Receptor Α1 Subunit Research Articles

Identification of intraneuronal amyloid beta oligomers in locus coeruleus neurons of Alzheimer's patients and their potential impact on inhibitory neurotransmitter receptors and neuronal excitability.

Amyloid β-oligomers (AβO) are potent modulators of Alzheimer's pathology, yet their impact on one of the earliest brain regions to exhibit signs of the condition, the locus coeruleus (LC), remains to be determined. Of particular importance is whether AβO impact the spontaneous excitability of LC neurons. This parameter determines brain-wide noradrenaline (NA) release, and thus NA-mediated brain functions, including cognition, emotion and immune function, which are all compromised in Alzheimer's patients. Therefore, the aim of the study was to determine the expression profile of AβO in the LC of Alzheimer's patients and to probe their potential impact on the molecular and functional correlates of LC excitability, using a mouse model of increased Aβ production (APP-PSEN1). Immunohistochemistry and confocal microscopy, using AβO-specific antibodies, confirmed LC AβO expression both intraneuronally and extracellularly in both Alzheimer's and APP-PSEN1 samples. Patch clamp electrophysiology recordings revealed that APP-PSEN1 LC neuronal hyperexcitability accompanied this AβO expression profile, arising from a diminished inhibitory effect of GABA due to impaired expression and function of the GABA-A receptor (GABAA R) α3 subunit. This altered LC α3-GABAA R expression profile overlapped with AβO expression in samples from both APP-PSEN1 mice and Alzheimer's patients. Finally, strychnine-sensitive glycine receptors (GlyRs) remained resilient to Aβ-induced changes and their activation reversed LC hyperexcitability. The data suggest a close association between AβO and α3-GABAA Rs in the LC of Alzheimer's patients, and their potential to dysregulate LC activity, thereby contributing to the spectrum of pathology of the LC-NA system in this condition.

Identification of the Aedes aegypti nAChR gene family and molecular target of spinosad.

Spinosad is an insecticide with unique mode of action (MOA) of disrupting nicotinic acetylcholine receptor and is efficacious against many insect species. Mutations in the nicotinic acetylcholine receptor (nAChR) α6 subunit have been identified that are associated with levels of spinosad resistance, but the molecular characterization of the nAChR gene family and a causative association between nAChR α6 and resistance to spinosad in Aedes aegypti, a primary vector of many arboviruses, have not yet been reported. In this study, we identified 10 candidate nAChR subunits in Ae. Aegypti, nAChRα1-α9 and nAChRβ1, showing similarly orthologous relationships with Anopheles gambiae. With the application of the CRISPR/Cas9 genome editing system, we introduced a 32-bp deletion at the 5' end of the Aaeα6 (Ae. aegypti nAChR α6) gene in a homozygous mutant strain (Aaeα6-KO). The mutation produced two successive pre-mature stop codons, resulting in loss of function in the target receptor. The Aaeα6-KO mutant strain exhibited a 320-fold level of resistance to spinosad compared with wildtype. A recessive mode of inheritance for spinosad resistance was found in the Aaeα6-KO strain. CRISPR/Cas9 introduced truncated Aaeα6 receptor in Ae. aegypti resulted in an increased level of resistance to spinosad, suggesting that the conserved nAChR α6 subunit is the target for spinosad insecticide. © 2020 Society of Chemical Industry.

Sex specific correlation between GABAergic disruption in the dorsal hippocampus and flurothyl seizure susceptibility after neonatal hypoxic-ischemic brain injury

Since neonatal hypoxia-ischemia (HI) disrupts the hippocampal (Hp) GABAergic network in the mouse and Hp injury in this model correlates with flurothyl seizure susceptibility only in male mice, we hypothesized that GABAergic disruption correlates with flurothyl seizure susceptibility in a sex-specific manner. C57BL6 mice were exposed to HI (Vannucci model) versus sham procedures at P10, randomized to normothermia (NT) or therapeutic hypothermia (TH), and subsequently underwent flurothyl seizure testing at P18. Only in male mice, Hp atrophy correlated with seizure susceptibility. The number of Hp parvalbumin positive interneurons (PV+INs) decreased after HI in both sexes, but TH attenuated this deficit only in females. In males only, seizure susceptibility directly correlated with the number of PV+INs, but not somatostatin or calretinin expressing INs. Hp GABAB receptor subunit levels were decreased after HI, but unrelated to later seizure susceptibility. In contrast, Hp GABAA receptor α1 subunit (GABAARα1) levels were increased after HI. Adjusting the number of PV+ INs for their GABAARα1 expression strengthened the correlation with seizure susceptibility in male mice. Thus, we identified a novel Hp sex-specific GABA-mediated mechanism of compensation after HI that correlates with flurothyl seizure susceptibility warranting further study to better understand potential clinical translation.

