Glycinergic Inhibition Research Articles

Principal Neurons in the Anteroventral Cochlear Nucleus Express Cell-Type Specific Glycine Receptor α Subunits

Signal processing in the principal neurons of the anteroventral cochlear nucleus (AVCN) is modulated by glycinergic inhibition. The kinetics of IPSCs are specific to the target neurons. It remains unclear what glycine receptor subunits are involved in generating such target-specific IPSC kinetics in AVCN principal neurons. We investigated the expression patterns of glycine receptor α (GlyRα) subunits in AVCN using immunohistochemical labeling of four isoforms of GlyRα subunits (GlyRα1–α4), and found that AVCN neurons express GlyRα1 and GlyRα4, but not GlyRα2 and GlyRα3 subunits. To further identify the cell type-specific expression patterns of GlyRα subunits, we combined whole-cell patch clamp recording with immunohistochemistry by recording from all three types of AVCN principal neurons, characterizing the synaptic properties of their glycinergic inhibition, dye-filling the neurons, and processing the slice for immunostaining of different GlyRα subunits. We found that AVCN bushy neurons express both GlyRα1 and GlyRα4 subunits that underlie their slow IPSC kinetics, whereas both T-stellate and D-stellate neurons express only GlyRα1 subunit that underlies their fast IPSC kinetics. In conclusion, AVCN principal neurons express cell-type specific GlyRα subunits that underlie their distinct IPSC kinetics, which enables glycinergic inhibition from the same source to exert target cell-specific modulation of activity to support the unique physiological function of these neurons.

D-Amphetamine Exposure Differentially Disrupts Signaling Across Ontogeny in the Zebrafish.

Background: Prescriptive and illicit amphetamine (AMPH) use continues to increase along with the likelihood that during an individual's lifetime, the drug deleteriously influences the growth and connectivity of behavior circuits necessary for survival. Throughout ontogeny, neural circuits underlying these behaviors grow in complexity, gradually integrating many sensory inputs that trigger higher order coordinated motor responses. In the present study, we examine how AMPH disrupts the establishment of these circuits at critical neurodevelopmental periods, as well as the communication among established survival circuits. Materials and Methods: Zebrafish embryos (from 1 hpf) were raised in AMPH solutions, growth parameters and escape behavior were assessed at 24 and 48 hpf, and spinal cord tissues analyzed for differences in excitatory-inhibitory signaling balance among treatments. Adult fish were fed an acute dosage of AMPH over an 11-day conditioned place preference (PP) paradigm during which behaviors were recorded and brain tissues analyzed for alterations in dopaminergic signaling. Results: AMPH negatively affects embryonic growth and slows the execution of escape behavior, suggesting an imbalance in locomotor signaling. Although local spinal circuits provide primary escape modulation, no differences in inhibitory glycinergic, and excitatory glutamatergic signaling were measured among spinal neurons. AMPH also influenced place preference in adult zebrafish and resulted in the increased expression of dopamine signaling proteins (DRD1) in brain areas governing survival behaviors.

Subtle differences in synaptic transmission in medial nucleus of trapezoid body neurons between wild-type and Fmr1 knockout mice

In animal models for fragile X syndrome where the gene for fragile X mental retardation protein is knocked out (Fmr1 KO), neurotransmission in multiple brain regions shifts excitation/inhibition balance, resulting in hyperexcitability in neural circuits. Here, using whole-cell recordings from brainstem slices, we investigated synaptic transmission at the medial nucleus of trapezoid body (MNTB, a critical nucleus in the brainstem sound localization circuit), in Fmr1 KO and wild-type (WT) mice 2–3 weeks of age in both sexes. Surprisingly, neither synaptic excitation nor inhibition in KO neurons was significantly changed. The synaptic strength, kinetics, and short-term plasticity of synaptic excitation remained largely unaltered. Subtle differences were observed in response patterns, with KO neurons displaying less all-or-none eEPSCs. Similarly, synaptic inhibition mediated by glycine and GABA remains largely unchanged, except for a slower kinetics of mixed sIPSCs. In pharmacologically isolated glycinergic and GABAergic inhibition, no significant differences in synaptic strength and kinetics were detected between the two genotypes. These results demonstrate that at the cellular level synaptic transmission at MNTB is largely unaffected in Fmr1 KO mice by 2–3 weeks after birth, suggesting the existence of compensatory mechanisms that maintain the inhibitory output of MNTB to its targets in the auditory brainstem.

