RTT mice display breathing irregularities similar to human patients. Insufficient inhibitory neurotransmission in the brainstem respiratory network is thought to contribute to the breathing problems in RTT. In the pontine Kölliker‐Fuse (KF) area of RTT mice, there are fewer GABAergic axon terminals and injection of a GABA reuptake antagonist alleviates breathing problems in vivo. However, no one has examined the physiology of GABA or glycine synapses in the KF of RTT mice. In the present work, we tested the hypothesis that GABAergic and glycinergic synapses are deficient in the KF of RTT mice. We performed whole‐cell voltage clamp recordings from KF neurons contained in brain slices of MeCP2‐deficient RTT mice and wild type littermates. The frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in the KF neurons of male RTT mice was reduced compared to wild type (WT) littermates. In contrast, female RTT mice showed no change in sIPSC frequency in the KF compared to control, despite having breathing abnormalities. The amplitude of sIPSCs in the KF of female mice were smaller compared to WT littermates, whereas male RTT mice showed no change in amplitude compared to WT littermates. These results spotlight the importance of studying respiratory controlling KF neurons in both male and female RTT mice, and suggest that reductions in IPSC frequency and amplitude are not sufficient to explain the breathing irregularities observed in RTT in females and males, respectively.

Developmental changes in GABAergic and glycinergic synaptic transmission to rat motoneurons innervating jaw-closing and jaw-opening muscles

Trigeminal motoneurons (MNs) innervating the jaw-closing and jaw-opening muscles receive numerous inhibitory synaptic inputs from GABAergic and glycinergic neurons, which are essential for oromotor functions, such as the orofacial reflex, suckling, and mastication. The properties of the GABAergic and glycinergic inputs of these MNs undergo developmental alterations during the period in which their feeding behavior proceeds from suckling to mastication; however, the detailed characteristics of the developmental patterns of GABAergic and glycinergic transmission in these neurons remain to be elucidated. This study was conducted to investigate developmental changes in miniature inhibitory postsynaptic currents (mIPSCs) in masseter (jaw-closing) and digastric (jaw-opening) MNs using brainstem slice preparations obtained from Wistar rats on postnatal day (P)2–5, P9–12, and P14–17. The frequency and amplitude of glycinergic mIPSCs substantially increased with age in both the masseter and digastric MNs. The rise time and decay time of glycinergic mIPSCs in both MNs decreased during development. In contrast, the frequency of GABAergic components in masseter MNs was higher at P2–5 than at P14–17, whereas that in the digastric MNs remained unchanged throughout the postnatal period. The proportion of currents mediated by GABA-glycine co-transmission was higher at P2–5, and then it decreased with age in both MNs. These results suggest that characteristics related to the development of inhibitory synaptic inputs differ between jaw-closing and jaw-opening MNs and between GABAergic and glycinergic currents. These distinct developmental characteristics may contribute to the development of feeding behaviors.

S2777 A Rare Case of Progressive Encephalomyelitis With Rigidity and Myoclonus Related to Hepatitis E Virus

Introduction: We present an unusual case of progressive encephalomyelitis with rigidity and myoclonus (PERM) caused by hepatitis E viral illness. Case Description/Methods: A 42-year-old male with a past medical history of left nephrectomy donation, hypertension, and chronic kidney disease presents with sudden onset of numbness of his tongue. He complains of intermittent dizziness (positional), hearing changes (including voices and music) as well as double/blurry vision, global fatigue, and visual hallucinations. The patient denies any extremity weakness, difficulty swallowing, or headache. No facial droop is noted, but right eye droop is noted. The patient is ambulating with a steady gait. The patient has elevated liver enzymes (AST 178, ALT 616) with elevated alkaline phosphatase (ALP 323). The patient has no recent exposures or ingestion. He is diagnosed with PERM following the onset of symptoms managed with high-dose steroid therapy and plasmapheresis. Symptoms persisted with dysphagia requiring feeding tube placement, tracheostomy, along with progressively worsening transaminase levels with ALP 742, AST 342, ALT 1,062. Hepatology was consulted with negative autoimmune work-up, hepatitis B and C screening. Hepatitis E IgM was obtained and found to be positive. A liver biopsy was obtained showing mild portal tract inflammation with ductal reaction, focal pericentral macrophages, and hepatocyte dropout reflective of viral hepatitis. He was recommended to start ribavirin 200 mg twice daily given persistently elevated transaminase levels. Repeat evaluation with HEV testing after two months of therapy was negative. Treatment for the PERM included steroids, intravenous immunoglobulin, plasmapheresis, and rituximab with improvement in symptoms. Most recent liver enzymes were normal on ribavirin treatment. Discussion: PERM is a rare disorder characterized by muscular rigidity, muscle spasms, abnormal posture, and is caused by inflammation in the brainstem and spinal cord. PERM can be diagnosed clinically or if the patient is positive for anti-HEV IgM and has symptomatology characteristic of stiff-person plus syndrome. Symptoms can be explained by the interruption of glycinergic synaptic transmission in the spinal cord and brainstem. Antibodies present against glycine receptors in the serum and CSF suggest antibody-mediated pathogenesis, possibly responsive to immunotherapy. Ribavirin is commonly prescribed for HEV clearance and amelioration of HEV-associated neurologic symptoms.