A Benzodiazepine Ligand with Improved GABAA Receptor α5-Subunit Selectivity Driven by Interactions with Loop C.

The family of GABAA receptors is an important drug target group in the treatment of sleep disorders, anxiety, epileptic seizures, and many others. The most frequent GABAA receptor subtype is composed of two α-, two β-, and one γ2-subunit, whereas the nature of the α-subunit critically determines the properties of the benzodiazepine binding site of those receptors. Nearly all of the clinically relevant drugs target all GABAA receptor subtypes equally. In the past years, however, drug development research has focused on studying α5-containing GABAA receptors. Beyond the central nervous system, α5-containing GABAA receptors in airway smooth muscles are considered as an emerging target for bronchial asthma. Here, we investigated a novel compound derived from the previously described imidazobenzodiazepine SH-053-2'F-R-CH3 (SH53d-ester). Although SH53d-ester is only moderately selective for α5-subunit-containing GABAA receptors, the derivative SH53d-acid shows superior (>40-fold) affinity selectivity and is a positive modulator. Using two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes and radioligand displacement assays with human embryonic kidney 293 cells, we demonstrated that an acid group as substituent on the imidazobenzodiazepine scaffold leads to large improvements of functional and binding selectivity for α5β3γ2 over other αxβ3γ2 GABAA receptors. Atom level structural studies provide hypotheses for the improved affinity to this receptor subtype. Mutational analysis confirmed the hypotheses, indicating that loop C of the GABAA receptor α-subunit is the dominant molecular determinant of drug selectivity. Thus, we characterize a promising novel α5-subunit-selective drug candidate. SIGNIFICANCE STATEMENT: In the current study we present the detailed pharmacological characterization of a novel compound derived from the previously described imidazobenzodiazepine SH-053-2'F-R-CH3. We describe its superior (>40-fold) affinity selectivity for α5-containing GABAA receptors and show atom-level structure predictions to provide hypotheses for the improved affinity to this receptor subtype. Mutational analysis confirmed the hypotheses, indicating that loop C of the GABAA receptor α-subunit is the dominant molecular determinant of drug selectivity.

Expression and Regulation of the GABAA Receptor/STEP61 Signaling Pathway in Cerebral Cortical Neurons Treated with Emulsified Isoflurane In Vitro.

Emulsified isoflurane (EISO) is an intravenous anesthetic. However, researchers have not clearly determined how emulsified isoflurane affects the central nervous system during the process of anesthesia. The aim of this study was to explore changes in the gamma-aminobutyric acid type A receptor subunit (GABAA), 61 kD isoform of striatal-enriched protein phosphatase (STEP61) signaling pathway, and epigenetic regulation in cortical neurons after treatment with emulsified isoflurane. After immunological identification, the isolated neurons were randomly divided into three groups: the blank group (Con), intralipid treatment group (FE), and emulsified isoflurane treatment group (EISO). Neuron viability was assayed using cell counting kit-8 (CCK-8). The expression levels of target nucleic acids, proteins, and corresponding ligands were detected. Using real-time polymerase chain reaction (PCR) to assess the promoter methylation of ion channel proteins in the cerebral cortex of rats anesthetized with EISO, we observed changes in promoter methylation of the genes encoding gamma-aminobutyric acid type A receptor α1 subunit (GABAAα1), N-methyl-d-aspartate receptor subunit 1 (NMDAR1), and mu opioid receptor 1 (OPRM1), accompanied by changes in the levels of their messenger ribonucleic acids (mRNAs) and proteins. The levels of ligands for these receptors were also altered. EISO altered the methylation rate of the promoter region of channel protein-coding genes involved in the GABAA/STEP61 signaling pathway in cerebral cortical neurons to regulate gene expression. The ligands for the receptors were also changed.

Effects of GABAA Receptor α3 Subunit Epilepsy Mutations on Inhibitory Synaptic Signaling.

