Glycinergic Transmission Research Articles

Interactions Involving Glycine and Other Amino Acid Neurotransmitters: Focus on Transporter-Mediated Regulation of Release and Glycine-Glutamate Crosstalk.

Glycine plays a pivotal role in the Central Nervous System (CNS), being a major inhibitory neurotransmitter as well as a co-agonist of Glutamate at excitatory NMDA receptors. Interactions involving Glycine and other neurotransmitters are the subject of different studies. Functional interactions among neurotransmitters include the modulation of release through release-regulating receptors but also through transporter-mediated mechanisms. Many transporter-mediated interactions involve the amino acid transmitters Glycine, Glutamate, and GABA. Different studies published during the last two decades investigated a number of transporter-mediated interactions in depth involving amino acid transmitters at the nerve terminal level in different CNS areas, providing details of mechanisms involved and suggesting pathophysiological significances. Here, this evidence is reviewed also considering additional recent information available in the literature, with a special (but not exclusive) focus on glycinergic neurotransmission and Glycine-Glutamate interactions. Some possible pharmacological implications, although partly speculative, are also discussed. Dysregulations in glycinergic and glutamatergic transmission are involved in relevant CNS pathologies. Pharmacological interventions on glycinergic targets (including receptors and transporters) are under study to develop novel therapies against serious CNS pathological states including pain, schizophrenia, epilepsy, and neurodegenerative diseases. Although with limitations, it is hoped to possibly contribute to a better understanding of the complex interactions between glycine-mediated neurotransmission and other major amino acid transmitters, also in view of the current interest in potential drugs acting on "glycinergic" targets.

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.

GPR39 regulated spinal glycinergic inhibition and mechanical inflammatory pain.

G protein-coupled receptor 39 (GPR39) senses the change of extracellular divalent zinc ion and signals through multiple G proteins to a broad spectrum of downstream effectors. Here, we found that GPR39 was prevalent at inhibitory synapses of spinal cord somatostatin-positive (SOM+) interneurons, a mechanosensitive subpopulation that is critical for the conveyance of mechanical pain. GPR39 complexed specifically with inhibitory glycine receptors (GlyRs) and helped maintain glycinergic transmission in a manner independent of G protein signalings. Targeted knockdown of GPR39 in SOM+ interneurons reduced the glycinergic inhibition and facilitated the excitatory output from SOM+ interneurons to spinoparabrachial neurons that engaged superspinal neural circuits encoding both the sensory discriminative and affective motivational domains of pain experience. Our data showed that pharmacological activation of GPR39 or augmenting GPR39 interaction with GlyRs at the spinal level effectively alleviated the sensory and affective pain induced by complete Freund's adjuvant and implicated GPR39 as a promising therapeutic target for the treatment of inflammatory mechanical pain.

Disinhibition does not play a role in endomorphin-2-induced changes in inspiratory motoneuron output produced by in vitro neonatal rat preparations

Low level activation of mu-opioid receptors (MORs) in neonatal rat brainstem-spinal cord preparations increases inspiratory burst amplitude recorded on cervical spinal roots. We tested whether: (1) MOR activation with an endogenous ligand, such as endomorphin-2, increases inspiratory burst amplitude, (2) disinhibition of GABAergic or glycinergic inhibitory synaptic transmission is involved, and (3) inflammation alters endomorphin-2 effects. Using neonatal rat (P0-P3) brainstem-spinal cord preparations, bath-applied endomorphin-2 (10–200 nM) increased inspiratory burst amplitude and decreased burst frequency. Blockade of GABAA receptors (picrotoxin), glycine receptors (strychnine), or both (picrotoxin and strychnine) did not abolish endomorphin-2-induced effects. In preparations isolated from neonatal rats injected 3 h previously with lipopolysaccharide (LPS, 0.1 mg/kg), endomorphin-2 continued to decrease burst frequency but abolished the burst amplitude increase. Collectively, these data indicate that disinhibition of inhibitory synaptic transmission is unlikely to play a role in endomorphin-2-induced changes in inspiratory motor output, and that different mechanisms underlie the endomorphin-2-induced increases in inspiratory burst amplitude and decreases in burst frequency.