Development of γ-aminobutyric acid-, glycine-, and glutamate-immunopositive boutons on the rat genioglossal motoneurons.

Detailed information about the development of excitatory and inhibitory synapses on the genioglossal (GG) motoneuron may help to understand the mechanism of fine control of GG motoneuron firing and the coordinated tongue movement during postnatal development. For this, we investigated the development of γ-aminobutyric acid (GABA)-immunopositive (GABA +), glycine + (Gly +), and glutamate + (Glut +) axon terminals (boutons) on the somata of rat GG motoneurons at a postnatal day 2 (P2), P6 and P18 by retrograde labeling of GG motoneurons with horseradish peroxidase, electron microscopic postembedding immunogold staining with GABA, Gly, and Glut antisera, and quantitative analysis. The number of boutons per GG motoneuron somata and the mean length of bouton apposition, measures of bouton size and synaptic covering percentage, were significantly increased from P2/P6 to P18. The number and fraction of GABA + only boutons of all boutons decreased significantly, whereas those of Gly + only boutons increased significantly from P2/P6 to P18, suggesting developmental switch from GABAergic to glycinergic synaptic transmission. The fraction of mixed GABA +/Gly + boutons of all boutons was the highest among inhibitory bouton types throughout the postnatal development. The fractions of excitatory and inhibitory boutons of all boutons remained unchanged during postnatal development. These findings reveal a distinct developmental pattern of inhibitory synapses on the GG motoneurons different from that on spinal or trigeminal motoneurons, which may have an important role in the regulation of the precise and coordinated movements of the tongue during the maturation of the oral motor system.

Identification of the hypertension drug niflumic acid as a glycine receptor inhibitor

Glycine is one of the major neurotransmitters in the brainstem and the spinal cord. Glycine binds to and activates glycine receptors (GlyRs), increasing Cl− conductance at postsynaptic sites. This glycinergic synaptic transmission contributes to the generation of respiratory rhythm and motor patterns. Strychnine inhibits GlyR by binding to glycine-binding site, while picrotoxin blocks GlyR by binding to the channel pore. We have previously reported that bath application of strychnine to zebrafish embryos causes bilateral muscle contractions in response to tactile stimulation. To explore the drug-mediated inhibition of GlyRs, we screened a chemical library of ~ 1,000 approved drugs and pharmacologically active molecules by observing touch-evoked response of zebrafish embryos in the presence of drugs. We found that exposure of zebrafish embryos to nifedipine (an inhibitor of voltage-gated calcium channel) or niflumic acid (an inhibitor of cyclooxygenase 2) caused bilateral muscle contractions just like strychnine-treated embryos showed. We then assayed strychnine, picrotoxin, nifedipine, and niflumic acid for concentration-dependent inhibition of glycine-mediated currents of GlyRs in oocytes and calculated IC50s. The results indicate that all of them concentration-dependently inhibit GlyR in the order of strychnine > picrotoxin > nifedipine > niflumic acid.

Identification and Interaction Analysis of Key Genes and MicroRNAs in Systemic Sclerosis by Bioinformatics Approaches.

