Nursing students' perceived stress, self-efficacy, control and evaluation of a course in systematic clinical observation, physical assessment and decision-making: An observational study in Norway.

AIM: Simulation-based nursing education interventions have a strong educational effect on psychomotoric skills, but students may experience physiological stress and anxiety during simulation. The aims of this study were to explore (1) nursing students' perceived stress, self-efficacy, control and evaluation before and after simulation as part a structured course in physical assessment, (2) whether factors such as gender, age or previous work experience were associated with perceived stress during simulation and (3) nursing students evaluation of the course. DESIGN: An observational, cross-sectional study before and after simulation and a course in physical assessment. METHODS: We utilized "the Self-Assessment Manikin for measuring emotion" before and after simulation, a questionnaire to identify symptoms of stress after simulation, and a questionnaire to evaluate the physical assessment course. RESULTS: A total of 59 students participated. Students perceived stress before simulation but reported a lower degree of activation, a more positive mood, increased feeling of control and self-efficacy after the simulation. They also felt more secure about their assessments. Even though students reported of several symptoms of stress before simluation, the course increased students' self-reported competence and feeling of security.

Multivariate statistical and comparison analysis of chemical constituents in Arenaria kansuensis Maxim. from different regions in Qinghai-Tibet Plateau.

INTRODUCTION: Arenaria kansuensis Maxim. (AKM) is one of the most valued medicinal and edible herbs widely used in Qinghai-Tibet Plateau and there is also a large number of AKM bioactive constituents for health benefits of human beings. However, few works have referred to phytochemical content, fingerprint analysis and quality control of AKM. Therefore, the establishment of validated analytical methods is urgently needed for fingerprint comparison and quantitative analysis of AKM multicomponent. OBJECTIVES: To determine quantitatively and compare the phytochemical constituents of AKM located at different areas. METHODOLOGY: The chemical constituents in AKM samples were separated, identified, and quantified by high-performance liquid chromatography (HPLC) with a diode array detector. The discrimination and separation models for the chemical constituents were developed by chemometric analysis. RESULTS: The flavones and ß-carboline alkaloids were rich in AKM herbs, and the overall pattern of phytochemical profiles was the same, while the significant differences were detected in the total flavonoids, total ß-carboline alkaloids and individual contents, especially the predominant compounds such as tricin and arenarine B. This demonstrated that ecogeographical origin gave an important impact on phytochemical compositions which could be considered as reliable parameters for classifying the AKM resources. Moreover, the contents of AKM constituents were higher in July and/or August than other months of the year, and there were no significant differences in the main phytochemical contents between cultivated and wild AKM herbs. CONCLUSION: This study could provide credible data and method for geographical origin trace, comprehensive evaluation and further utilization of AKM resources.

GABAA receptors in GtoPdb v.2021.3.