Protein Kinase A Is Responsible for the Presynaptic Inhibition of Glycinergic and Glutamatergic Transmissions by Xenon in Rat Spinal Cord and Hippocampal CA3 Neurons.

The effects of a general anesthetic xenon (Xe) on spontaneous, miniature, electrically evoked synaptic transmissions were examined using the "synapse bouton preparation," with which we can clearly evaluate pure synaptic responses and accurately quantify pre- and postsynaptic transmissions. Glycinergic and glutamatergic transmissions were investigated in rat spinal sacral dorsal commissural nucleus and hippocampal CA3 neurons, respectively. Xe presynaptically inhibited spontaneous glycinergic transmission, the effect of which was resistant to tetrodotoxin, Cd2+, extracellular Ca2+, thapsigargin (a selective sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor), SQ22536 (an adenylate cyclase inhibitor), 8-Br-cAMP (membrane-permeable cAMP analog), ZD7288 (an hyperpolarization-activated cyclic nucleotide-gated channel blocker), chelerythrine (a PKC inhibitor), and KN-93 (a CaMKII inhibitor) while being sensitive to PKA inhibitors (H-89, KT5720, and Rp-cAMPS). Moreover, Xe inhibited evoked glycinergic transmission, which was canceled by KT5720. Like glycinergic transmission, spontaneous and evoked glutamatergic transmissions were also inhibited by Xe in a KT5720-sensitive manner. Our results suggest that Xe decreases glycinergic and glutamatergic spontaneous and evoked transmissions at the presynaptic level in a PKA-dependent manner. These presynaptic responses are independent of Ca2+ dynamics. We conclude that PKA can be the main molecular target of Xe in the inhibitory effects on both inhibitory and excitatory neurotransmitter release. SIGNIFICANCE STATEMENT: Spontaneous and evoked glycinergic and glutamatergic transmissions were investigated using the whole-cell patch clamp technique in rat spinal sacral dorsal commissural nucleus and hippocampal CA3 neurons, respectively. Xenon (Xe) significantly inhibited glycinergic and glutamatergic transmission presynaptically. As a signaling mechanism, protein kinase A was responsible for the inhibitory effects of Xe on both glycine and glutamate release. These results may help understand how Xe modulates neurotransmitter release and exerts its excellent anesthetic properties.

N-methyl-d-aspartate receptors and glycinergic transmission, respectively, mediate muscle relaxation and immobility of pentobarbital in mice

Pentobarbital-induced anesthesia is believed to be mediated by enhancement of the inhibitory action of γ-aminobutyric acid (GABA)ergic neurons in the central nervous system. However, it is unclear whether all components of anesthesia induced by pentobarbital, such as muscle relaxation, unconsciousness, and immobility in response to noxious stimuli, are mediated only through GABAergic neurons. Thus, we examined whether the indirect GABA and glycine receptor agonists gabaculine and sarcosine, respectively, the neuronal nicotinic acetylcholine receptor antagonist mecamylamine, or the N-methyl-d-aspartate receptor channel blocker MK-801 could enhance pentobarbital-induced components of anesthesia. Muscle relaxation, unconsciousness, and immobility were evaluated by grip strength, the righting reflex, and loss of movement in response to nociceptive tail clamping, respectively, in mice. Pentobarbital reduced grip strength, impaired the righting reflex, and induced immobility in a dose-dependent manner. The change in each behavior induced by pentobarbital was roughly consistent with that in electroencephalographic power. A low dose of gabaculine, which significantly increased endogenous GABA levels in the central nervous system but had no effect on behaviors alone, potentiated muscle relaxation, unconsciousness, and immobility induced by low pentobarbital doses. A low dose of MK-801 augmented only the masked muscle-relaxing effects of pentobarbital among these components. Sarcosine enhanced only pentobarbital-induced immobility. Conversely, mecamylamine had no effect on any behavior. These findings suggest that each component of anesthesia induced by pentobarbital is mediated through GABAergic neurons and that pentobarbital-induced muscle relaxation and immobility may partially be associated with N-methyl-d-aspartate receptor antagonism and glycinergic neuron activation, respectively.

µ-Opioid Receptor Activation Reduces Glutamate Release in the PreBötzinger Complex in Organotypic Slice Cultures.