Systemic sclerosis (SSc) is a highly heterogeneous autoimmune disease with a high mortality rate. However, the cellular and molecular mechanisms of SSc remain unclear. Here, we identified the key hub genes and microRNAs (miRNAs) that modulate the occurrence and development of SSc. We downloaded the microarray dataset GSE95065 from the Gene Expression Omnibus (GEO) database and then analyzed the data by using GEO2R. The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used for functional pathway enrichment analyses of differentially expressed genes (DEGs), and Cytoscape software was used to generate the protein-protein interaction (PPI) network. In addition, OmicsNet was used to predict the miRNAs for the hub genes of SSc. As a result, 783 DEGs were identified, of which 770 genes (142 up-regulated genes and 628 down-regulated genes) were matched to the genes in SSc skin samples. Gene Ontology (GO) analyses by DAVID indicated that the up-regulated genes were mainly involved in immune response, and the down-regulated genes were greatly enriched in glycinergic synaptic transmission. In the PPI network, 22 nodes were selected as key genes, including several members of the chemokine family. Furthermore, after uploading these key genes to the OmicsNet tool, we found that hsa-miR-26b-5p might target CXCL9 and CXCL13. Moreover, we demonstrated that the hsa-miR-26b-5p inhibitor might inhibit fibrosis in TGF-β-activated fibroblasts, which would be a promising target for SSc therapy.

Capsaicin causes robust reduction in glycinergic transmission to rat hypoglossal motor neurons via a TRPV1-independent mechanism.

The effect of capsaicin on glycinergic synaptic transmission to juvenile rat hypoglossal motor neurons in acute brainstem slices was evaluated in the presence of TTX. Capsaicin caused a robust decrease in miniature IPSC frequency, amplitude, and half-width, showing that this effect is independent of action potential generation. In the presence of capsazepine, a classic TRPV1 antagonist, capsaicin was still able to reduce spontaneous inhibitory postsynaptic current (IPSC) amplitude and frequency. We further investigated whether the effect of capsaicin on glycinergic transmission to hypoglossal motor neurons is pre- or postsynaptic in nature by recording pairs of evoked IPSCs. Interestingly, capsaicin also reduced evoked IPSC amplitude without affecting paired-pulse ratio, indicating a postsynaptic mechanism of action. Significant reduction was also observed in evoked IPSC half-width, rise time, and decay tau. We also show that capsaicin does not have any effect on either transient (It) or sustained (Is) potassium currents. Finally, we also show that the hyperpolarization-activated cationic current (Ih) also remains unchanged after capsaicin application. NEW & NOTEWORTHY Capsaicin reduces the amplitude of quantal and evoked glycinergic inhibitory neurotransmission to brainstem motor neurons without altering activity-dependent transmitter release. This effect of capsaicin is not due to activation of TRPV1 receptors, as it is not blocked by capsazepine, a TRPV1 receptor antagonist. Capsaicin does not alter voltage-dependent potassium current or the hyperpolarization-activated cationic current in brainstem motor neurons.

Ubiquitination and inhibition of glycine receptor by HUWE1 in spinal cord dorsal horn

Glycine receptors (GlyRs) are pentameric proteins that consist of α (α1-α4) subunits and/or β subunit. In the spinal cord of adult animals, the majority of inhibitory glycinergic neurotransmission is mediated by α1 subunit-containing GlyRs. The reduced glycinergic inhibition (disinhibition) is proposed to increase the excitabilities and spontaneous activities of spinal nociceptive neurons during pathological pain. However, the molecular mechanisms by which peripheral lesions impair GlyRs-α1-mediated synaptic inhibition remain largely unknown. Here we found that activity-dependent ubiquitination of GlyRs-α1 subunit might contribute to glycinergic disinhibition after peripheral inflammation. Our data showed that HUWE1 (HECT, UBA, WWE domain containing 1), an E3 ubiquitin ligase, located at spinal synapses and specifically interacted with GlyRs-α1 subunit. By ubiquitinating GlyRs-α1, HUWE1 reduced the surface expression of GlyRs-α1 through endocytic pathway. In the dorsal horn of Complete Freund's Adjuvant-injected mice, shRNA-mediated knockdown of HUWE1 blunted GlyRs-α1 ubiquitination, potentiated glycinergic synaptic transmission and attenuated inflammatory pain. These data implicated that ubiquitin modification of GlyRs-α1 represented an important way for peripheral inflammation to reduce spinal glycinergic inhibition and that interference with HUWE1 activity generated analgesic action by resuming GlyRs-α1-mediated synaptic transmission.