The GABAA receptor is a ligand-gated ion channel of the Cys-loop family that includes the nicotinic acetylcholine, 5-HT3 and strychnine-sensitive glycine receptors. GABAA receptor-mediated inhibition within the CNS occurs by fast synaptic transmission, sustained tonic inhibition and temporally intermediate events that have been termed 'GABAA, slow' [45]. GABAA receptors exist as pentamers of 4TM subunits that form an intrinsic anion selective channel. Sequences of six α, three ß, three γ, one δ, three ρ, one ε, one π and one θ GABAA receptor subunits have been reported in mammals [278, 235, 236, 283]. The π-subunit is restricted to reproductive tissue. Alternatively spliced versions of many subunits exist (e.g. α4- and α6- (both not functional) α5-, ß2-, ß3- and γ2), along with RNA editing of the α3 subunit [71]. The three ρ-subunits, (ρ1-3) function as either homo- or hetero-oligomeric assemblies [359, 50]. Receptors formed from ρ-subunits, because of their distinctive pharmacology that includes insensitivity to bicuculline, benzodiazepines and barbiturates, have sometimes been termed GABAC receptors [359], but they are classified as GABA A receptors by NC-IUPHAR on the basis of structural and functional criteria [16, 235, 236]. Many GABAA receptor subtypes contain α-, ß- and γ-subunits with the likely stoichiometry 2α.2ß.1γ [168, 235]. It is thought that the majority of GABAA receptors harbour a single type of α- and ß - subunit variant. The α1ß2γ2 hetero-oligomer constitutes the largest population of GABAA receptors in the CNS, followed by the α2ß3γ2 and α3ß3γ2 isoforms. Receptors that incorporate the α4- α5-or α 6-subunit, or the ß1-, γ1-, γ3-, δ-, ε- and θ-subunits, are less numerous, but they may nonetheless serve important functions. For example, extrasynaptically located receptors that contain α6- and δ-subunits in cerebellar granule cells, or an α4- and δ-subunit in dentate gyrus granule cells and thalamic neurones, mediate a tonic current that is important for neuronal excitability in response to ambient concentrations of GABA [209, 272, 83, 19, 288]. GABA binding occurs at the ß+/α- subunit interface and the homologous γ+/α- subunits interface creates the benzodiazepine site. A second site for benzodiazepine binding has recently been postulated to occur at the α+/ß- interface ([254]; reviewed by [282]). The particular α-and γ-subunit isoforms exhibit marked effects on recognition and/or efficacy at the benzodiazepine site. Thus, receptors incorporating either α4- or α6-subunits are not recognised by 'classical' benzodiazepines, such as flunitrazepam (but see [356]). The trafficking, cell surface expression, internalisation and function of GABAA receptors and their subunits are discussed in detail in several recent reviews [52, 140, 188, 316] but one point worthy of note is that receptors incorporating the γ2 subunit (except when associated with α5) cluster at the postsynaptic membrane (but may distribute dynamically between synaptic and extrasynaptic locations), whereas as those incorporating the δ subunit appear to be exclusively extrasynaptic. NC-IUPHAR [16, 235, 3, 2] class the GABAA receptors according to their subunit structure, pharmacology and receptor function. Currently, eleven native GABAA receptors are classed as conclusively identified (i.e., α1ß2γ2, α1ßγ2, α3ßγ2, α4ßγ2, α4ß2δ, α4ß3δ, α5ßγ2, α6ßγ2, α6ß2δ, α6ß3δ and ρ) with further receptor isoforms occurring with high probability, or only tentatively [235, 236]. It is beyond the scope of this Guide to discuss the pharmacology of individual GABAA receptor isoforms in detail; such information can be gleaned in the reviews [16, 95, 168, 173, 143, 278, 216, 235, 236] and [9, 10]. Agents that discriminate between α-subunit isoforms are noted in the table and additional agents that demonstrate selectivity between receptor isoforms, for example via ß-subunit selectivity, are indicated in the text below. The distinctive agonist and antagonist pharmacology of ρ receptors is summarised in the table and additional aspects are reviewed in [359, 50, 145, 223]. Several high-resolution cryo-electron microscopy structures have been described in which the full-length human α1ß3γ2L GABAA receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA (γ-aminobutyric acid), and the classical benzodiazepines alprazolam and diazepam [198].

Flavonoid compounds isolated from Tibetan herbs, binding to GABAA receptor with anxiolytic property.

ETHNOPHARMACOLOGICAL RELEVANCE: Previously, the phytochemical constituents of Biebersteinia heterostemon Maxim (BHM) and Arenaria kansuensis Maxim (AKM) were studied and the evaluation of anxiolytic effect based on their extracts was also investigated. The two traditional Tibetan herbs, BHM and AKM, have been widely used in Qinghai-Tibet Plateau for cardiopulmonary disorders and neuropsychiatric diseases. The anxiolytic activities of a number of agents mediated by α2/3-containing GABAA receptors (GABAARs) have been demonstrated through the genetic and pharmacological studies. Flavonoids, such as flavones and flavanols, are a class of ligands that act at GABAARs and exhibit anxiolytic effects in vivo. Here, the flavonoids are the predominant constituents isolated from BHM and AKM. And our purpose is to investigate structure-activity relationships of the flavonoid compounds with binding to BZ-S of GABAAR complexes, and to search for anxiolytic constituents that lack undesirable-effects such as sedation and myorelaxation. MATERIALS AND METHODS: The flavonoid constituents were separated and purified through the repeatedly silica gel or/and C18 column chromatography. The affinities of the compounds for BZ-S of GABAARs were detected by the radioreceptor binding assay with bovine cerebellum membranes, in which the different recombinant subunits-containing GABAARs were expressed in HEK 293T cells. The behavior tests, including elevated plus maze, locomotor activity, holeboard, rotarod and horizontal wire, were used to determine and evaluate the anxiolytic, sedative, and myorelaxant effects of these flavonoids. RESULTS: Eleven total flavonoid compounds were obtained from the Tibetan herbs (BHM and AKM). The flavones with 6-and/or 8-OMe possessed the most potent binding affinity to GABAARs, which were based on the result of structure-activity relationships analysis. Demethoxysudachitin (DMS, Ki = 0.59 µM), a flavone that binds to recombinant α1-3/5 subunit-containing GABAARs, was isolated from BHM, and exhibited high anxiolytic activity, without inducing sedation and myorelaxation. Moreover, the anxiolytic effect of DMS was antagonized by flumazenil, suggesting that a mode of action was mediated via the BZ-S of GABAARs. CONCLUSIONS: This present study indicated that the flavones, especially DMS, are novel GABAAR ligands and therapeutic potential candidates for anxiety.