The inspiratory rhythm generator, located in the brainstem preBötzinger complex (preBötC), is dependent on glutamatergic signaling and is affected profoundly by opioids. Here, we used organotypic slice cultures of the newborn mouse brainstem of either sex in combination with genetically encoded sensors for Ca2+, glutamate, and GABA to visualize Ca2+, glutamatergic and GABAergic signaling during spontaneous rhythm and in the presence of DAMGO. During spontaneous rhythm, the glutamate sensor SF-iGluSnFR.A184S revealed punctate synapse-like fluorescent signals along dendrites and somas in the preBötC with decay times that were prolonged by the glutamate uptake blocker (TFB-TBOA). The GABA sensor iGABASnFR showed a more diffuse fluorescent signal during spontaneous rhythm. Rhythmic Ca2+- and glutamate transients had an inverse relationship between the spontaneous burst frequency and the burst amplitude of the Ca2+ and glutamate signals. A similar inverse relationship was observed when bath applied DAMGO reduced spontaneous burst frequency and increased the burst amplitude of Ca2+, glutamate, and GABA transient signals. However, a hypoxic challenge reduced both burst frequency and Ca2+ transient amplitude. Using a cocktail that blocked glutamatergic, GABAergic, and glycinergic transmission to indirectly measure the release of glutamate/GABA in response to an electrical stimulus, we found that DAMGO reduces the release of glutamate in the preBötC but has no effect on GABA release. This suggest that the opioid mediated slowing of respiratory rhythm involves presynaptic reduction of glutamate release, which would impact the ability of the network to engage in recurrent excitation, and may result in the opioid-induced slowing of inspiratory rhythm.SIGNIFICANCE STATEMENT Opioids slow down breathing rhythm by affecting neurons in the preBötzinger complex (preBötC) and other brainstem regions. Here, we used cultured slices of the preBötC to better understand this effect by optically recording Ca2+, glutamate, and GABA transients during preBötC activity. Spontaneous rhythm showed an inverse relationship between burst frequency and burst amplitude in the Ca2+ and glutamate signals. Application of the opioid DAMGO slowed the rhythm, with a concomitant increase in Ca2+, glutamate, and GABA signals. When rhythm was blocked pharmacologically, DAMGO reduced the presynaptic release of glutamate, but not GABA. These data suggest the mechanism of action of opioids involves presynaptic reduction of glutamate release, which may play an important role in the opioid-induced slowing of inspiratory rhythm.

Kappa opioids inhibit the GABA/glycine terminals of rostral ventromedial medulla projections in the superficial dorsal horn of the spinal cord.

Descending projections from neurons in the rostral ventromedial medulla (RVM) make synapses within the superficial dorsal horn (SDH) of the spinal cord that are involved in the modulation of nociception, the development of chronic pain and itch, and an important analgesic target for opioids. This projection is primarily inhibitory, but the relative contribution of GABAergic and glycinergic transmission is unknown and there is limited knowledge about the SDH neurons targeted. Additionally, the details of how spinal opioids mediate analgesia remain unclear, and no study has investigated the opioid modulation of this synapse. We address this using ex vivo optogenetic stimulation of RVM fibres in conjunction with whole‐cell patch‐clamp recordings from the SDH in spinal cord slices. We demonstrate that both GABAergic and glycinergic neurotransmission is employed and show that SDH target neurons have diverse morphological and electrical properties, consistent with both inhibitory and excitatory interneurons. Then, we describe a subtype of SDH neurons that has a glycine‐dominant input, indicating that the quality of descending inhibition across cells is not uniform. Finally, we discovered that the kappa‐opioid receptor agonist U69593 presynaptically suppressed most RVM‐SDH synapses. By contrast, the mu‐opioid receptor agonist DAMGO acted both pre‐ and postsynaptically at a subset of synapses, and the delta‐opioid receptor agonist deltorphin II had little effect. These data provide important mechanistic information about a descending control pathway that regulates spinal circuits. This information is necessary to understand how sensory inputs are shaped and develop more reliable and effective alternatives to current opioid analgesics.

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 Formation of the GABAergic and Glycinergic Networks in the Mouse Spinal Cord.

