Gephyrin is thought to play a critical role in clustering glycine receptors at synapses within the central nervous system (CNS). The main in vivo evidence for this comes from Gephyrin (Gphn)-null mice, where glycine receptors are depleted from synaptic regions. However, these mice die at birth, possibly due to impaired molybdenum cofactor (MoCo) synthesis, an essential role Gephyrin assumes throughout an animal. This complicates the interpretation of synaptic phenotypes in Gphn-null mice and raises the question whether the synaptic and enzymatic functions of Gephyrin can be investigated separately. Here, we generated a gephyrinb zebrafish mutant, vo84, that almost entirely lacks Gephyrin staining in the spinal cord. gephyrinbvo84 mutants exhibit normal gross morphology at both larval and adult stages. In contrast to Gphn-null mice, gephyrinbvo84 mutants exhibit normal motor activity and MoCo-dependent enzyme activity. Instead, gephyrinbvo84 mutants display impaired rheotaxis and increased mortality in late development. To investigate what may mediate these defects in gephyrinbvo84 mutants, we examined the cell density of neurons and myelin in the spinal cord and found no obvious changes. Surprisingly, in gephyrinbvo84 mutants, glycine receptors are still present in the synaptic regions. However, their abundance is reduced, potentially contributing to the observed defects. These findings challenge the notion that Gephyrin is absolutely required to cluster glycine receptors at synapses and reveals a new role of Gephyrin in regulating glycine receptor abundance and rheotaxis. They also establish a powerful new model for studying the mechanisms underlying synaptic, rather than enzymatic, functions of Gephyrin.

UNASSIGNED: Antiglycine receptor (anti-GlyR) antibody mediates multiple immune-related diseases. This study aimed to summarize the clinical features to enhance our understanding of anti-GlyR antibody-related disease.
UNASSIGNED: By collecting clinical information from admitted patients positive for glycine receptor (GlyR) antibody, the clinical characteristics of a new patient positive for GlyR antibody were reported in this study. To obtain additional information regarding anti-GlyR antibody-linked illness, clinical data and findings on both newly reported instances in this study and previously published cases were merged and analyzed.
UNASSIGNED: A new case of anti-GlyR antibody-related progressive encephalomyelitis with rigidity and myoclonus (PERM) was identified in this study. A 20-year-old man with only positive cerebrospinal fluid anti-GlyR antibody had a good prognosis with first-line immunotherapy. The literature review indicated that the common clinical manifestations of anti-GlyR antibody-related disease included PERM or stiff-person syndrome (SPS) (n = 179, 50.1%), epileptic seizure (n = 94, 26.3%), and other neurological disorders (n = 84, 24.5%). Other neurological issues included demyelination, inflammation, cerebellar ataxia and movement disorders, encephalitis, acute psychosis, cognitive impairment or dementia, celiac disease, Parkinson\'s disease, neuropathic pain and allodynia, steroid-responsive deafness, hemiballism/tics, laryngeal dystonia, and generalized weakness included respiratory muscles. The group of PERM/SPS exhibited a better response to immunotherapy than others.
UNASSIGNED: The findings suggest the presence of multiple clinical phenotypes in anti-GlyR antibody-related disease. Common clinical phenotypes include PERM, SPS, epileptic seizure, and paraneoplastic disease. Patients with RERM/SPS respond well to immunotherapy.

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It has been recently established that GPR158, a class C orphan G protein-coupled receptor, serves as a metabotropic glycine receptor. GPR158 is highly expressed in the nucleus accumbens (NAc), a major input structure of the basal ganglia that integrates information from cortical and subcortical structures to mediate goal-directed behaviors. However, whether glycine modulates neuronal activity in the NAc through GPR158 activation has not been investigated yet. Using whole-cell patch-clamp recordings, we found that glycine-dependent activation of GPR158 increased the firing rate of NAc medium spiny neurons (MSNs) while it failed to significantly affect the excitability of cholinergic interneurons (CIN). In MSNs GPR158 activation reduced the latency to fire, increased the action potential half-width, and reduced action potential afterhyperpolarization, effects that are all consistent with negative modulation of potassium M-currents, that in the central nervous system are mainly carried out by Kv7/KCNQ-channels. Indeed, we found that the GPR158-induced increase in MSN excitability was associated with decreased M-current amplitude, and selective pharmacological inhibition of the M-current mimicked and occluded the effects of GPR158 activation. In addition, when the protein kinase A (PKA) or extracellular signal-regulated kinase (ERK) signaling was pharmacologically blocked, modulation of MSN excitability by GPR158 activation was suppressed. Moreover, GPR158 activation increased the phosphorylation of ERK and Kv7.2 serine residues. Collectively, our findings suggest that GPR158/PKA/ERK signaling controls MSN excitability via Kv7.2 modulation. Glycine-dependent activation of GPR158 may significantly affect MSN firing in vivo, thus potentially mediating specific aspects of goal-induced behaviors.