Modulation of Hippocampal GABAergic Neurotransmission and Gephyrin Levels by Dihydromyricetin Improves Anxiety.

Anxiety disorders are the most common mental illness in the U.S. and are estimated to consume one-third of the country's mental health spending. Although anxiolytic therapies are available, many patients exhibit treatment-resistance, relapse, or substantial side effects. An urgent need exists to explore the underlying mechanisms of chronic anxiety and to develop alternative therapies. Presently, we identified dihydromyricetin (DHM), a flavonoid that has anxiolytic properties in a mouse model of isolation-induced anxiety. Socially isolated mice demonstrated increased anxiety levels and reduced exploratory behavior measured by elevated plus-maze and open-field tests. Socially isolated mice showed impaired GABAergic neurotransmission, including reduction in GABAA receptor-mediated extrasynaptic tonic currents, as well as amplitude and frequency of miniature inhibitory postsynaptic currents measured by whole-cell patch-clamp recordings from hippocampal slices. Furthermore, intracellular ATP levels and gephyrin expression decreased in anxious animals. DHM treatment restored ATP and gephyrin expression, GABAergic transmission and synaptic function, as well as decreased anxiety-like behavior. Our findings indicate broader roles for DHM in anxiolysis, GABAergic neurotransmission, and synaptic function. Collectively, our data suggest that reduction in intracellular ATP and gephyrin contribute to the development of anxiety, and represent novel treatment targets. DHM is a potential candidate for pharmacotherapy for anxiety disorders.

Nurses' perspectives on technical skill requirements in primary and tertiary healthcare services.

Aim: To explore nurses' perspectives on technical skill requirements in primary and tertiary healthcare services and their perspectives on where students should learn these skills. Design: The study was conducted in Norway and had a cross-sectional, multicentre, descriptive design. Methods: We used a questionnaire to explore registered nurses and intellectual disability nurses' perspectives on technical skill requirements and learning (N = 437). Results: All the skills included in the university college curricula were reported to be required, and most skills should from the respondents' perspective be learned in the university college. There were significant differences between registered nurses and intellectual disability nurses about their perspectives on skill learning, but no significant differences between respondents in the interface between hospital and municipality wards. Conclusions: Results from this study indicate that nurses need extensive technical skills in both primary and tertiary health care. Findings also indicate a need to modify the university college curricula.

Development and validation of a questionnaire to assess healthcare personnel competence in cardiac arrest and resuscitation in pregnancy.