Missense mutations T166M, Q242L, T336M, and Y474C in the GABAA receptor (GABAAR) α3 subunit gene are associated with epileptic seizures, dysmorphic features, intellectual disability, and developmental delay. When incorporated into GABAARs expressed in oocytes, all mutations are known to reduce GABA-evoked whole-cell currents. However, their impact on the properties of inhibitory synaptic currents (IPSCs) is unknown, largely because it is difficult to establish, much less control, the stoichiometry of GABAAR expressed in native neuronal synapses. To circumvent this problem, we employed a HEK293 cell-neuron co-culture expression system that permits the recording of IPSCs mediated by a pure population of GABAARs with a defined stoichiometry. We first demonstrated that IPSCs mediated by α3-containing GABAARs (α3β3γ2) decay significantly slower than those mediated by α1-containing isoforms (α1β2γ2 or α1β3γ2). GABAAR α3 mutations did not affect IPSC peak amplitudes or 10–90% rise times, but three of the mutations affected IPSC decay. T336M significantly accelerated the IPSC decay rate whereas T166M and Y474C had the opposite effect. The acceleration of IPSC decay kinetics caused by the T366M mutation was returned to wild-type-like values by the anti-epileptic medication, midazolam. Quantification experiments in HEK293 cells revealed a significant reduction in cell-surface expression for all mutants, in agreement with previous oocyte data. Taken together, our results show that impaired surface expression and altered IPSC decay rates could both be significant factors underlying the pathologies associated with these mutations.

Effects of adolescent intermittent ethanol on hippocampal expression of glutamate homeostasis and astrocyte‐neuronal tethering proteins in male and female rats

Adolescent alcohol drinking is widely recognized as a significant public health problem, and evidence is accumulating that sufficient levels of consumption during this critical period of brain development have an enduring impact on neural and behavioral function. Recent studies have indicated that adolescent intermittent ethanol (AIE) exposure alters astrocyte function, astrocyte-neuronal interactions, and related synaptic regulation and activity. However, few of those studies have included female animals, and a broader assessment of AIE effects on the proteins mediating astrocyte-mediated glutamate dynamics and synaptic function is needed. We measured synaptic membrane expression of several such proteins in the dorsal and ventral regions of the hippocampal formation (DH, VH) from male and female rats exposed to AIE or adolescent intermittent water. In the DH, AIE caused elevated expression of glutamate transporter 1 (GLT-1) in both males and females, elevated postsynaptic density 95 expression in females only, and diminished NMDA receptor subunit 2A expression in males only. AIE and sex interactively altered ephrin receptor A4 (EphA4) expression in the DH. In the VH, AIE elevated expression of the cystine/glutamate antiporter and the glutamate aspartate transporter 1 (GLAST) in males only. Compared to males, female animals expressed lower levels of GLT-1 in the DH and greater levels of ephrin receptor B6 (EphB6) in the VH, in the absence of AIE effects. These results support the growing literature indicating that adolescent alcohol exposure produces long-lasting effects on astrocyte function and astrocyte-neuronal interactions. The sex and subregion specificity of these effects have mechanistic implications for our understanding of AIE effects generally.

Interaction between GABAA receptor α1 and β2 subunits at the N-terminal peripheral regions is crucial for receptor binding and gating

Pentameric ligand gated ion channels (pLGICs) are crucial in electrochemical signaling but exact molecular mechanisms of their activation remain elusive. So far, major effort focused on the top-down molecular pathway between the ligand binding site and the channel gate. However, recent studies revealed that pLGIC activation is associated with coordinated subunit twisting in the membrane plane. This suggests a key role of intersubunit interactions but the underlying mechanisms remain largely unknown. Herein, we investigated a “peripheral” subunit interface region of GABAA receptor where structural modeling indicated interaction between N-terminal α1F14 and β2F31 residues. Our experiments underscored a crucial role of this interaction in ligand binding and gating, especially preactivation and opening, showing that the intersubunit cross-talk taking place outside (above) the top-down pathway can be strongly involved in receptor activation. Thus, described here intersubunit interaction appears to operate across a particularly long distance, affecting vast portions of the macromolecule.

Spontaneous motor-behavior abnormalities in two Drosophila models of neurodevelopmental disorders