The effects of intra-hippocampal manipulation of glycine receptors on the reconsolidation of recent and late long-term spatial memory were evaluated and assessed in the Morris water maze. The results obtained from the intra-hippocampal infusion of glycine and taurine demonstrated that taurine at a 100 nmol/side dose impaired the reconsolidation of recent and late long-term spatial memory. In comparison, at a dose of 10 nmol/side, it only affected the reconsolidation of late long-term spatial memory, reinforcing that there are differences between molecular mechanisms underlying recent and late long-term memory reconsolidation. On the other hand, glycine impaired the reconsolidation of early and late spatial memory when infused at a dose of 10 nmol/side, but not at a dose of 100 nmol/side, unless it is co-infused with an allosteric site antagonist of the NMDA receptor. Altogether these results show that glycine acting in situ in the hippocampal CA1 region exerts a pharmacological effect on U-curve, which can be explained by its concomitant action on its ionotropic receptor GlyR and on its NMDA receptor co-agonist site.

OBJECTIVE: To explore the clinical and genetic characteristics of three children with Hyperekplexia.
METHODS: Three children who were diagnosed with Hyperekplexia at the Third Affiliated Hospital of Zhengzhou University between June 2018 and March 2020 were selected as the study subjects. Clinical data of the three children were collected. All children were subjected to whole exome sequencing. Pathogenicity of candidate variants were verified by Sanger sequencing and bioinformatic analysis.
RESULTS: The three children were all males, and had presented exaggerated startle reflexes and generalized stiffness in response to unexpected auditory or tactile stimulation, or had frequent traumatic falls following exaggerated startle. All children had shown positive nose-tapping reflex, though EEG and cranial MRI exams were all negative. Whole exome sequencing revealed that two children had harbored homozygous variants of the GLRB gene, of which the c.1017_c.1018insAG (p.G340Rfs*14) was unreported previously. The third child had harbored compound heterozygous variants of the GLRA1 gene, among which the c.1262T>A (p.IIe421Asn) variant showed an unreported autosomal recessive inheritance. All children had responded well to clonazepam treatment.
CONCLUSIONS: Patients with Hyperekplexia have typical clinical manifestations. Early clinical identification and genetic analysis can facilitate their diagnosis.

Glycine receptors are pentameric ligand-gated ion channels that conduct chloride ions across postsynaptic membranes to facilitate fast inhibitory neurotransmission. In addition to gating by the glycine agonist, interactions with lipids and other compounds in the surrounding membrane environment modulate their function, but molecular details of these interactions remain unclear, in particular, for cholesterol. Here, we report coarse-grained simulations in a model neuronal membrane for three zebrafish glycine receptor structures representing apparent resting, open, and desensitized states. We then converted the systems to all-atom models to examine detailed lipid interactions. Cholesterol bound to the receptor at an outer-leaflet intersubunit site, with a preference for the open and desensitized versus resting states, indicating that it can bias receptor function. Finally, we used short atomistic simulations and iterative amino acid perturbations to identify residues that may mediate allosteric gating transitions. Frequent cholesterol contacts in atomistic simulations clustered with residues identified by perturbation analysis and overlapped with mutations influencing channel function and pathology. Cholesterol binding at this site was also observed in a recently reported pig heteromeric glycine receptor. These results indicate state-dependent lipid interactions relevant to allosteric transitions of glycine receptors, including specific amino acid contacts applicable to biophysical modeling and pharmaceutical design.