Developmental Changes in the Inhibition of Glycinergic Synaptic Currents by Niflumic Acid in Hypoglossal Motoneurons.

Mammalian brainstem hypoglossal motoneurones (HMs) receive powerful synaptic glycinergic inputs and are involved in a variety of motor functions, including respiration, chewing, sucking, swallowing, and phonation. During the early postnatal development, subunit composition of chloride-permeable glycine receptors (GlyRs) changes leading to a decrease of “fetal” alpha2 and elevation of “adult” alpha1 GlyR subunits. It has been recently demonstrated that niflumic acid (NFA), a member of the fenamate class of non-steroidal anti-inflammatory drugs, is an efficient subunits-specific blocker of GlyRs. At a heterologous expression of different GlyR subunits it has been shown that blocking potency of NFA is more than one order higher for alpha2 GlyRs than for receptors formed by alpha1 subunit. To reveal the action of NFA on the synaptic activity we analyzed here the effects of NFA on the glycinergic inhibitory post-synaptic currents in the HMs from mouse brainstem slices. In the whole-cell patch clamp configuration, the amplitude and the frequency of glycinergic synaptic currents from two age groups have been analyzed: “neonate” (P2–P4) and “juvenile” (P7–P12). Addition of NFA in the presence of antagonists of glutamate and GABA receptors caused a decrease in the mean amplitude and frequency of synaptic events. The degree of the inhibition induced by NFA decreased with the postnatal development, being higher on the motoneurons from “neonate” brainstem slices in comparison with the “juvenile” age group. Analysis of the pair-pulse facilitation suggests the post-synaptic origin of NFA action. These observations provide evidence on the developmental changes in the inhibition by NFA of glycinergic synaptic transmission, which reflects increase in the alpha1 and decrease in the alpha2 GlyR subunits expression in synapses to hypoglossal motoneurons during the early stages of postnatal life.

A neuronal role of the Alanine-Serine-Cysteine-1 transporter (SLC7A10, Asc-1) for glycine inhibitory transmission and respiratory pattern

The Alanine-Serine-Cysteine-1 transporter (SLC7A10, Asc-1) has been shown to play a role in synaptic availability of glycine although the exact mechanism remains unclear. We used electrophysiological recordings and biochemical experiments to investigate the role of Asc-1 transporter in glycinergic transmission in the brainstem respiratory network. Using both the Asc-1 substrate and transportable inhibitor D-isoleucine (D-Ile), and the non-transportable Asc-1 blocker Lu AE00527 (Lu), we found that D-Ile reduces glycinergic transmission and increases glycine release via hetero-exchange, whereas Lu has no acute effect on glycinergic synaptic transmission. Furthermore, D-Ile increases the frequency and reduces amplitude of the phrenic nerve activity in the arterially-perfused working heart brainstem preparation. These results suggest a role of Asc-1 in modulating presynaptic glycine levels that can impact on the respiratory network.

Developmental nicotine exposure alters glycinergic neurotransmission to hypoglossal motoneurons in neonatal rats.

We tested the hypothesis that nicotine exposure in utero and after birth [developmental nicotine exposure (DNE)] disrupts development of glycinergic synaptic transmission to hypoglossal motoneurons (XIIMNs). Glycinergic spontaneous and miniature inhibitory postsynaptic currents (sIPSC/mIPSC) were recorded from XIIMNs in brain stem slices from 1- to 5-day-old rat pups of either sex, under baseline conditions and following stimulation of nicotinic acetylcholine (ACh) receptors with nicotine (i.e., an acute nicotine challenge). Under baseline conditions, there were no significant effects of DNE on the amplitude or frequency of either sIPSCs or mIPSCs. In addition, DNE did not alter the magnitude of the whole cell current evoked by bath application of glycine, consistent with an absence of change in postsynaptic glycine-mediated conductance. An acute nicotine challenge (bath application of 0.5 μM nicotine) increased sIPSC frequency in the DNE cells, but not control cells. In contrast, nicotine challenge did not change mIPSC frequency in either control or DNE cells. In addition, there were no significant changes in the amplitude of either sIPSCs or mIPSCs in response to nicotine challenge. The increased frequency of sIPSCs in response to an acute nicotine challenge in DNE cells reflects an enhancement of action potential-mediated input from glycinergic interneurons to hypoglossal motoneurons. This could lead to more intense inhibition of hypoglossal motoneurons in response to exogenous nicotine or endogenous ACh. The former would occur with smoking or e-cigarette use while the latter occurs with changes in sleep state and with hypercapnia. NEW & NOTEWORTHY Here we show that perinatal nicotine exposure does not impact baseline glycinergic neurotransmission to hypoglossal motoneurons but enhances glycinergic inputs to hypoglossal motoneurons in response to activation of nicotinic acetylcholine (ACh) receptors with acute nicotine. Given that ACh is the endogenous ligand for nicotinic ACh receptors, the latter reveals a potential mechanism whereby perinatal nicotine exposure alters motor function under conditions where ACh release increases, such as the transition from non-rapid-eye movement to rapid-eye movement sleep, and during hypercapnia.