Mutations in hundreds of genes cause neurodevelopmental disorders with abnormal motor behavior alongside cognitive deficits. Boys with fragile X syndrome (FXS), a leading monogenic cause of intellectual disability, often display repetitive behaviors, a core feature of autism. By direct observation and manual analysis, we characterized spontaneous-motor-behavior phenotypes of Drosophila dfmr1 mutants, an established model for FXS. We recorded individual 1-day-old adult flies, with mature nervous systems and prior to the onset of aging, in small arenas. We scored behavior using open-source video-annotation software to generate continuous activity timelines, which were represented graphically and quantitatively. Young dfmr1 mutants spent excessive time grooming, with increased bout number and duration; both were rescued by transgenic wild-type dfmr1+ . By two grooming-pattern measures, dfmr1-mutant flies showed elevated repetitions consistent with perseveration, which is common in FXS. In addition, the mutant flies display a preference for grooming posterior body structures, and an increased rate of grooming transitions from one site to another. We raise the possibility that courtship and circadian rhythm defects, previously reported for dfmr1 mutants, are complicated by excessive grooming. We also observed significantly increased grooming in CASK mutants, despite their dramatically decreased walking phenotype. The mutant flies, a model for human CASK-related neurodevelopmental disorders, displayed consistently elevated grooming indices throughout the assay, but transient locomotory activation immediately after placement in the arena. Based on published data identifying FMRP-target transcripts and functional analyses of mutations causing human genetic neurodevelopmental disorders, we propose the following proteins as candidate mediators of excessive repetitive behaviors in FXS: CaMKIIα, NMDA receptor subunits 2A and 2B, NLGN3, and SHANK3. Together, these fly-mutant phenotypes and mechanistic insights provide starting points for drug discovery to identify compounds that reduce dysfunctional repetitive behaviors.

Electroacupuncture Ameliorates CUMS-induced Depression-like Behavior: Involvement of the Glutamatergic System and Apoptosis in Rats.

Converging evidence indicates that the glutamatergic system and glia are directly implicated in the pathophysiology of depression. Clinical studies indicate that electroacupuncture (EA) has anti-depressant-like effects with low side effects for depression. However, the underlying antidepressant mechanism of acupuncture remains obscure. Chronic unpredictable mild stress (CUMS)-induced depressive rats were used to induce depressive-like behavior and evaluated by the weight change, open field test, sucrose preference test, and novelty suppressed feeding test. EA, NMDA receptor subunit 2A antagonist (NR2A RA) or NMDA receptor subunit 2B antagonist (NR2B RA) was used for comparison. Highperformance liquid chromatography (HPLC) was performed to detect the content of hippocampal glutamate, while western blot was performed for the hippocampal protein expression levels of calcium/calmodulin-dependent protein kinase II (CaMKII), Bax, caspase 3 and B-cell lymphoma-2 (Bcl-2). The distribution of glutamate ionotropic receptor NMDA type subunit 2A (NR2A), neuronal nuclear protein (NeuN), glutamate ionotropic receptor NMDA type subunit 2B (NR2B), and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence. Significant depression behavior (reduced body weight and sucrose preference, increased feeding and immobility time) was produced in CUMS-induced depressive rats, which was reversed significantly by EA. EA decreased hippocampal glutamate level. EA led to a significant decrease in expression levels of Bax, caspase 3, and CaMK II accompanied by increased Bcl-2 expression levels. Furthermore, EA significantly increased NR2A expression level as well as decreased NR2B expression level in the hippocampus. EA ameliorated depression-like behavior in CUMS rats, which might be mediated, at least in part, by regulating the glutamate, NMDA receptors, and apoptosis in the hippocampus.

The Neurotrophic Function of Glucagon-Like Peptide-1 Promotes Human Neuroblastoma Differentiation via the PI3K-AKT Axis.