BACKGROUND: Cardiac arrest is rare in pregnancy, and up-to date competence can be difficult to assess and maintain. The objective of this study was to develop and validate a questionnaire to assess healthcare personnel experiences, self-assessed competence and perception of role and resposibility related to cardiac arrest and cardio-pulmonary resuscitation (CPR) in pregnancy. METHODS: The study had a cross-sectional design, developing and validating a questionnaire: the Competence in cardiac arrest and CPR in pregnancy (ComCA-P). Development and validation of the ComCA-P was conducted in three stages: 1) Literature review and expert group panel inputs, 2) a pilot study and 3) a cross-sectional questionnaire study. In stage one, the ComCA-P was developed over several iterations between the researchers, including inputs from an expert group panel consisting of highly competent professionals (n = 11). In stage two, the questionnaire was piloted in a group of healthcare personnel with relevant competence (n = 16). The ComCA-P was then used in a baseline study including healthcare personnel potentially involved in CPR in pregnancy (n = 527) in six hospital wards. Based on these data, internal consistency, intra-class correlations, and confirmatory factor analysis were utilized to validate the questionnaire. RESULTS: The expert group and pilot study participants evaluated the appropriateness, relevance and accuracy to be high. Formulation of the items was considered appropriate, with no difficulties identified related to content- or face validity. Cronbach's alpha was 0.8 on the thematic area self-assessment, and 0.73 on the theoretical knowledge area of the ComCA-P. On both the self-assessed competence items and the teoretical knowledge items, Kaiser-Meyer-Olkin was 0.8. Moreover, the Bertletts' test of sphericity was greater than the critical value for chi-square, and significant (p < .0001). CONCLUSIONS: Findings indicate that the ComCA-P is a valid questionnaire that can be used to assess healthcare personnel competence in cardiac arrest and resuscitation in pregnancy.

Correction to: Mechanisms underlying the EEG biomarker in Dup15q syndrome.

[This corrects the article DOI: 10.1186/s13229-019-0280-6.].

Mechanisms underlying the EEG biomarker in Dup15q syndrome.

Background: Duplications of 15q11.2-q13.1 (Dup15q syndrome), including the paternally imprinted gene UBE3A and three nonimprinted gamma-aminobutyric acid type-A (GABAA) receptor genes, are highly penetrant for neurodevelopmental disorders such as autism spectrum disorder (ASD). To guide targeted treatments of Dup15q syndrome and other forms of ASD, biomarkers are needed that reflect molecular mechanisms of pathology. We recently described a beta EEG phenotype of Dup15q syndrome, but it remains unknown which specific genes drive this phenotype. Methods: To test the hypothesis that UBE3A overexpression is not necessary for the beta EEG phenotype, we compared EEG from a reference cohort of children with Dup15q syndrome (n = 27) to (1) the pharmacological effects of the GABAA modulator midazolam (n = 12) on EEG from healthy adults, (2) EEG from typically developing (TD) children (n = 14), and (3) EEG from two children with duplications of paternal 15q (i.e., the UBE3A-silenced allele). Results: Peak beta power was significantly increased in the reference cohort relative to TD controls. Midazolam administration recapitulated the beta EEG phenotype in healthy adults with a similar peak frequency in central channels (f = 23.0 Hz) as Dup15q syndrome (f = 23.1 Hz). Both paternal Dup15q syndrome cases displayed beta power comparable to the reference cohort. Conclusions: Our results suggest a critical role for GABAergic transmission in the Dup15q syndrome beta EEG phenotype, which cannot be explained by UBE3A dysfunction alone. If this mechanism is confirmed, the phenotype may be used as a marker of GABAergic pathology in clinical trials for Dup15q syndrome.

Flavonoids isolated from Tibetan medicines, binding to GABAA receptor and the anticonvulsant activity.

BACKGROUND: Our previous studies on Asterothamnus centrali-asiaticus Novopokr. (ACN) and Arenaria kansuensis Maxim. (AKM) had led to the isolation of some phytochemical constituents and evaluation of anticonvulsant effect based on their extracts. ACN and AKM have been widely used in traditional Tibetan herbs for neuropsychiatric diseases and cardiopulmonary disorders. PURPOSE: The purpose is to investigate structure-activity relationships of flavonoids isolated from ACN and AKM, for binding to the benzodiazepine site (BZ-S) of γ-aminobutyric acid type A (GABAA) receptor complex, and to search for anticonvulsant compounds without undesirable effects such as myorelaxation and sedation. STUDY DESIGN AND METHODS: The affinities of these flavonoids for the BZ-S of GABAA receptors were determined by [3H]flunitrazepam binding to mouse cerebellum membranes in vitro. And the anticonvulsant, myorelaxant and sedative effects were determined by pentylenetetrazol (PTZ)-induced seizure and electrogenic seizure protection, rotarod test and locomotor activity test, respectively. RESULTS: Fifteen and thirteen flavonoids were isolated from ACN and AKM, respectively. Structure-activity relationships analysis indicated that 6-and/or 8-OMe flavones exhibited the most potent binding affinity to GABAA receptors. Furthermore, 2',4',5,7-tetrahydroxy-5',6-dimethoxyflavone (DMF, IC50 value of 0.10 µM), a flavone isolated from ACN, presented high anticonvulsant activity against chemical-induced seizures and electrogenic seizures, without myorelaxation and sedation. CONCLUSION: This study suggested that these flavones, especially DMF, are new BZ receptor ligands and prospective therapeutic candidates for seizures.