Autism-associated neuroligin-4 mutation selectively impairs glycinergic synaptic transmission in mouse brainstem synapses.

In human patients, loss-of-function mutations of the postsynaptic cell-adhesion molecule neuroligin-4 were repeatedly identified as monogenetic causes of autism. In mice, neuroligin-4 deletions caused autism-related behavioral impairments and subtle changes in synaptic transmission, and neuroligin-4 was found, at least in part, at glycinergic synapses. However, low expression levels precluded a comprehensive analysis of neuroligin-4 localization, and overexpression of neuroligin-4 puzzlingly impaired excitatory but not inhibitory synaptic function. As a result, the function of neuroligin-4 remains unclear, as does its relation to other neuroligins. To clarify these issues, we systematically examined the function of neuroligin-4, focusing on excitatory and inhibitory inputs to defined projection neurons of the mouse brainstem as central model synapses. We show that loss of neuroligin-4 causes a profound impairment of glycinergic but not glutamatergic synaptic transmission and a decrease in glycinergic synapse numbers. Thus, neuroligin-4 is essential for the organization and/or maintenance of glycinergic synapses.

Second Messengers in the Presynaptic Regulation of Glycinergic Synapses in Frog Motoneurons

The possible pathways mediating the inhibitory influences of two types of presynaptic metabotropic receptors – group III metabotropic glutamate receptors (mGluRIII) and GABAB receptors (GABABR) – on miniature glycinergic activity in motoneurons in the isolated spinal cord of the frog Rana ridibunda were studied. The glycinergic component is dominant in the inhibitory miniature activity of motoneurons [3]. Baclofen and LAP4, which are selective agonists of GABABR and mGluRIII, inhibited miniature glycinergic activity, decreasing frequency by 48.0 ± 5.6 (n = 8) and 48.5 ± 8.6% (n = 5), respectively. The amplitude of miniature potentials did not change significantly, which is evidence for a presynaptic mechanism of modulation of synaptic transmission. Studies of the stages in the mediation of the influences of the metabotropic receptors studied here on the process of glycine release from presynaptic terminals were performed by applying their selective agonists in conditions of previous selective blockade of the components mediating modulation of glycinergic synaptic transmission. The effects of mGluRIII were shown to be mediated tghe feedback via adenylate cyclase (AC), the next stage being mediated by protein kinase A (PKA), as their selective blockade virtually eliminated by effect of activation of mGluRIII. The effects of GABABR on miniature glycinergic activity did not involve AC activity, as its blocker SQ22536 did not alter the inhibitory effect of baclofen. The effects of GABABR were mediated by negative feedback via phospholipase C (PLC), as its prior exclusion (inhibitor U73122) prevented the effect of application of baclofen. However, the next possible stage in this pathway – protein kinase C (PKC) – did not have a role in mediating the influences of GABABR, as the PKC blocker (GF109302X) had no influences on the effect of baclofen. The common component in mediating the effects of mGluRIII and GABABR on miniature glycinergic activity may consist of inositol triphosphate receptors (IP3R), which regulate calcium release from intraterminal depots and, according to published data, are linked with both PKA or cAMP and with PLC. In our experiments, 2-APB, an IP3R antagonist, decreased miniature glycinergic activity frequency by 26 ± 8% (n = 7) and prevented the inhibitory effects of application of both mGluRIII and GABABR agonists. This stage of mediation of the influences of mGluRIII and GABABR appears to involve collision and interaction between these (crosstalk), as demonstrated by our previous studies [2].

Ethanol-induced enhancement of inhibitory synaptic transmission in the rat spinal substantia gelatinosa.

