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Pharmacological stimulation/antagonism of astrocyte glio-peptide octadecaneuropeptide signaling alters ventromedial hypothalamic nucleus (VMN) counterregulatory γ-aminobutyric acid (GABA) and nitric oxide transmission. The current research used newly developed capillary zone electrophoresis-mass spectrometry methods to investigate hypoglycemia effects on VMN octadecaneuropeptide content, along with gene knockdown tools to determine if octadecaneuropeptide signaling regulates these transmitters during eu- and/or hypoglycemia. Hypoglycemia caused dissimilar adjustments in the octadecaneuropeptide precursor, i.e., diazepam-binding-inhibitor and octadecaneuropeptide levels in dorsomedial versus ventrolateral VMN. Intra-VMN diazepam-binding-inhibitor siRNA administration decreased baseline 67 and 65 kDa glutamate decarboxylase mRNA levels in GABAergic neurons laser-microdissected from each location, but only affected hypoglycemic transcript expression in ventrolateral VMN. This knockdown therapy imposed dissimilar effects on eu- and hypoglycemic glucokinase and 5’-AMP-activated protein kinase-alpha1 (AMPKα1) and -alpha2 (AMPKα2) gene profiles in dorsomedial versus ventrolateral GABAergic neurons. Diazepam-binding-inhibitor gene silencing up-regulated baseline (dorsomedial) or hypoglycemic (ventrolateral) nitrergic neuron neuronal nitric oxide synthase mRNA profiles. Baseline nitrergic cell glucokinase mRNA was up- (ventrolateral) or down- (dorsomedial) regulated by diazepam-binding-inhibitor siRNA, but knockdown enhanced hypoglycemic profiles in both sites. Nitrergic nerve cell AMPKα1 and -α2 transcripts exhibited division-specific responses to this genetic manipulation during eu- and hypoglycemia. Results document the utility of capillary zone electrophoresis-mass spectrometric tools for quantification of ODN in small-volume brain tissue samples. Data show that hypoglycemia has dissimilar effects on ODN signaling in the two major neuroanatomical divisions of the VMN and that this glio-peptide imposes differential control of glucose-regulatory neurotransmission in the VMNdm versus VMNvl during eu- and hypoglycemia. Graphical Abstract This is a visual representation of the abstract.

Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABA A ) receptors to dampen neuronal activity in the brain 1 – 5 . However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABA A receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABA A receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABA A receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain. Cryo-electron microscopy structures of GABA A receptors bound to intravenous anaesthetics and benzodiazepines reveal both common and distinct transmembrane binding sites, and show that the mechanisms of action of anaesthetics partially overlap with those of benzodiazepines.

A new target for analgesia The chronic pain that accompanies inflammatory disease and nerve injury is often resistant to conventional analgesics, hence the interest in establishing the mechanisms that induce this pain. Growing evidence points to a loss of synaptic inhibition in the spinal cord as a critical factor in chronic pain. This suggests that a rational therapy should aim at a reversal of this disinhibition. New work using 'knock-in' mice has identified specific GABA A receptor subtypes as keys to this spinal pain. The experimental drug L-838,417, which targets these receptor subtypes, is effective in rat models of inflammatory and neuropathic pain, yet was devoid of sedation, motor impairment and tolerance development expected with less specific GABA agonists. Chronic pain is an unwelcome consequence of inflammatory disease and nerve injury that currently has no effective treatment. This paper shows that specific pharmacological inhibition of α2 and α3 subunits of GABA A receptors is an effective therapy for inflammatory and neuropathic pain in rats. Inflammatory diseases and neuropathic insults are frequently accompanied by severe and debilitating pain, which can become chronic and often unresponsive to conventional analgesic treatment 1 , 2 . A loss of synaptic inhibition in the spinal dorsal horn is considered to contribute significantly to this pain pathology 3 , 4 , 5 , 6 , 7 . Facilitation of spinal γ-aminobutyric acid (GABA)ergic neurotransmission through modulation of GABA A receptors should be able to compensate for this loss 8 , 9 . With the use of GABA A -receptor point-mutated knock-in mice in which specific GABA A receptor subtypes have been selectively rendered insensitive to benzodiazepine-site ligands 10 , 11 , 12 , we show here that pronounced analgesia can be achieved by specifically targeting spinal GABA A receptors containing the α2 and/or α3 subunits. We show that their selective activation by the non-sedative (‘α1-sparing’) benzodiazepine-site ligand L-838,417 (ref. 13 ) is highly effective against inflammatory and neuropathic pain yet devoid of unwanted sedation, motor impairment and tolerance development. L-838,417 not only diminished the nociceptive input to the brain but also reduced the activity of brain areas related to the associative-emotional components of pain, as shown by functional magnetic resonance imaging in rats. These results provide a rational basis for the development of subtype-selective GABAergic drugs for the treatment of chronic pain, which is often refractory to classical analgesics.