Simple SummaryThe study demonstrated that the treatment with GLP-1 of SH-SY5Y human neuroblastoma cells increased the expression of AMPA receptors, NMDA receptors, dopamine receptors, synaptic proteins-synapsin 1, synaptophysin, and postsynaptic density protein 95, but not muscular and nicotinic acetylcholine receptors. In addition, the biomarker of dividing neuronal cells, vimentin, was decreased after treatment with GLP-1. Tuj1 immunostaining images showed that GLP-1 induced neurite processes and the development of neuronal morphologies. The GLP-1-differentiated neurons were able to be induced to generate action potentials by single cell patch-clamp. Our results also suggested that the PI3K-AKT axis is the dominant signaling pathway promoting the differentiation of SH-SY5Y cells into mature and functional neurons in response to GLP-1 receptor activation. The sequential treatment of retinoic acid and GLP-1 within a serum-free medium is able to trigger the differentiation of SH-SY5Y cells into morphologically and physiologically mature glutamatergic and dopaminergic neurons.Background: Neurons are terminally-differentiated cells that generally develop from neuronal stem cells stimulated by various neurotrophic factors such as NGF, BDNF, NT3, and NT-4. Neurotrophic factors have multiple functions for neurons, including enabling neuronal development, growth, and protection. Glucagon-like peptide-1 (GLP-1) is an intestinal-secreted incretin that enhances cellular glucose up-take to decrease blood sugar levels. However, many studies suggest that the function of GLP-1 is not limited to the regulation of blood sugar levels. Instead, it may also act as a neurotrophic factor with a role in ensuring neuronal survival and neurite outgrowth, as well as protecting synaptic plasticity and memory formation. Methods: The SH-SY5Y cells were differentiated by sequential treatments of retinoic acid and GLP-1 treatment within polyethylenimine-coated dishes under serum-free Neurobasal medium. PI3K inhibitor (LY294002) and MEK inhibitor (U0126) were used to determine the signaling pathway in regulation of neuronal differentiation. Neuronal marker (TUJ1) and synaptic markers (synapsin 1, synaptophysin, and PSD95) as well as single cell patch-clamp were applied to determine maturity of neurons. Antibodies of AMPA receptor, NMDA receptor subunit 2A, dopamine receptor D1, muscarinic acetylcholine receptor 2, and nicotinic acetylcholine receptor α4 were used to examine the types of differentiated neurons. Results: Our study’s results demonstrated that the treatment with GLP-1 of SH-SY5Y human neuroblastoma cells increased the expression of AMPA receptors, NMDA receptors, dopamine receptors, synaptic proteins-synapsin 1, synaptophysin, and postsynaptic density protein 95, but not muscular and nicotinic acetylcholine receptors. In addition, the biomarker of dividing neuronal cells, vimentin, was decreased after treatment with GLP-1. Tuj1 immunostaining images showed that GLP-1 induced neurite processes and the development of neuronal morphologies. The GLP-1-differentiated neurons were able to be induced to generate action potentials by single cell patch-clamp. Our study also suggested that the PI3K-AKT axis is the dominant signaling pathway promoting the differentiation of SH-SY5Y cells into mature and functional neurons in response to GLP-1 receptor activation. Conclusions: The sequential treatment of retinoic acid and GLP-1 within a serum-free medium is able to trigger the differentiation of SH-SY5Y cells into morphologically and physiologically mature glutamatergic and dopaminergic neurons.

Combination of Isoflurane and Propofol as General Anesthesia During Orthopedic Surgery of Perioperative Cerebral Hypoperfusion Rats to Avoid Cognitive Impairment.

Background: Perioperative cerebral hypoperfusion (CH) is common, although the underlying mechanism of cognitive impairment that results due to perioperative cerebral hypoperfusion remains to be determined. Isoflurane anesthesia induces neuronal injury via endoplasmic reticulum (ER) stress, whereas a sub-anesthetic dose of propofol improves postoperative cognitive function. However, the effects of the combination of isoflurane plus propofol, which is a common aesthetic combination administered to patients, on ER stress and cognition remain unknown.Methods: We sought to determine the effects of isoflurane plus propofol on ER stress and cognitive function in rats insulted by cerebral hypoperfusion. Ligation of the bilateral common carotid arteries (CCA) was adopted to develop the cerebral hypoperfusion rat model. A second surgery, open reduction and internal fixation (ORIF), requiring general anesthesia, was performed 30 days later so that the effects of anesthetics on the cognitive function of CH rats could be assessed. Rats received isoflurane alone (1.9%), propofol alone (40 mg·kg−1·h−1) or a combination of isoflurane and propofol (1% and 20 mg·kg−1·h−1 or 1.4% and 10 mg·kg−1·h−1). Behavioral studies (contextual fear conditioning [FC] test), histological analyses (Nissl staining) and biochemical analyses (western blotting of the harvested rat brain tissues) were employed.Results: Hippocampus-dependent memory of rats in group IP1 (1% isoflurane plus 20 mg·kg−1·h−1 propofol) was not impaired, and expression level of γ-aminobutyric acid A type receptor α1 subunit, a key cognition-related protein, remained normal. ER stress alleviator, binding immunoglobulin protein, increased extremely while ER stress transcription factor, C/EBP homologous protein, showed no statistical difference compared with the control group. Numbers of surviving neurons confirmed the substantial neuronal damage caused by propofol or isoflurane alone.Conclusions: These data suggest that ER stress contributes to the underlying mechanism of cognitive impairment and that the combination of isoflurane and propofol did not aggravate cognitive impairment and ER stress in aging rats with CH that were further subjected to ORIF surgery.