Recent studies have shown that ethanol produces a widespread modulation of neuronal activity in the central nervous system. It is not fully understood, however, how ethanol changes nociceptive transmission. We investigated acute effects of ethanol on synaptic transmission in the substantia gelatinosa (lamina II of the spinal dorsal horn) and mechanical responses in the spinal dorsal horn. In substantia gelatinosa neurons, bath application of ethanol at low concentration (10 mM) did not change the frequency and amplitude of spontaneous inhibitory postsynaptic currents. At medium to high concentrations (20–100 mM), however, ethanol elicited a barrage of large amplitude spontaneous inhibitory postsynaptic currents. In the presence of tetrodotoxin, such enhancement of spontaneous inhibitory postsynaptic currents was not detected. In addition, ethanol (20–100 mM) increased the frequency of spontaneous discharge of vesicular GABA transporter-Venus-labeled neurons and suppressed the mechanical nociceptive response in wide-dynamic range neurons in the spinal dorsal horn. The present results suggest that ethanol may reduce nociceptive information transfer in the spinal dorsal horn by enhancement of inhibitory GABAergic and glycinergic synaptic transmission.

Defects of the Glycinergic Synapse in Zebrafish.

Glycine mediates fast inhibitory synaptic transmission. Physiological importance of the glycinergic synapse is well established in the brainstem and the spinal cord. In humans, the loss of glycinergic function in the spinal cord and brainstem leads to hyperekplexia, which is characterized by an excess startle reflex to sudden acoustic or tactile stimulation. In addition, glycinergic synapses in this region are also involved in the regulation of respiration and locomotion, and in the nociceptive processing. The importance of the glycinergic synapse is conserved across vertebrate species. A teleost fish, the zebrafish, offers several advantages as a vertebrate model for research of glycinergic synapse. Mutagenesis screens in zebrafish have isolated two motor defective mutants that have pathogenic mutations in glycinergic synaptic transmission: bandoneon (beo) and shocked (sho). Beo mutants have a loss-of-function mutation of glycine receptor (GlyR) β-subunit b, alternatively, sho mutant is a glycinergic transporter 1 (GlyT1) defective mutant. These mutants are useful animal models for understanding of glycinergic synaptic transmission and for identification of novel therapeutic agents for human diseases arising from defect in glycinergic transmission, such as hyperekplexia or glycine encephalopathy. Recent advances in techniques for genome editing and for imaging and manipulating of a molecule or a physiological process make zebrafish more attractive model. In this review, we describe the glycinergic defective zebrafish mutants and the technical advances in both forward and reverse genetic approaches as well as in vivo visualization and manipulation approaches for the study of the glycinergic synapse in zebrafish.

CNQX facilitates inhibitory synaptic transmission in rat hypoglossal nucleus

6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) is a most commonly used antagonist of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor in the central nervous system. During the past two decades, studies had demonstrated that CNQX could partially activate AMPA receptors that are located on the hippocampal and cerebellar interneurons, thus subsequently leading to the facilitation of inhibitory transmission. However, whether CNQX could enhance inhibitory synaptic transmission in the hypoglossal nucleus remains elusive. Here, using whole-cell patch-clamp recording in the brainstem slice, we showed that CNQX greatly increased both frequency and amplitude of spontaneous inhibitory postsynaptic currents in the hypoglossal motoneurons, whereas D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5), N-methyl-D-aspartate (NMDA) receptor antagonist, had no effect on inhibitory synaptic transmission. Application of bicuculline and strychnine further identified that CNQX not only increased GABAergic sIPSCs but also glycinergic one in these motoneurons. Similar enhancement of inhibitory transmission was observed with application of 6, 7-dinitroquinoxaline-2, 3-dione (DNQX), a quinoxaline derivative of CNQX, but not with application of GYKI 53655, a non-competitive antagonist of AMPA receptor. In the presence of tetradotoxin, the effect of CNQX on sIPSCs was abolished, suggesting that an increase in presynaptic interneuron spike firing rate induced by CNQX was responsible for the facilitation of sIPSCs. Taken together, these results demonstrated that the excitatory effect of CNQX on presynaptic interneurons triggered enhancement of both GABAergic and glycinergic synaptic transmission within the rat hypoglossal nucleus.