Inhibitory Actions of Tropeines on the α3 Glycine Receptor Function.

Glycine receptors (GlyRs) are chloride-permeable pentameric ligand-gated ion channels. The inhibitory activity of GlyRs is essential for many physiological processes, such as motor control and respiration. In addition, several pathological states, such as hyperekplexia, epilepsy, and chronic pain, are associated with abnormal glycinergic inhibition. Recent studies have pointed out that positive allosteric modulators targeting the GlyR α3 subunit (α3GlyR) displayed beneficial effects in chronic pain models. Interestingly, previous electrophysiological studies have shown that tropeines, which are a family of synthetic antagonists of the serotonin type 3 receptors (5-HT3Rs), potentiate the activity of GlyRs conformed by α1 subunits. However, despite its importance as a pharmacological target in chronic pain, it is currently unknown whether the α3GlyR function is modulated by tropeines. Using electrophysiological techniques and molecular docking simulations, here we show that tropeines are inhibitors of the α3GlyR function. Tropisetron, a prototypical tropeine, exerted concentration-dependent inhibitory effects on α3GlyRs at the low micromolar range. In addition, three other tropeines showed similar effects. Single-channel recordings show that tropisetron inhibition is associated with a decrease in the open probability of the ion channel. Molecular docking assays suggest that tropeines preferentially bind to an agonist-free, closed state of the ion channel. The tropeine binding occurs in a discrete pocket around the vicinity of the orthosteric site within the extracellular domain of α3GlyR. Thus, our results describe the pharmacological modulation of tropeines on α3GlyRs. These findings may contribute to the development of GlyR-selective tropeine derivatives for basic and/or clinical applications.

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.

Modulation of glycinergic inhibition on respiratory rhythmic hypoglossal bursting in the rat.

The hypoglossal nerve displays respiratory rhythmic bursting and is composed of preinspiratory and inspiratory activity which is important in maintaining upper airway patency. The present study was designed to examine the modulatory role of glycinergic inhibition in respiratory rhythmic hypoglossal bursting. The activity of the phrenic nerve, as well as the medial and lateral branches of the hypoglossal nerve, was recorded simultaneously in urethane-anesthetized and mechanically ventilated adult rats in response to moderate and high levels of sustained lung inflation. The results demonstrated that inspiratory activity of the phrenic nerve gradually reduced with increasing lung inflation. The burst amplitude and discharge onset of the hypoglossal nerve branches were enhanced during moderate lung inflation but inhibited by high levels of lung inflation. These lung volume-mediated respiratory reflexes were abolished following a bilateral cervical vagotomy. In addition, intravenous administration of a glycine receptor antagonist (strychnine, 1 μmole/kg) attenuated preceding onset of rhythmic hypoglossal bursting but enhanced inspiratory hypoglossal burst amplitude during the baseline. Moreover, both excitatory and inhibitory effects of lung inflation on hypoglossal nerve activity were attenuated following a glycine transmission blockade. These results suggest that glycinergic inhibition modulated rhythmic hypoglossal bursting and was involved in mediating lung volume-induced respiratory reflexes.

Inhibitory mechanisms shaping delay-tuned combination-sensitivity in the auditory cortex and thalamus of the mustached bat

Delay-tuned auditory neurons of the mustached bat show facilitative responses to a combination of signal elements of a biosonar pulse-echo pair with a specific echo delay. The subcollicular nuclei produce latency-constant phasic on-responding neurons, and the inferior colliculus produces delay-tuned combination-sensitive neurons, designated “FM-FM” neurons. The combination-sensitivity is a facilitated response to the coincidence of the excitatory rebound following glycinergic inhibition to the pulse (1st harmonic) and the short-latency response to the echo (2nd–4th harmonics). The facilitative response of thalamic FM-FM neurons is mediated by glutamate receptors (NMDA and non-NMDA receptors). Different from collicular FM-FM neurons, thalamic ones respond more selectively to pulse-echo pairs than individual signal elements. A number of differences in response properties between collicular and thalamic or cortical FM-FM neurons have been reported. However, differences between thalamic and cortical FM-FM neurons have remained to be studied. Here, we report that GABAergic inhibition controls the duration of burst of spikes of facilitative responses of thalamic FM-FM neurons and sharpens the delay tuning of cortical ones. That is, intra-cortical inhibition sharpens the delay tuning of cortical FM-FM neurons that is potentially broad because of divergent/convergent thalamo-cortical projections. Compared with thalamic neurons, cortical ones tend to show sharper delay tuning, longer response duration, and larger facilitation index. However, those differences are statistically insignificant.