Gamma-aminobutyric acid (GABA) and glycine act as inhibitory neurotransmitters. Three types of inhibitory neurons and terminals, GABAergic, GABA/glycine coreleasing, and glycinergic, are orchestrated in the spinal cord neural circuits and play critical roles in regulating pain, locomotive movement, and respiratory rhythms. In this study, we first describe GABAergic and glycinergic transmission and inhibitory networks, consisting of three types of terminals in the mature mouse spinal cord. Second, we describe the developmental formation of GABAergic and glycinergic networks, with a specific focus on the differentiation of neurons, formation of synapses, maturation of removal systems, and changes in their action. GABAergic and glycinergic neurons are derived from the same domains of the ventricular zone. Initially, GABAergic neurons are differentiated, and their axons form synapses. Some of these neurons remain GABAergic in lamina I and II. Many GABAergic neurons convert to a coreleasing state. The coreleasing neurons and terminals remain in the dorsal horn, whereas many ultimately become glycinergic in the ventral horn. During the development of terminals and the transformation from radial glia to astrocytes, GABA and glycine receptor subunit compositions markedly change, removal systems mature, and GABAergic and glycinergic action shifts from excitatory to inhibitory.

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.

Mediators of Neuropathic Pain; Focus on Spinal Microglia, CSF-1, BDNF, CCL21, TNF-α, Wnt Ligands, and Interleukin 1β.

Intractable neuropathic pain is a frequent consequence of nerve injury or disease. When peripheral nerves are injured, damaged axons undergo Wallerian degeneration. Schwann cells, mast cells, fibroblasts, keratinocytes and epithelial cells are activated leading to the generation of an “inflammatory soup” containing cytokines, chemokines and growth factors. These primary mediators sensitize sensory nerve endings, attract macrophages, neutrophils and lymphocytes, alter gene expression, promote post-translational modification of proteins, and alter ion channel function in primary afferent neurons. This leads to increased excitability and spontaneous activity and the generation of secondary mediators including colony stimulating factor 1 (CSF-1), chemokine C-C motif ligand 21 (CCL-21), Wnt3a, and Wnt5a. Release of these mediators from primary afferent neurons alters the properties of spinal microglial cells causing them to release tertiary mediators, in many situations via ATP-dependent mechanisms. Tertiary mediators such as BDNF, tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and other Wnt ligands facilitate the generation and transmission of nociceptive information by increasing excitatory glutamatergic transmission and attenuating inhibitory GABA and glycinergic transmission in the spinal dorsal horn. This review focusses on activation of microglia by secondary mediators, release of tertiary mediators from microglia and a description of their actions in the spinal dorsal horn. Attention is drawn to the substantial differences in the precise roles of various mediators in males compared to females. At least 25 different mediators have been identified but the similarity of their actions at sensory nerve endings, in the dorsal root ganglia and in the spinal cord means there is considerable redundancy in the available mechanisms. Despite this, behavioral studies show that interruption of the actions of any single mediator can relieve signs of pain in experimental animals. We draw attention this paradox. It is difficult to explain how inactivation of one mediator can relieve pain when so many parallel pathways are available.

Inhibition of Glycine Re-Uptake: A Potential Approach for Treating Pain by Augmenting Glycine-Mediated Spinal Neurotransmission and Blunting Central Nociceptive Signaling.

Among the myriad of cellular and molecular processes identified as contributing to pathological pain, disinhibition of spinal cord nociceptive signaling to higher cortical centers plays a critical role. Importantly, evidence suggests that impaired glycinergic neurotransmission develops in the dorsal horn of the spinal cord in inflammatory and neuropathic pain models and is a key maladaptive mechanism causing mechanical hyperalgesia and allodynia. Thus, it has been hypothesized that pharmacological agents capable of augmenting glycinergic tone within the dorsal horn may be able to blunt or block aberrant nociceptor signaling to the brain and serve as a novel class of analgesics for various pathological pain states. Indeed, drugs that enhance dysfunctional glycinergic transmission, and in particular inhibitors of the glycine transporters (GlyT1 and GlyT2), are generating widespread interest as a potential class of novel analgesics. The GlyTs are Na+/Cl−-dependent transporters of the solute carrier 6 (SLC6) family and it has been proposed that the inhibition of them presents a possible mechanism by which to increase spinal extracellular glycine concentrations and enhance GlyR-mediated inhibitory neurotransmission in the dorsal horn. Various inhibitors of both GlyT1 and GlyT2 have demonstrated broad analgesic efficacy in several preclinical models of acute and chronic pain, providing promise for the approach to deliver a first-in-class non-opioid analgesic with a mechanism of action differentiated from current standard of care. This review will highlight the therapeutic potential of GlyT inhibitors as a novel class of analgesics, present recent advances reported for the field, and discuss the key challenges associated with the development of a GlyT inhibitor into a safe and effective agent to treat pain.