Homotaurine, a safe blood-brain barrier permeable GABAA-R-specific agonist, ameliorates disease in mouse models of multiple sclerosis.

There is a need for treatments that can safely promote regulatory lymphocyte responses. T cells express GABA receptors (GABAA-Rs) and GABA administration can inhibit Th1-mediated processes such as type 1 diabetes and rheumatoid arthritis in mouse models. Whether GABAA-R agonists can also inhibit Th17-driven processes such as experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS), is an open question. GABA does not pass through the blood-brain barrier (BBB) making it ill-suited to inhibit the spreading of autoreactivity within the CNS. Homotaurine is a BBB-permeable amino acid that antagonizes amyloid fibril formation and was found to be safe but ineffective in long-term Alzheimer's disease clinical trials. Homotaurine also acts as GABAA-R agonist with better pharmacokinetics than that of GABA. Working with both monophasic and relapsing-remitting mouse models of EAE, we show that oral administration of homotaurine can (1) enhance CD8+CD122+PD-1+ and CD4+Foxp3+ Treg, but not Breg, responses, (2) inhibit autoreactive Th17 and Th1 responses, and (3) effectively ameliorate ongoing disease. These observations demonstrate the potential of BBB-permeable GABAA-R agonists as a new class of treatment to enhance CD8+ and CD4+ Treg responses and limit Th17 and Th1-medaited inflammation in the CNS.

The Role of Hospital Design in Reducing Anxiety for Pediatric Patients.

OBJECTIVES: To study the impact of hospital design on patient and family experiences during and after hospitalization. BACKGROUND: Hospitalization can be psychologically traumatic for children. Few research studies have studied the role of the design of the hospital environment in mitigating that traumatic experience. METHODS: The study employs a two-group posttest and follow-up design to compare the impact of hospitalization on child anxiety and parent stress. It compares the experiences of children (ages 3-17) hospitalized at a new facility designed to support child-centered care and with family-friendly features with an older facility that did not have these features. The new facility was a replacement of the old one, so that many challenges to comparison are addressed. RESULTS: Controlling for the facts of hospitalization, patient demographics, and the child's typical anxiety level, children in the new facility experienced less anxiety than in the old facility. The study does not provide evidence that the hospital design reduced the psychological sequelae of hospitalization. Parents and children found different features of the hospital to be restorative. CONCLUSIONS: The study supports the use of Ulrich's theory of supportive design to children's healthcare environments, though what is experienced as supportive design will vary by the developmental stage of the child.

Single Ethanol Withdrawal Regulates Extrasynaptic δ-GABAA Receptors Via PKCδ Activation.

Alcohol (ethanol, EtOH) is one of the most widely abused drugs with profound effects on brain function and behavior. GABAA receptors (GABAARs) are one of the major targets for EtOH in the brain. Temporary plastic changes in GABAARs after withdrawal from a single EtOH exposure occur both in vivo and in vitro, which may be the basis for chronic EtOH addiction, tolerance and withdrawal symptoms. Extrasynaptic δ-GABAAR endocytosis is implicated in EtOH-induced GABAAR plasticity, but the mechanisms by which the relative abundance and localization of specific GABAARs are altered by EtOH exposure and withdrawal remain unclear. In this study, we investigated the mechanisms underlying rapid regulation of extrasynaptic δ-GABAAR by a single EtOH withdrawal in cultured rat hippocampal neurons. Thirty-minutes EtOH (60 mM) exposure increased extrasynaptic tonic current (Itonic) amplitude without affecting synaptic GABAAR function in neurons. In contrast, at 30 min after withdrawal, Itonic amplitude and responsiveness to acute EtOH were both reduced. Similar results occurred in neurons with okadaic acid (OA) or phorbol 12,13-dibutyrate (PDBu) exposure. Protein kinase C (PKC) inhibition prevented the reduction of Itonic amplitude and the tolerance to acute EtOH, as well as the reduction of GABAAR-δ subunit abundance induced by a single EtOH withdrawal. Moreover, EtOH withdrawal selectively increased PKCδ level, whereas PKCδ inhibition specifically rescued the EtOH-induced alterations in GABAAR-δ subunit level and δ-GABAAR function. Together, we provided strong evidence for the important roles of PKCδ in the rapid regulation of extrasynaptic δ-GABAAR induced by a single EtOH withdrawal.