Glycinergic neurotransmission in the rostral ventrolateral medulla controls the time course of baroreflex-mediated sympathoinhibition.

To maintain appropriate blood flow to various tissues of the body under a variety of physiological states, autonomic nervous system reflexes regulate regional sympathetic nerve activity and arterial blood pressure. Our data obtained in anaesthetized rats revealed that glycine released in the rostral ventrolateral medulla (RVLM) plays a critical role in maintaining arterial baroreflex sympathoinhibition. Manipulation of brainstem nuclei with known inputs to the RVLM (nucleus tractus solitarius and caudal VLM) unmasked tonic glycinergic inhibition in the RVLM. Whole-cell, patch clamp recordings demonstrate that both GABA and glycine inhibit RVLM neurons. Potentiation of neurotransmitter release from the active synaptic inputs in the RVLM produced saturation of GABAergic inhibition and emergence of glycinergic inhibition. Our data suggest that GABA controls threshold excitability, wherreas glycine increases the strength of inhibition under conditions of increased synaptic activity within the RVLM. The arterial baroreflex is a rapid negative-feedback system that compensates changes in blood pressure by adjusting the output of presympathetic neurons in the rostral ventrolateral medulla (RVLM). GABAergic projections from the caudal VLM (CVLM) provide a primary inhibitory input to presympathetic RVLM neurons. Although glycine-dependent regulation of RVLM neurons has been proposed, its role in determining RVLM excitability is ill-defined. The present study aimed to determine the physiological role of glycinergic neurotransmission in baroreflex function, identify the mechanisms for glycine release, and evaluate co-inhibition of RVLM neurons by GABA and glycine. Microinjection of the glycine receptor antagonist strychnine (4mm, 100nL) into the RVLM decreased the duration of baroreflex-mediated inhibition of renal sympathetic nerve activity (control= 12± 1min; RVLM-strychnine= 5.1± 1min), suggesting that RVLM glycine plays a critical role in regulating the time course of sympathoinhibition. Blockade of output from the nucleus tractus solitarius and/or disinhibition of the CVLM unmasked tonic glycinergic inhibition of the RVLM. To evaluate cellular mechanisms, RVLM neurons were retrogradely labelled (prior injection of pseudorabies virus PRV-152) and whole-cell, patch clamp recordings were obtained in brainstem slices. Under steady-state conditions GABAergic inhibition of RVLM neurons predominated and glycine contributed less than 25% of the overall inhibition. By contrast, stimulation of synaptic inputs in the RVLM decreased GABAergic inhibition to 53%; and increased glycinergic inhibition to 47%. Thus, under conditions of increased synaptic activity in the RVLM, glycinergic inhibition is recruited to strengthen sympathoinhibition.

The postnatal development of ultrasonic vocalization-associated breathing is altered in glycine transporter 2-deficient mice.