A proline-rich motif in the large intracellular loop of the glycine receptor α1 subunit interacts with the Pleckstrin homology domain of collybistin

IntroductionThe inhibitory glycine receptor (GlyR), a mediator of fast synaptic inhibition, is located and held at neuronal synapses through the anchoring proteins gephyrin and collybistin. Stable localization of neurotransmitter receptors is essential for synaptic function. In case of GlyRs, only beta subunits were known until now to mediate synaptic anchoring. ObjectivesWe identified a poly-proline II helix (PPII) in position 365–373 of the intra-cellular TM3-4 loop of the human GlyRα1 subunit as a novel potential synaptic anchoring site. The potential role of the PPII helix as synaptic anchoring site was tested. MethodsGlycine receptors and collybistin variants were generated and recombinantly expressed in HEK293 cells and cultured neurons. Receptor function was assessed using patch-clamp electrophysiology, protein-protein interaction was studied using co-immuno-precipitation and pulldown experiments. ResultsRecombinantly expressed collybistin bound to isolated GlyRα1 TM3-4 loops in GST-pulldown assays. When the five proline residues P365A, P366A, P367A, P369A, P373A (GlyRα1P1-5A) located in the GlyRα1-PPII helix were replaced by alanines, the PPII secondary structure was disrupted. Recombinant GlyRα1P1-5A mutant subunits displayed normal cell surface expression and wildtype-like ion channel function, but binding to collybistin was abolished. The GlyRα1-collybistin interaction was independently confirmed by o-immunoprecipitation assays using full-length GlyRα1 subunits. Surprisingly, the interaction was not mediated by the SH3 domain of collybistin, but by its Pleckstrin homology (PH) domain. The mutation GlyRα1P366L, identified in a hyperekplexia patient, is also disrupting the PPII helix, and caused reduced collybistin binding. ConclusionOur data suggest a novel interaction between α1 GlyR subunits and collybistin, which is physiologically relevant in vitro and in vivo and may contribute to postsynaptic anchoring of glycine receptors.

Low-Dose Nicotine Activates EGFR Signaling via α5-nAChR and Promotes Lung Adenocarcinoma Progression.

Nicotine in tobacco smoke is considered carcinogenic in several malignancies including lung cancer. The high incidence of lung adenocarcinoma (LAC) in non-smokers, however, remains unexplained. Although LAC has long been less associated with smoking behavior based on previous epidemiological correlation studies, the effect of environmental smoke contributing to low-dose nicotine exposure in non-smoking population could be underestimated. Here we provide experimental evidence of how low-dose nicotine promotes LAC growth in vitro and in vivo. Screening of nicotinic acetylcholine receptor subunits in lung cancer cell lines demonstrated a particularly high expression level of nicotinic acetylcholine receptor subunit α5 (α 5-nAChR) in LAC cell lines. Clinical specimen analysis revealed up-regulation of α 5-nAChR in LAC tumor tissues compared to non-tumor counterparts. In LAC cell lines α 5-nAChR interacts with epidermal growth factor receptor (EGFR), positively regulates EGFR pathway, enhances the expression of epithelial-mesenchymal transition markers, and is essential for low-dose nicotine-induced EGFR phosphorylation. Functionally, low-dose nicotine requires α 5-nAChR to enhance cell migration, invasion, and proliferation. Knockdown of α 5-nAChR inhibits the xenograft tumor growth of LAC. Clinical analysis indicated that high level of tumor α 5-nAChR is correlated with poor survival rates of LAC patients, particularly in those expressing wild-type EGFR. Our data identified α 5-nAChR as an essential mediator for low-dose nicotine-dependent LAC progression possibly through signaling crosstalk with EGFR, supporting the involvement of environmental smoke in tumor progression in LAC patients.

PSD-95 deficiency alters GABAergic inhibition in the prefrontal cortex

Postsynaptic Density Protein-95 (PSD-95) is a major scaffolding protein in the excitatory synapses in the brain and a critical regulator of synaptic maturation for NMDA and AMPA receptors. PSD-95 deficiency has been linked to cognitive and learning deficits implicated in neurodevelopmental disorders such as autism and schizophrenia. Previous studies have shown that PSD-95 deficiency causes a significant reduction in the excitatory response in the hippocampus. However, little is known about whether PSD-95 deficiency will affect gamma-aminobutyric acid (GABA)ergic inhibitory synapses. Using a PSD-95 transgenic mouse model (PSD-95+/−), we studied how PSD-95 deficiency affects GABAA receptor expression and function in the medial prefrontal cortex (mPFC) during adolescence. Our results showed a significant increase in the GABAA receptor subunit α1. Correspondingly, there are increases in the frequency and amplitude in spontaneous inhibitory postsynaptic currents (sIPSCs) in pyramidal neurons in the mPFC of PSD-95+/− mice, along with a significant increase in evoked IPSCs, leading to a dramatic shift in the excitatory-to-inhibitory balance in PSD-95 deficient mice. Furthermore, PSD-95 deficiency promotes inhibitory synapse function via upregulation and trafficking of NLGN2 and reduced GSK3β activity through tyr-216 phosphorylation. Our study provides novel insights on the effects of GABAergic transmission in the mPFC due to PSD-95 deficiency and its potential link with cognitive and learning deficits associated with neuropsychiatric disorders.