Effects of Interleukin-1β in Glycinergic Transmission at the Central Amygdala

Interleukin-1β (IL-1β) is an important cytokine that modulates peripheral and central pain sensitization at the spinal level. Among its effects, it increases spinal cord excitability by reducing inhibitory Glycinergic and GABAergic neurotransmission. In the brain, IL-1β is released by glial cells in regions associated with pain processing during neuropathic pain. It also has important roles in neuroinflammation and in regulating NMDA receptor activity required for learning and memory. The modulation of glycine-mediated inhibitory activity via IL-1β may play a critical role in the perception of different levels of pain. The central nucleus of the amygdala (CeA) participates in receiving and processing pain information. Interestingly, this nucleus is enriched in the regulatory auxiliary glycine receptor (GlyR) β subunit (βGlyR); however, no studies have evaluated the effect of IL-1β on glycinergic neurotransmission in the brain. Hence, we hypothesized that IL-1β may modulate GlyR-mediated inhibitory activity via interactions with the βGlyR subunit. Our results show that the application of IL-1β (10 ng/ml) to CeA brain slices has a biphasic effect; transiently increases and then reduces sIPSC amplitude of CeA glycinergic currents. Additionally, we performed molecular docking, site-directed mutagenesis, and whole-cell voltage-clamp electrophysiological experiments in HEK cells transfected with GlyRs containing different GlyR subunits. These data indicate that IL-1β modulates GlyR activity by establishing hydrogen bonds with at least one key amino acid residue located in the back of the loop C at the ECD domain of the βGlyR subunit. The present results suggest that IL-1β in the CeA controls glycinergic neurotransmission, possibly via interactions with the βGlyR subunit. This effect could be relevant for understanding how IL-1β released by glia modulates central processing of pain, learning and memory, and is involved in neuroinflammation.

Glycinergic Transmission in the Presence and Absence of Functional GlyT2: Lessons From the Auditory Brainstem.

Synaptic transmission is controlled by re-uptake systems that reduce transmitter concentrations in the synaptic cleft and recycle the transmitter into presynaptic terminals. The re-uptake systems are thought to ensure cytosolic concentrations in the terminals that are sufficient for reloading empty synaptic vesicles (SVs). Genetic deletion of glycine transporter 2 (GlyT2) results in severely disrupted inhibitory neurotransmission and ultimately to death. Here we investigated the role of GlyT2 at inhibitory glycinergic synapses in the mammalian auditory brainstem. These synapses are tuned for resilience, reliability, and precision, even during sustained high-frequency stimulation when endocytosis and refilling of SVs probably contribute substantially to efficient replenishment of the readily releasable pool (RRP). Such robust synapses are formed between MNTB and LSO neurons (medial nucleus of the trapezoid body, lateral superior olive). By means of patch-clamp recordings, we assessed the synaptic performance in controls, in GlyT2 knockout mice (KOs), and upon acute pharmacological GlyT2 blockade. Via computational modeling, we calculated the reoccupation rate of empty release sites and RRP replenishment kinetics during 60-s challenge and 60-s recovery periods. Control MNTB-LSO inputs maintained high fidelity neurotransmission at 50 Hz for 60 s and recovered very efficiently from synaptic depression. During 'marathon-experiments' (30,600 stimuli in 20 min), RRP replenishment accumulated to 1,260-fold. In contrast, KO inputs featured severe impairments. For example, the input number was reduced to ~1 (vs. ~4 in controls), implying massive functional degeneration of the MNTB-LSO microcircuit and a role of GlyT2 during synapse maturation. Surprisingly, neurotransmission did not collapse completely in KOs as inputs still replenished their small RRP 80-fold upon 50 Hz | 60 s challenge. However, they totally failed to do so for extended periods. Upon acute pharmacological GlyT2 inactivation, synaptic performance remained robust, in stark contrast to KOs. RRP replenishment was 865-fold in marathon-experiments, only ~1/3 lower than in controls. Collectively, our empirical and modeling results demonstrate that GlyT2 re-uptake activity is not the dominant factor in the SV recycling pathway that imparts indefatigability to MNTB-LSO synapses. We postulate that additional glycine sources, possibly the antiporter Asc-1, contribute to RRP replenishment at these high-fidelity brainstem synapses.