Newborn mice produce ultrasonic vocalization to communicate with their mother. The neuronal glycine transporter (GlyT2) is required for efficient loading of synaptic vesicles in glycinergic neurons. Mice lacking GlyT2 develop a phenotype that resembles human hyperekplexia and the mice die in the second postnatal week. In the present study, we show that GlyT2-knockout mice do not acquire adult ultrasonic vocalization-associated breathing patterns. Despite the strong impairment of glycinergic inhibition, they can produce sufficient expiratory airflow to produce ultrasonic vocalization. Because mouse ultrasonic vocalization is a valuable read-out in translational research, these data are highly relevant for a broad range of research fields. Mouse models are instrumental with respect to determining the genetic basis and neural foundations of breathing regulation. To test the hypothesis that glycinergic synaptic inhibition is required for normal breathing and proper post-inspiratory activity, we analysed breathing and ultrasonic vocalization (USV) patterns in neonatal mice lacking the neuronal glycine transporter (GlyT2). GlyT2-knockout (KO) mice have a profound reduction of glycinergic synaptic currents already at birth, develop a severe motor phenotype and survive only until the second postnatal week. At this stage, GlyT2-KO mice are smaller, have a reduced respiratory rate and still display a neonatal breathing pattern with active expiration for the production of USV. By contrast, wild-type mice acquire different USV-associated breathing patterns that depend on post-inspiratory control of air flow. Nonetheless, USVs per se remain largely indistinguishable between both genotypes. We conclude that GlyT2-KO mice, despite the strong impairment of glycinergic inhibition, can produce sufficient expiratory airflow to produce ultrasonic vocalization.

Propofol-Induced Seizure

Approximately 30% of ambulatory anesthesia services for diagnostic or therapeutic procedures in the USA are performed under Monitored Anesthesia Care (MAC). Propofol is a widely used agent for MAC because of its rapid, short-acting sedative/hypnotic effects. This case report is about seizure like activity in the recovery period attributed to propofol noted in a lady who underwent MAC for endoscopic evaluation. A 39 year old female with history of hypertension, persistent GERD symptoms and melena presented for EGD/ Colonoscopy under MAC. In the anesthetic room, pre-oxygenation was performed and then induced with 400mg of propofol. She had an EGD and colonoscopy which she tolerated well. In the post recovery area, she was noted to have generalized jerky body movements, rolled her eyes to the back and became unconscious. Vital signs were stable. She was noted to be post ictal with gradual improvement in mental status. Electrolytes and blood glucose level were in normal ranges. Ct head performed did not show any acute event. Evaluation by neurology revealed a normal neurological exam. Neurology recommended an EEG and a follow up in the neuro clinic. Anti-epileptic drugs were not prescribed. Propofol is a widely used agent for anesthesia, sedation and as an anticonvulsant in status epilepticus. Given its short half-life even after prolonged infusions, it is widely used in MAC. MAC results in less physiologic disturbance and allows for more rapid recovery than general anesthesia.Propofol has been noted to induce abnormal neuromuscular events during induction, maintenance or emergence from propofol anesthesia and sedation. Unresponsiveness, myoclonus, opisthotonus, dystonia, ataxia and seizures have all been described. Several hypotheses have been put forward to explain the paradoxical response to propofol including: desensitization of inhibitory receptors leading to refractoriness in the inhibitory glycinergic and GABAergic pathways and imbalance between cholinergic and dopaminergic activity at the level of basal ganglia. Our patient had no previous history of seizures.There is no clear consensus regarding the prevention and identifying patients at risk for such adverse events. Seizure like phenomenon has been reported with propofol administration in both healthy and epileptic patients. Propofol, though a great agent for MAC is a known cause of seizure like phenomenon. More needs to be known about the mechanism of this complication and identifying patients at risk.

Ultrastructural basis of strong unitary inhibition in a binaural neuron.

Neurons of the lateral superior olive (LSO) in the brainstem receive powerful glycinergic inhibition that originates from the contralateral ear, and that plays an important role in sound localization. We investigated the ultrastructural basis for strong inhibition of LSO neurons using serial block face scanning electron microscopy. The soma and the proximal dendrite of an LSO neuron are surrounded by a high density of inhibitory axons, whereas excitatory axons are much sparser. A given inhibitory axon establishes contacts via several large axonal thickenings, called varicosities, which typically elaborate several active zones (range 1-11). The number of active zones across inhibitory axon segments is variable. These data thus provide an ultrastructural correlate for the strong and multiquantal, but overall variable, unitary IPSC amplitude observed for inhibitory inputs to LSO neuron. Binaural neurons in the lateral superior olive (LSO) integrate sound information arriving from each ear, and powerful glycinergic inhibition of these neurons plays an important role in this process. In the present study, we investigated the ultrastructural basis for strong inhibitory inputs onto LSO neurons using serial block face scanning electron microscopy. We reconstructed axon segments that make contact with the partially reconstructed soma and proximal dendrite of a mouse LSO neuron at postnatal day 18. Using functional measurements and the Sr2+ method, we find a constant quantal size but a variable quantal content between 'weak' and 'strong' unitary IPSCs. A 3-D reconstruction of a LSO neuron and its somatic synaptic afferents reveals how a large number of inhibitory axons intermingle in a complex fashion on the soma and proximal dendrite of an LSO neuron; a smaller number of excitatory axons was also observed. A given inhibitory axon typically contacts an LSO neuron via several large varicosities (average diameter 3.7μm), which contain several active zones (range 1-11). The number of active zones across individual axon segments was highly variable. These data suggest that the variable unitary IPSC amplitude is caused by a variable number of active zones between inhibitory axons that innervate a given LSO neuron. The results of the present study show that relatively large multi-active zone varicosities, which can be repeated many times in a given presynaptic axon, provide the ultrastructural basis for the strong multiquantal inhibition received by LSO neurons.