Enriched environment ameliorates memory impairments in rats after postsurgery sleep deprivation

Postsurgery sleep deprivation is a common complication that severely deteriorates the quality of life of patients. Here we aim to investigate the effects and mechanism of enriched environment in ameliorating sleep deprivation and memory impairments. Hernia repair surgery was performed on rats to induce sleep deprivation. Enriched environment (EE) was used to treat rats with sleep deprivation, and open field and Y-maze tests were performed to compare behavioral parameters of sleep deprivation rats with or without EE treatments to those of normal rats. To understand the mechanism, neurotrophic and growth factors including BDNF, NGF, NT-3 and GDNF were analyzed using enzyme-linked immunosorbent assay (ELISA). AMPAR subunits, including GluA1-A3, and GABAA receptor α1 subunit expression in hippocampus tissues were assessed using western blot. EE restored normal levels of anxiety index and freezing behavior in open field test and level of alternation in Y-maze test, suggesting the reduction of anxiolytic effects and spatial memory impairment induced by sleep deprivation. EE increased BDNF levels and reduced NT-3 levels in sleep deprivation rats. GluA1/GluA2 ratio was increased by EE. GABAA receptor α1 subunit expression was decreased by EE. EE is effective in ameliorating the detrimental effects of sleep deprivation in spatial memory impairment, and restoring normal levels of neurotrophic factors, which are potentially mediated by attenuating the changes in AMPAR subunit expression and reducing GABAA receptor α1 subunit expression. These data provide supporting evidences for the use of EE to treat adverse outcomes of sleep deprivation induced by surgery.

Ethanol consumption and sedation are altered in mice lacking the glycine receptor α2 subunit.

The precise mechanism/s of action of ethanol, although studied for many years, are not well understood. Like other drugs of abuse, ethanol affects dopamine levels in the nucleus accumbens (nAc), an important region of the mesolimbic system, causing a reinforcing effect. It has been shown that glycine receptors (GlyRs) present in the nAc are potentiated by clinically relevant concentrations of ethanol, where α1 and α2 are the predominant subunits expressed. Using a combination of electrophysiology and behavioural assays, we studied the involvement of GlyR α2 subunits on the effects of low and high doses of ethanol, as well as on consumption using mice lacking the GlyR α2 subunit (male Glra2-/Y and female Glra2-/- ). GlyR α2 subunits exist in accumbal neurons, since the glycine-evoked currents and glycinergic miniature inhibitory postsynaptic currents (mIPSCs) in Glra2-/Y mice were drastically decreased. In behavioural studies, differences in ethanol consumption and sedation were observed between wild-type (WT) and Glra2 knockout (KO) mice. Using the drinking in the dark (DID) paradigm, we found that Glra2-/Y mice presented a binge-like drinking behaviour immediately when exposed to ethanol rather than the gradual consumption seen in WT animals. Interestingly, the effect of knocking out Glra2 in female (Glra2-/- ) mice was less evident, since WT female mice already showed higher DID. The differences in ethanol consumption between WT and KO mice provide additional evidence supporting the conclusion that GlyRs are biologically relevant targets for the sedative and rewarding properties of ethanol.

Partial and full deletion of nicotinic acetylcholine receptor α4 and β2 subunits reduces sensitivity to acute nicotine administration and development of tolerance following chronic nicotine administration.