GABAA receptor: Positive and negative allosteric modulators.

gamma-Aminobutyric acid (GABA)-mediated inhibitory neurotransmission and the gene products involved were discovered during the mid-twentieth century. Historically, myriad existing nervous system drugs act as positive and negative allosteric modulators of these proteins, making GABA a major component of modern neuropharmacology, and suggesting that many potential drugs will be found that share these targets. Although some of these drugs act on proteins involved in synthesis, degradation, and membrane transport of GABA, the GABA receptors Type A (GABAAR) and Type B (GABABR) are the targets of the great majority of GABAergic drugs. This discovery is due in no small part to Professor Norman Bowery. Whereas the topic of GABABR is appropriately emphasized in this special issue, Norman Bowery also made many insights into GABAAR pharmacology, the topic of this article. GABAAR are members of the ligand-gated ion channel receptor superfamily, a chloride channel family of a dozen or more heteropentameric subtypes containing 19 possible different subunits. These subtypes show different brain regional and subcellular localization, age-dependent expression, and potential for plastic changes with experience including drug exposure. Not only are GABAAR the targets of agonist depressants and antagonist convulsants, but most GABAAR drugs act at other (allosteric) binding sites on the GABAAR proteins. Some anxiolytic and sedative drugs, like benzodiazepine and related drugs, act on GABAAR subtype-dependent extracellular domain sites. General anesthetics including alcohols and neurosteroids act at GABAAR subunit-interface trans-membrane sites. Ethanol at high anesthetic doses acts on GABAAR subtype-dependent trans-membrane domain sites. Ethanol at low intoxicating doses acts at GABAAR subtype-dependent extracellular domain sites. Thus GABAAR subtypes possess pharmacologically specific receptor binding sites for a large group of different chemical classes of clinically important neuropharmacological agents. This article is part of the "Special Issue Dedicated to Norman G. Bowery".

On merging Acer sections Rubra and Hyptiocarpa: Molecular and morphological evidence.

In this study, we expanded Acer sect. Rubra Pax to include A. sect. Hyptiocarpa Fang. Traditionally, section Rubra comprises two iconic species, Acer rubrum Linnaeus (red maple) and A. saccharinum Linnaeus (silver maple), of eastern North American forests as well as the rare Japanese montane species, A. pycnanthum K. Koch. Section Hyptiocarpa consists of A. laurinum Hasskarl and A. pinnatinervium Merrill, which occur in subtropical and tropical regions of southwestern China to southeast Asia. Here, we confirm prior phylogenetic results showing the close relationship between sects. Rubra and Hyptiocarpa, and we use scanning electron microscopy to demonstrate that leaves of species within these sections have similar arrangements of cuticular waxes, which account for the silvery color of their abaxial surfaces. We describe that the sections also share labile sex expression; inflorescences that range from compound racemose thyrses, to racemes or umbels and that may have undergone evolutionary reduction; and several features of their fruits, such as seed locules without keels, basal portion of wings straight, acute attachment angle between mericarps, and production of some mericarps that are seedless and partially developed at maturity. Our expansion of sect. Rubra to include sect. Hyptiocarpa better elucidates the biogeographic and evolutionary history of these species. Additionally, we show that A. laurinum and A. pinnatinervium have intergrading morphology and are probably synonymous, but we note that further studies are required to conclude their taxonomic status.