GlyT1 determines the glycinergic phenotype of amacrine cells in the mouse retina.

The amino acid glycine acts as a neurotransmitter at both inhibitory glycinergic and excitatory glutamatergic synapses predominantly in caudal regions of the central nervous system but also in frontal brain regions and the retina. After its presynaptic release and binding to postsynaptic receptors at caudal glycinergic synapses, two high-affinity glycine transporters GlyT1 and GlyT2 remove glycine from the extracellular space. Glycinergic neurons express GlyT2, which is essential for the presynaptic replenishment of the transmitter, while glial-expressed GlyT1 was shown to control the extracellular glycine concentration. Here we show that GlyT1 expressed by glycinergic amacrine cells of the retina does not only contribute to the control of the extracellular glycine concentration in the retina but is also essential for the maintenance of the glycinergic transmitter phenotype of this cell population. Specifically, loss of GlyT1 from the glycinergic AII amacrine cells impairs AII-mediated glycinergic neurotransmission and alters regulation of the extracellular glycine concentration, without changes in the overall distribution and/or size of glycinergic synapses. Taken together, our results suggest that GlyT1 expressed by amacrine cells in the retina combines functions covered by neuronal GlyT2 and glial GlyT1 at caudal glycinergic synapses.

Activity of novel lipid glycine transporter inhibitors on synaptic signalling in the dorsal horn of the spinal cord.

Inhibitory neurotransmission plays an important role in controlling excitability within nociceptive circuits of the spinal cord dorsal horn. Loss of inhibitory signalling is thought to contribute to the development of pathological pain. Preclinical studies suggest that increasing inhibitory glycinergic signalling is a good therapeutic strategy for treating pain. One approach to increase synaptic glycine is to inhibit the activity of the glycine transporter 2 (GlyT2) on inhibitory nerve terminals. These transporters are involved in regulating glycine concentrations and recycling glycine into presynaptic terminals. Inhibiting activity of GlyT2 increases synaptic glycine, which decreases excitability in nociceptive circuits and provides analgesia in neuropathic and inflammatory pain models. We investigated the effects of reversible and irreversible GlyT2 inhibitors on inhibitory glycinergic and NMDA receptor-mediated excitatory neurotransmission in the rat dorsal horn. The effect of these drugs on synaptic signalling was determined using patch-clamp electrophysiology techniques to measure glycine- and NMDA-mediated postsynaptic currents in spinal cord slices in vitro. We compared activity of four compounds that increase glycinergic tone with a corresponding increase in evoked glycinergic postsynaptic currents. These compounds did not deplete synaptic glycine release over time. Interestingly, none of these compounds increased glycine-mediated excitatory signalling through NMDA receptors. The results suggest that these compounds preferentially inhibit GlyT2 over G1yT1 with no potentiation of the glycine receptor and without inducing spillover from inhibitory to excitatory synapses. GlyT2 inhibitors increase inhibitory neurotransmission in the dorsal horn and have potential as pain therapeutics. This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