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.

Cerebellar and vestibular nuclear synapses in the inferior olive have distinct release kinetics and neurotransmitters.

The inferior olive (IO) is composed of electrically-coupled neurons that make climbing fiber synapses onto Purkinje cells. Neurons in different IO subnuclei are inhibited by synapses with wide ranging release kinetics. Inhibition can be exclusively synchronous, asynchronous, or a mixture of both. Whether the same boutons, neurons or sources provide these kinetically distinct types of inhibition was not known. We find that in mice the deep cerebellar nuclei (DCN) and vestibular nuclei (VN) are two major sources of inhibition to the IO that are specialized to provide inhibitory input with distinct kinetics. DCN to IO synapses lack fast synaptotagmin isoforms, release neurotransmitter asynchronously, and are exclusively GABAergic. VN to IO synapses contain fast synaptotagmin isoforms, release neurotransmitter synchronously, and are mediated by combined GABAergic and glycinergic transmission. These findings indicate that VN and DCN inhibitory inputs to the IO are suited to control different aspects of IO activity.

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.

Modulation of mechanosensory vibrissal responses in the trigeminocervical complex by stimulation of the greater occipital nerve in a rat model of trigeminal neuropathic pain

BackgroundStimulation of the occipital or trigeminal nerves has been successfully used to treat chronic refractory neurovascular headaches such as migraine or cluster headache, and painful neuropathies. Convergence of trigeminal and occipital sensory afferents in the ‘trigeminocervical complex’ (TCC) from cutaneous, muscular, dural, and visceral sources is a key mechanism for the input-induced central sensitization that may underlie the altered nociception. Both excitatory (glutamatergic) and inhibitory (GABAergic and glycinergic) mechanisms are involved in modulating nociception in the spinal and medullary dorsal horn neurons, but the mechanisms by which nerve stimulation effects occur are unclear. This study was aimed at investigating the acute effects of electrical stimulation of the greater occipital nerve (GON) on the responses of neurons in the TCC to the mechanical stimulation of the vibrissal pad.MethodsAdult male Wistar rats were used. Neuronal recordings were obtained in laminae II-IV in the TCC in control, sham and infraorbital chronic constriction injury (CCI-IoN) animals. The GON was isolated and electrically stimulated. Responses to the stimulation of vibrissae by brief air pulses were analyzed before and after GON stimulation. In order to understand the role of the neurotransmitters involved, specific receptor blockers of NMDA (AP-5), GABAA (bicuculline, Bic) and Glycine (strychnine, Str) were applied locally.ResultsGON stimulation produced a facilitation of the response to light facial mechanical stimuli in controls, and an inhibition in CCI-IoN cases. AP-5 reduced responses to GON and vibrissal stimulation and blocked the facilitation of GON on vibrissal responses found in controls. The application of Bic or Str significantly reduced the facilitatory effect of GON stimulation on the response to vibrissal stimulation in controls. However, the opposite effect was found when GABAergic or Glycinergic transmission was prevented in CCI-IoN cases.ConclusionsGON stimulation modulates the responses of TCC neurons to light mechanical input from the face in opposite directions in controls and under CCI-IoN. This modulation is mediated by GABAergic and Glycinergic mechanisms. These results will help to elucidate the neural mechanisms underlying the effectiveness of nerve stimulation in controlling painful craniofacial disorders, and may be instrumental in identifying new therapeutic targets for their prevention and treatment.