Role of GABAA receptors in alcohol use disorders suggested by chronic intermittent ethanol (CIE) rodent model.

GABAergic inhibitory transmission is involved in the acute and chronic effects of ethanol on the brain and behavior. One-dose ethanol exposure induces transient plastic changes in GABAA receptor subunit levels, composition, and regional and subcellular localization. Rapid down-regulation of early responder δ subunit-containing GABAA receptor subtypes mediating ethanol-sensitive tonic inhibitory currents in critical neuronal circuits corresponds to rapid tolerance to ethanol's behavioral responses. Slightly slower, α1 subunit-containing GABAA receptor subtypes mediating ethanol-insensitive synaptic inhibition are down-regulated, corresponding to tolerance to additional ethanol behaviors plus cross-tolerance to other GABAergic drugs including benzodiazepines, anesthetics, and neurosteroids, especially sedative-hypnotic effects. Compensatory up-regulation of synaptically localized α4 and α2 subunit-containing GABAA receptor subtypes, mediating ethanol-sensitive synaptic inhibitory currents follow, but exhibit altered physio-pharmacology, seizure susceptibility, hyperexcitability, anxiety, and tolerance to GABAergic positive allosteric modulators, corresponding to heightened alcohol withdrawal syndrome. All these changes (behavioral, physiological, and biochemical) induced by ethanol administration are transient and return to normal in a few days. After chronic intermittent ethanol (CIE) treatment the same changes are observed but they become persistent after 30 or more doses, lasting for at least 120 days in the rat, and probably for life. We conclude that the ethanol-induced changes in GABAA receptors represent aberrant plasticity contributing critically to ethanol dependence and increased voluntary consumption. We suggest that the craving, drug-seeking, and increased consumption in the rat model are tied to ethanol-induced plastic changes in GABAA receptors, importantly the development of ethanol-sensitive synaptic GABAA receptor-mediating inhibitory currents that participate in maintained positive reward actions of ethanol on critical neuronal circuits. These probably disinhibit nerve endings of inhibitory GABAergic neurons on dopamine reward circuit cells, and limbic system circuits mediating anxiolysis in hippocampus and amygdala. We further suggest that the GABAA receptors contributing to alcohol dependence in the rat and presumably in human alcohol use disorders (AUD) are the ethanol-induced up-regulated subtypes containing α4 and most importantly α2 subunits. These mediate critical aspects of the positive reinforcement of ethanol in the dependent chronic user while alleviating heightened withdrawal symptoms experienced whenever ethanol is absent. The speculative conclusions based on firm observations are readily testable.

Decreased surface expression of the δ subunit of the GABAA receptor contributes to reduced tonic inhibition in dentate granule cells in a mouse model of fragile X syndrome.

While numerous changes in the GABA system have been identified in models of Fragile X Syndrome (FXS), alterations in subunits of the GABAA receptors (GABAARs) that mediate tonic inhibition are particularly intriguing. Considering the key role of tonic inhibition in controlling neuronal excitability, reduced tonic inhibition could contribute to FXS-associated disorders such as hyperactivity, hypersensitivity, and increased seizure susceptibility. The current study has focused on the expression and function of the δ subunit of the GABAAR, a major subunit involved in tonic inhibition, in granule cells of the dentate gyrus in the Fmr1 knockout (KO) mouse model of FXS. Electrophysiological studies of dentate granule cells revealed a marked, nearly four-fold, decrease in tonic inhibition in the Fmr1 KO mice, as well as reduced effects of two δ subunit-preferring pharmacological agents, THIP and DS2, supporting the suggestion that δ subunit-containing GABAARs are compromised in the Fmr1 KO mice. Immunohistochemistry demonstrated a small but statistically significant decrease in δ subunit labeling in the molecular layer of the dentate gyrus in Fmr1 KO mice compared to wildtype (WT) littermates. The discrepancy between the large deficits in GABA-mediated tonic inhibition in granule cells in the Fmr1 KO mice and only modest reductions in immunolabeling of the δ subunit led to studies of surface expression of the δ subunit. Cross-linking experiments followed by Western blot analysis demonstrated a small, non-significant decrease in total δ subunit protein in the hippocampus of Fmr1 KO mice, but a four-fold decrease in surface expression of the δ subunit in these mice. No significant changes were observed in total or surface expression of the α4 subunit protein, a major partner of the δ subunit in the forebrain. Postembedding immunogold labeling for the δ subunit demonstrated a large, three-fold, decrease in the number of symmetric synapses with immunolabeling at perisynaptic locations in Fmr1 KO mice. While α4 immunogold particles were also reduced at perisynaptic locations in the Fmr1 KO mice, the labeling was increased at synaptic sites. Together these findings suggest that, in the dentate gyrus, altered surface expression of the δ subunit, rather than a decrease in δ subunit expression alone, could be limiting δ subunit-mediated tonic inhibition in this model of FXS. Finding ways to increase surface expression of the δ subunit of the GABAAR could be a novel approach to treatment of hyperexcitability-related alterations in FXS.