NOVEL ROLE OF GLYCINE IN CONTROL OF SYMPATHETIC OUTFLOW

The arterial baroreflex is one of the essential negative‐feedback mechanisms that compensates rapid changes in blood pressure (BP) through inhibition of presympathetic neurons in the rostral ventrolateral medulla (RVLM). There is general consensus that GABAergic projections originating from the caudal VLM (CVLM) provide the primary inhibitory input that controls the excitability of presympathetic RVLM neurons and sympathetic output. However, glycine, another inhibitory neurotransmitter, is also present in the RVLM. In anesthetized rats, we found that RVLM glycine played a critical role in arterial baroreflex function by controlling the time course of baroreflex mediated inhibition of renal sympathetic nerve activity. Blockade of glycine receptors in the RVLM, following an increase in BP, increased recovery of renal sympathetic nerve activity to baseline levels (5.1 ± 1.0 vs 11.7 ± 1.3 min, N=6, P<0.01) in comparison to control. In addition, tonic glycinergic inhibition of the RVLM was unmasked following blockade of output from the nucleus tractus solitaries and/or during disinhibition of the CVLM. Our whole‐cell, patch‐clamp recordings were obtained from brainstem slices containing presympathetic neurons in the RVLM labeled with PRV‐152. We confirmed that in steady state conditions GABAergic inhibition of RVLM neurons predominated and glycine contributed less than 25% of overall inhibition. In contrast, activation of the network produced saturation of GABAergic inhibition and glycinergic inhibition became prominent. Our findings demonstrated that blockade of glycine receptors in the RVLM decreased duration of baroreflex sympathoinhibition; increase of BP and activation of the network unmasked glycinergic inhibition of the RVLM; and release of glycine in the RVLM depended on the activity of presynaptic inhibitory inputs.Support or Funding InformationNational Institutes of Health R01 HL98602 (Cheryl M. Heesch) and HL122829 (Andrei V. Derbenev). Australian Research Council Future Fellowship FT170100363 and the National Health and Medical Research Council of Australia GNT 1079680 (Song T. Yao). High Blood Pressure Research Council of Australia, the Rebecca L Cooper Medical Foundation, and the Victorian Government through the Operational Infrastructure Scheme (Willian S. Korim).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Diverse inhibitory and excitatory mechanisms shape temporal tuning in transient OFF α ganglion cells in the rabbit retina.

Neurons combine excitatory and inhibitory signals to perform computations. In the retina, interactions between excitation and inhibition enable neurons to detect specific visual features. We describe how several excitatory and inhibitory mechanisms work together to allow transient OFF α ganglion cells in the rabbit retina to respond selectively to high temporal frequencies and thus detect faster image motion. The weightings of these different mechanisms change with the contrast and spatiotemporal properties of the visual input, and thereby support temporal tuning in α cells over a range of visual conditions. The results help us understand how ganglion cells selectively integrate excitatory and inhibitory signals to extract specific information from the visual input. The 20 to 30 types of ganglion cell in the mammalian retina represent parallel signalling pathways that convey different information to the brain. α ganglion cells are selective for high temporal frequencies in visual inputs, which makes them particularly sensitive to rapid motion. Although α ganglion cells have been studied in several species, the synaptic basis for their selective temporal tuning remains unclear. Here, we analyse excitatory synaptic inputs to transient OFF α ganglion cells (t-OFF α GCs) in the rabbit retina. We show that convergence of excitatory and inhibitory synaptic inputs within the bipolar cell terminals presynaptic to the t-OFF α GCs shifts the temporal tuning to higher temporal frequencies. GABAergic inhibition suppresses the excitatory input at low frequencies, but potentiates it at high frequencies. Crossover glycinergic inhibition and sodium channel activity in the presynaptic bipolar cells also potentiate high frequency excitatory inputs. We found differences in the spatial and temporal properties, and contrast sensitivities of these mechanisms. These differences in stimulus selectivity allow these mechanisms to generate bandpass temporal tuning of t-OFF α GCs over a range of visual conditions.

Heteromeric α/βglycine receptors regulate excitability in parvalbumin-expressing dorsal horn neurons through phasic and tonic glycinergic inhibition.