Presynaptic facilitation of glycinergic mIPSC is reduced in mice lacking α3 glycine receptor subunits

Glycinergic neurons provide an important mechanism to control excitation of motoneurons in the brainstem and a reduction or loss of glycinergic inhibition can be deleterious by leading to hyperexcitation such as in hyperekplexia or neurodegeneration and neuronal death as in amyotrophic lateral sclerosis (ALS). Second messenger systems that change cyclic AMP and lead to phosphorylation of the α3 subunit of the glycine receptor (GlyR α3) have been shown to be potent modulators of synaptic inhibition in the spinal cord and brain stem. In this study we analyzed the role of GlyR α3 in synaptic inhibition to the hypoglossal nucleus using Glra3 (the gene encoding the glycine receptor α3 subunit) knockout mice. We observed that baseline glycinergic synaptic transmission to nucleus of hypoglossal motoneurons is rather normal in Glra3 knockout mice. Interestingly, we found that the modulation of synaptic transmission by cAMP-mediated pathways appeared to be reduced in Glra3 knockout mice. In the second postnatal week the forskolin-induced increase of miniature inhibitory postsynaptic potential (mIPSC) frequency was significantly larger in control as compared to Glra3 knockout mice suggesting that presynaptic glycine release in the hypoglossal nucleus is partially depending on GlyR α3.

Progressive encephalomyelitis with rigidity and myoclonus (PERM): brucellosis as a possible triggering factor and long-term follow-up therapy with rituximab.

Progressive encephalomyelitis with rigidity and myoclonus (PERM) is a severe syndrome that presents with autonomic features, hyperekplexia (brainstem myoclonus or excessive startle), painful spasms and breathing problems [Carvajal-Gonzalez et al. 2014]. Symptoms can be explained by the disruption of the inhibitory glycinergic synaptic transmission, which is prominent in the spinal cord and brainstem [Carvajal-Gonzalez et al. 2014]. The documented presence in the serum and cerebrospinal fluid (CSF) of antibodies against glycine receptors suggests an antibody-mediated pathogenesis with possible good response to immunotherapies [Dalakas et al. 2001; Kosmidis and Dalakas, 2010]. The precise immunopathogenetic mechanism and the triggering factors of PERM have not yet been clarified. Brucella spp. is a pathogen able to invade the central nervous system (CNS) and cause an inflammatory response with activation of microglia and macrophages, apoptosis and increased expression of antigen-presenting molecules. Neurobrucellosis often manifests with meningoencephalitis and seems that T-cell mediated mechanisms in concert with microglia activation play a key role [Seidel et al. 2003]. We present a case of PERM preceded by a meningoencephalitis caused by Brucella spp. infection and highlight response to immunotherapy, especially rituximab, many months after disease onset. The possibility that neurobrucellosis may share some common immunopathogenic mechanisms with PERM is discussed.

Mechanisms of Spontaneous Electrical Activity in the Developing Cerebral Cortex – Subplate Zone

In a developing brain, neurogenesis, migration, neurodifferentiation and synapse formation are regulated by spontaneous calcium and electrical activity. The aim of this work is to investigate the mechanisms contributing towards the generation of spontaneous electrical activity in the mammalian cortical mantle. We are focused on a group of neurons located in the subplate zone called the subplate (SP) neurons. One unique property of SP neurons, in both rodent and human cerebral cortex, is spontaneous electrical activity comprising of highly irregular plateau depolarizations crowned with action potentials (UP states). SP neurons govern the path-finding of incoming axonal projections and establishment of cortical connections and cortical columns. The selective disruption of SP neurons has been implicated in mental retardation and schizophrenia. We performed whole-cell recordings, immunolabeling and calcium imaging from positively identified SP neurons in acute brain slices obtained from the newborn mice pups (P01 – P06). The spontaneously-occurring outbursts of electrical activity (UP states) were challenged with drugs that block voltage-gated and ligand-gated membrane conductances. We found that the spontaneous activity in SP neurons was not exclusively mediated by glutamatergic, GABAergic, or glycinergic synaptic transmission. A significant portion of spontaneous depolarizing currents is linked to intact functioning of connexin-based pores and purinergic receptors. Our results suggest that spontaneous flickering of connexin hemichannels and release of ATP contribute to the generation of spontaneous electrical activity in the subplate zone of developing mammalian cerebral cortex. We think that connexin hemichannels located on both neurons and glia are involved.