α2 Subunit-Containing GABAA Receptor Subtypes Are Upregulated and Contribute to Alcohol-Induced Functional Plasticity in the Rat Hippocampus.

Alcohol (EtOH) intoxication causes changes in the rodent brain γ-aminobutyric acid receptor (GABAAR) subunit composition and function, playing a crucial role in EtOH withdrawal symptoms and dependence. Building evidence indicates that withdrawal from acute EtOH and chronic intermittent EtOH (CIE) results in decreased EtOH-enhanced GABAAR δ subunit-containing extrasynaptic and EtOH-insensitive α1ßγ2 subtype synaptic GABAARs but increased synaptic α4ßγ2 subtype, and increased EtOH sensitivity of GABAAR miniature postsynaptic currents (mIPSCs) correlated with EtOH dependence. Here we demonstrate that after acute EtOH intoxication and CIE, upregulation of hippocampal α4ßγ2 subtypes, as well as increased cell-surface levels of GABAAR α2 and γ1 subunits, along with increased α2ß1γ1 GABAAR pentamers in hippocampal slices using cell-surface cross-linking, followed by Western blot and coimmunoprecipitation. One-dose and two-dose acute EtOH treatments produced temporal plastic changes in EtOH-induced anxiolysis or withdrawal anxiety, and the presence or absence of EtOH-sensitive synaptic currents correlated with cell surface peptide levels of both α4 and γ1(new α2) subunits. CIE increased the abundance of novel mIPSC patterns differing in activation/deactivation kinetics, charge transfer, and sensitivity to EtOH. The different mIPSC patterns in CIE could be correlated with upregulated highly EtOH-sensitive α2ßγ subtypes and EtOH-sensitive α4ßγ2 subtypes. Naïve α4 subunit knockout mice express EtOH-sensitive mIPSCs in hippocampal slices, correlating with upregulated GABAAR α2 (and not α4) subunits. Consistent with α2, ß1, and γ1 subunits genetically linked to alcoholism in humans, our findings indicate that these new α2-containing synaptic GABAARs could mediate the maintained anxiolytic response to EtOH in dependent individuals, rat or human, contributing to elevated EtOH consumption.

Use of Mycelium and Detached Leaves in Bioassays for Assessing Resistance to Boxwood Blight.

Boxwood blight caused by Calonectria pseudonaviculata is a newly emergent disease of boxwood (Buxus spp. L.) in the United States that causes leaf drop, stem lesions, and plant death. A rapid and reliable laboratory assay that enables screening hundreds of boxwood genotypes for resistance to boxwood blight is needed to enable breeding and selection of resistant cultivars. Using eight boxwood cultivars with differing susceptibilities, we examined parameters for a screening assay comparing whole plant inoculation with detached leaf inoculation, use of mycelium versus spores as the inoculum, comparison of times of the year for inoculation, and comparison of two leaf inoculation methods. Inoculation of detached leaves gave comparable results to inoculation of whole plants when compared across genotypes, although the detached leaf assay resulted in greater percentages of symptom expression. The time of year of plant inoculation (spring, summer, or winter) did not affect the relative expression of symptoms among the most resistant and susceptible genotypes. Inoculating plants with mycelium was as effective as spore inoculation for causing disease symptoms and allowed us to distinguish the more resistant genotypes, yet mycelium inoculation was much easier to prepare in large quantities for multiple assays.