Key points Spinal parvalbumin‐expressing interneurons have been identified as a critical source of inhibition to regulate sensory thresholds by gating mechanical inputs in the dorsal horn.This study assessed the inhibitory regulation of the parvalbumin‐expressing interneurons, showing that synaptic and tonic glycinergic currents dominate, blocking neuronal or glial glycine transporters enhances tonic glycinergic currents, and these manipulations reduce excitability.Synaptically released glycine also enhanced tonic glycinergic currents and resulted in decreased parvalbumin‐expressing interneuron excitability.Analysis of the glycine receptor properties mediating inhibition of parvalbumin neurons, as well as single channel recordings, indicates that heteromeric α/β subunit‐containing receptors underlie both synaptic and tonic glycinergic currents.Our findings indicate that glycinergic inhibition provides critical control of excitability in parvalbumin‐expressing interneurons in the dorsal horn and represents a pharmacological target to manipulate spinal sensory processing. The dorsal horn (DH) of the spinal cord is an important site for modality‐specific processing of sensory information and is essential for contextually relevant sensory experience. Parvalbumin‐expressing inhibitory interneurons (PV+ INs) have functional properties and connectivity that enables them to segregate tactile and nociceptive information. Here we examine inhibitory drive to PV+ INs using targeted patch‐clamp recording in spinal cord slices from adult transgenic mice that express enhanced green fluorescent protein in PV+ INs. Analysis of inhibitory synaptic currents showed glycinergic transmission is the dominant form of phasic inhibition to PV+ INs. In addition, PV+ INs expressed robust glycine‐mediated tonic currents; however, we found no evidence for tonic GABAergic currents. Manipulation of extracellular glycine by blocking either, or both, the glial and neuronal glycine transporters markedly decreased PV+ IN excitability, as assessed by action potential discharge. This decreased excitability was replicated when tonic glycinergic currents were increased by electrically activating glycinergic synapses. Finally, we show that both phasic and tonic forms of glycinergic inhibition are mediated by heteromeric α/β glycine receptors. This differs from GABAA receptors in the dorsal horn, where different receptor stoichiometries underlie phasic and tonic inhibition. Together these data suggest both phasic and tonic glycinergic inhibition regulate the output of PV+ INs and contribute to the processing and segregation of tactile and nociceptive information. The shared stoichiometry for phasic and tonic glycine receptors suggests pharmacology is unlikely to be able to selectively target each form of inhibition in PV+ INs.

Tonotopic alterations in inhibitory input to the medial nucleus of the trapezoid body in a mouse model of Fragile X syndrome.

Hyperexcitability and the imbalance of excitation/inhibition are one of the leading causes of abnormal sensory processing in Fragile X syndrome (FXS). The precise timing and distribution of excitation and inhibition is crucial for auditory processing at the level of the auditory brainstem, which is responsible for sound localization ability. Sound localization is one of the sensory abilities disrupted by loss of the Fragile X Mental Retardation 1 (Fmr1) gene. Using triple immunofluorescence staining we tested whether there were alterations in the number and size of presynaptic structures for the three primary neurotransmitters (glutamate, glycine, and GABA) in the auditory brainstem of Fmr1 knockout mice. We found decreases in either glycinergic or GABAergic inhibition to the medial nucleus of the trapezoid body (MNTB) specific to the tonotopic location within the nucleus. MNTB is one of the primary inhibitory nuclei in the auditory brainstem and participates in the sound localization process with fast and well-timed inhibition. Thus, a decrease in inhibitory afferents to MNTB neurons should lead to greater inhibitory output to the projections from this nucleus. In contrast, we did not see any other significant alterations in balance of excitation/inhibition in any of the other auditory brainstem nuclei measured, suggesting that the alterations observed in the MNTB are both nucleus and frequency specific. We furthermore show that glycinergic inhibition may be an important contributor to imbalances in excitation and inhibition in FXS and that the auditory brainstem is a useful circuit for testing these imbalances.