Alterations in cognitive and non-cognitive cerebral functions characterize Alzheimer\'s disease (AD). Cortical and hippocampal impairments related to extracellular accumulation of Aβ in AD animal models have been extensively investigated. However, recent reports have also implicated intracellular Aβ in limbic regions, such as the nucleus accumbens (nAc). Accumbal neurons express high levels of inhibitory glycine receptors (GlyRs) that are allosterically modulated by ethanol and have a role in controlling its intake. In the present study, we investigated how GlyRs in the 2xTg mice (AD model) affect nAc functions and ethanol intake behavior. Using transgenic and control aged-matched litter mates, we found that the GlyRα2 subunit was significantly decreased in AD mice (6-month-old). We also examined intracellular calcium dynamics using the fluorescent calcium protein reporter GCaMP in slice photometry. We also found that the calcium signal mediated by GlyRs, but not GABAAR, was also reduced in AD neurons. Additionally, ethanol potentiation was significantly decreased in accumbal neurons in the AD mice. Finally, we performed drinking in the dark (DID) experiments and found that 2xTg mice consumed less ethanol on the last day of DID, in agreement with a lower blood ethanol concentration. 2xTg mice also showed lower sucrose consumption, indicating that overall food reward was altered. In conclusion, the data support the role of GlyRs in nAc neuron excitability and a decreased glycinergic activity in the 2xTg mice that might lead to impairment in reward processing at an early stage of the disease.

OBJECTIVE: Glycine receptors (GlyRs) are potentiated by physiologically relevant concentrations of ethanol, and mutations in the intracellular loop of α1 and α2 subunits reduced the effect of the drug. Knock-in (KI) mice having these individual mutations revealed that α1 and α2 subunits played a role in ethanol-induced sedation and ethanol intake. In this study, we wanted to examine if the effects of stacking both mutations in a 2xKI mouse model (α1/α2) generated by a selective breeding strategy further impacted cellular and behavioral responses to ethanol.
METHODS: We used electrophysiological recordings to examine ethanol\'s effect on GlyRs and evaluated ethanol-induced neuronal activation using c-Fos immunoreactivity and the genetically encoded calcium indicator GCaMP6s in the nucleus accumbens (nAc). We also examined ethanol-induced behavior using open field, loss of the righting response, and drinking in the dark (DID) paradigm.
RESULTS: Ethanol did not potentiate GlyRs nor affect neuronal excitability in the nAc from 2xKI. Moreover, ethanol decreased the Ca2+ signal in WT mice, whereas there were no changes in the signal in 2xKI mice. Interestingly, there was an increase in c-Fos baseline in the 2xKI mice in the absence of ethanol. Behavioral assays showed that 2xKI mice recovered faster from a sedative dose of ethanol and had higher ethanol intake on the first test day of the DID test than WT mice. Interestingly, an open-field assay showed that 2xKI mice displayed less anxiety-like behavior than WT mice.
CONCLUSIONS: The results indicate that α1 and α2 subunits are biologically relevant targets for regulating sedative effects and ethanol consumption.

N-methyl-d-aspartate receptors (NMDARs) and other ionotropic glutamate receptors (iGluRs) mediate most of the excitatory signaling in the mammalian brains in response to the neurotransmitter glutamate. Uniquely, NMDARs composed of GluN1 and GluN3 are activated exclusively by glycine, the neurotransmitter conventionally mediating inhibitory signaling when it binds to pentameric glycine receptors. The GluN1-3 NMDARs are vital for regulating neuronal excitability, circuit function, and specific behaviors, yet our understanding of their functional mechanism at the molecular level has remained limited. Here, we present cryo-electron microscopy structures of GluN1-3A NMDARs bound to an antagonist, CNQX, and an agonist, glycine. The structures show a 1-3-1-3 subunit heterotetrameric arrangement and an unprecedented pattern of GluN3A subunit orientation shift between the glycine-bound and CNQX-bound structures. Site-directed disruption of the unique subunit interface in the glycine-bound structure mitigated desensitization. Our study provides a foundation for understanding the distinct structural dynamics of GluN3 that are linked to the unique function of GluN1-3 NMDARs.