The diversity of nicotinic cholinergic receptor (nAChR) subunits underlies the complex responses to nicotine. Mice differing in the expression of α4 and β2 subunits, which are most widely expressed in brain, were evaluated for the responses to acute nicotine administration on Y-maze crossings and rears, open-field locomotion and body temperature following chronic treatment with nicotine (0, 0.25, 1.0 and 4.0 mg/kg/h). Deletion or partial deletion of the α4, β2 or both nAChR subunits reduced the sensitivity of mice to acute nicotine administration. This reduced sensitivity was gene dose-dependent. Modification of α4 subunit expression elicited a greater reduction in sensitivity than the modification of β2 subunit expression. No measurable tolerance was observed for mice of any genotype following chronic treatment with 0.25 mg/kg/h nicotine. Modest tolerance was noted following treatment with 1.0 mg/kg/h. Greater tolerance was observed following treatment with 4.0 mg/kg/h. The extent of tolerance differed among the mice depending on genotype: wild-type (α4 and β2) developed measurable tolerance for all four tests. Heterozygotes (α4, β2 and α4/β2) developed tolerance for only Y-maze crossings and body temperature. Null mutants (α4 and β2) did not become tolerant. However, following chronic treatment with 4.0 mg/kg/h nicotine, wild type, α4 and α4 mice displayed increased Y-maze crossings following acute administration of 0.5 mg/kg nicotine that may reflect the activity of α6β2*-nAChR. These results confirm the importance of the α4 and β2 nAChR subunits in mediating acute and chronic effects of nicotine on locomotion and body temperature in the mouse.

Targeting Nitric Oxide Production in Microglia with Novel Imidazodiazepines for Nonsedative Pain Treatment.

The goal of this research is the identification of new treatments for neuropathic pain. We characterized the GABAergic system of immortalized mouse and human microglia using electrophysiology and qRT-PCR. Cells from both species exhibited membrane current changes in response to γ-aminobutyric acid, with an EC50 of 260 and 1940 nM, respectively. Human microglia expressed high levels of the γ-aminobutyric acid type A receptor (GABAAR) α3 subunit, which can assemble with β1 and γ2/δ subunits to form functional GABAARs. Mouse microglia contained α2, α3, and α5, in addition to β1-3, γ1-2, and δ, mRNA, enabling a more diverse array of GABAARs than human microglia. Benzodiazepines are well-established modulators of GABAAR activity, prompting a screen of a library of diverse benzodiazepines in microglia for cellular effects. Several active compounds were identified by reduction of nitric oxide (NO) in interferon gamma and lipopolysaccharide activated microglia. However, further investigation with GABAAR antagonists flumazenil, picrotoxin, and bicuculline demonstrated that GABAARs were not linked to the NO response. A screen of 48 receptors identified the κ-opioid receptor and to a lesser extent the μ-opioid receptor as molecular targets, with opioid receptor antagonist norbinaltorphimine reversing benzodiazepine induced reduction of microglial NO. Functional assays identified the downregulation of inducible NO synthase as the mode of action of imidazodiazepines MP-IV-010 and GL-IV-03. Like other κ-opioid receptor agonists, GL-IV-03 reduced the agitation response in both phases of the formalin nociception test. However, unlike other κ-opioid receptor agonists, MP-IV-010 and GL-IV-03 did not impair sensorimotor coordination in mice. Thus, MP-IV-010 and GL-IV-03 represent a new class of nonsedative drug candidates for inflammatory pain.

GABAA Receptor/STEP61 Signaling Pathway May Be Involved in Emulsified Isoflurane Anesthesia in Rats.

(1) Background: Emulsified isoflurane (EISO) is a type of intravenous anesthetic. How emulsified isoflurane works in the brain is still unclear. The aim of this study was to explore whether epigenetic mechanisms affect anesthesia and to evaluate the anesthetic effects of emulsified isoflurane in rats. (2) Methods: Rats were randomly divided into four groups (n = 8/group): The tail vein was injected with normal saline 0.1 mL·kg−1·min−1 for the control (Con) group, with intralipid for the fat emulsion (FE) group, with EISO at 60 mg·kg−1·min−1 for the high-concentration (HD) group, and 45 mg·kg−1·min−1 for the low-concentration (LD) group. The consciousness state, motor function of limbs, and response to nociceptive stimulus were observed after drug administration. (3) Results: Using real-time polymerase chain reaction (PCR) to assess the promoter methylation of ion channel proteins in the cerebral cortex of rats anesthetized by EISO, we demonstrated that the change in the promoters’ methylation of the coding genes for gamma-aminobutyric acid A receptor α1 subunit (GABAAα1), N-methyl-D-aspartate receptor subunit 1 (NMDAR1), and mu opioid receptor 1 (OPRM1) was accompanied by the change in messenger ribonucleic acid (mRNA) and protein expression by these genes. (4) Conclusion: These data suggest that the epigenetic factors’ modulation might offer a novel approach to explore the anesthetic mechanism of EISO.