Opposite, bidirectional shifts in excitation and inhibition in specific types of dorsal horn interneurons are associated with spasticity and pain post-SCI

Spasticity, a common complication after spinal cord injury (SCI), is frequently accompanied by chronic pain. The physiological origin of this pain (critical to its treatment) remains unknown, although spastic motor dysfunction has been related to the hyperexcitability of motoneurons and to changes in spinal sensory processing. Here we show that the pain mechanism involves changes in sensory circuits of the dorsal horn (DH) where nociceptive inputs integrate for pain processing. Spasticity is associated with the DH hyperexcitability resulting from an increase in excitation and disinhibition occurring in two respective types of sensory interneurons. In the tonic-firing inhibitory lamina II interneurons, glutamatergic drive was reduced while glycinergic inhibition was potentiated. In contrast, excitatory drive was boosted to the adapting-firing excitatory lamina II interneurons while GABAergic and glycinergic inhibition were reduced. Thus, increased activity of excitatory DH interneurons coupled with the reduced excitability of inhibitory DH interneurons post-SCI could provide a neurophysiological mechanism of central sensitization and chronic pain associated with spasticity.

Recreational concentrations of alcohol enhance synaptic inhibition of cerebellar unipolar brush cells via pre- and postsynaptic mechanisms.

Variation in cerebellar sensitivity to alcohol/ethanol (EtOH) is a heritable trait associated with alcohol use disorder in humans and high EtOH consumption in rodents, but the underlying mechanisms are poorly understood. A recently identified cellular substrate of cerebellar sensitivity to EtOH, the GABAergic system of cerebellar granule cells (GCs), shows divergent responses to EtOH paralleling EtOH consumption and motor impairment phenotype. Although GCs are the dominant afferent integrator in the cerebellum, such integration is shared by unipolar brush cells (UBCs) in vestibulocerebellar lobes. UBCs receive both GABAergic and glycinergic inhibition, both of which may mediate diverse neurological effects of EtOH. Therefore, the impact of recreational concentrations of EtOH (~10-50 mM) on GABAA receptor (GABAAR)- and glycine receptor (GlyR)-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) of UBCs in cerebellar slices was characterized. Sprague-Dawley rat (SDR) UBCs exhibited sIPSCs mediated by GABAARs, GlyRs, or both, and EtOH dose-dependently (10, 26, 52 mM) increased their frequency and amplitude. EtOH increased the frequency of glycinergic and GABAergic sIPSCs and selectively enhanced the amplitude of glycinergic sIPSCs. This GlyR-specific enhancement of sIPSC amplitude resulted from EtOH actions at presynaptic Golgi cells and via protein kinase C-dependent direct actions on postsynaptic GlyRs. The magnitude of EtOH-induced increases in UBC sIPSC activity varied across SDRs and two lines of mice, in parallel with their respective alcohol consumption/motor impairment phenotypes. These data indicate that Golgi cell-to-UBC inhibitory synapses are targets of EtOH, which acts at pre- and postsynaptic sites, via Golgi cell excitation and direct GlyR enhancement.NEW & NOTEWORTHY Genetic variability in cerebellar alcohol/ethanol sensitivity (ethanol-induced ataxia) predicts ethanol consumption phenotype in rodents and humans, but the cellular and molecular mechanisms underlying genetic differences are largely unknown. Here it is demonstrated that recreational concentrations of alcohol (10-30 mM) enhance glycinergic and GABAergic inhibition of unipolar brush cells through increases in glycine/GABA release and postsynaptic enhancement of glycine receptor-mediated responses. Ethanol effects varied across rodent genotypes parallel to ethanol consumption and motor sensitivity phenotype.

Organization of Circadian Behavior Relies on Glycinergic Transmission

The small ventral lateral neurons (sLNvs) constitute a central circadian pacemaker in the Drosophila brain. They organize daily locomotor activity, partly through the release of the neuropeptide pigment-dispersing factor (PDF), coordinating the action of the remaining clusters required for network synchronization. Despite extensive efforts, the basic principles underlying communication among circadian clusters remain obscure. We identified classical neurotransmitters released by sLNvs through disruption of specific transporters. Adult-specific RNAi-mediated downregulation of the glycine transporter or impairment of glycine synthesis in LNv neurons increased period length by nearly an hour without affecting rhythmicity of locomotor activity. Electrophysiological recordings showed that glycine reduces spiking frequency in circadian neurons. Interestingly, downregulation of glycine receptor subunits in specific sLNv targets impaired rhythmicity, revealing involvement of glycine in information processing within the network. These data identify glycinergic inhibition of specific targets as a cue that contributes to the synchronization of the circadian network.