Data are accumulating on the hydrolytic activity of serum albumin towards esters and organophosphates. Previously, with the help of the technology of proton nuclear magnetic resonance (1H NMR) spectroscopy, we observed the yield of acetate in the solution of bovine serum albumin and p-nitrophenyl acetate (NPA). Thus, we showed that albumin possesses true esterase activity towards NPA. Then, using the methods of molecular docking and molecular dynamics, we established site Sudlow I as the catalytic center of true esterase activity of albumin. In the present work, to expand our understanding of the molecular mechanisms of albumin pseudoesterase and true esterase activity, we investigated—in experiments in vitro and in silico—the interaction of anticoagulant warfarin (WRF, specific ligand of site Sudlow I) and benzodiazepine diazepam (DIA, specific ligand of site Sudlow II) with albumins of different species, and determined how the binding of WRF and DIA affects the hydrolysis of NPA by albumin. It was found that the characteristics of the binding modes of WRF in site Sudlow I and DIA in site Sudlow II of human (HSA), bovine (BSA), and rat (RSA) albumins have species differences, which are more pronounced for site Sudlow I compared to site Sudlow II, and less pronounced between HSA and RSA compared to BSA. WRF competitively inhibits true esterase activity of site Sudlow I towards NPA and does not affect the functioning of site Sudlow II. Diazepam can slow down true esterase activity of site Sudlow I in noncompetitive manner. It was concluded that site Sudlow I is more receptive to allosteric modulation compared to site Sudlow II.

Acyl coenzyme A binding protein (ACBP), also known as diazepam binding inhibitor (DBI) is a multifunctional protein with an intracellular action (as ACBP), as well as with an extracellular role (as DBI). The plasma levels of soluble ACBP/DBI are elevated in human obesity and reduced in anorexia nervosa. Accumulating evidence indicates that genetic or antibody-mediated neutralization of ACBP/DBI has anorexigenic effects, thus inhibiting food intake and inducing lipo-catabolic reactions in mice. A number of anorexiants have been withdrawn from clinical development because of their side effects including an increase in depression and suicide. For this reason, we investigated the psychiatric impact of ACBP/DBI in mouse models and patient cohorts. Intravenously (i.v.) injected ACBP/DBI protein conserved its orexigenic function when the protein was mutated to abolish acyl coenzyme A binding, but lost its appetite-stimulatory effect in mice bearing a mutation in the γ2 subunit of the γ-aminobutyric acid (GABA) A receptor (GABA A R). ACBP/DBI neutralization by intraperitoneal (i.p.) injection of a specific mAb blunted excessive food intake in starved and leptin-deficient mice, but not in ghrelin-treated animals. Neither i.v. nor i.p. injected anti-ACBP/DBI antibody affected the behavior of mice in the dark–light box and open-field test. In contrast, ACBP/DBI increased immobility in the forced swim test, while anti-ACBP/DBI antibody counteracted this sign of depression. In patients diagnosed with therapy-resistant bipolar disorder or schizophrenia, ACBP/DBI similarly correlated with body mass index (BMI), not with the psychiatric diagnosis. Patients with high levels of ACBP/DBI were at risk of dyslipidemia and this effect was independent from BMI, as indicated by multivariate analysis. In summary, it appears that ACBP/DBI neutralization has no negative impact on mood and that human depression is not associated with alterations in ACBP/DBI concentrations.

A wide variety of mechanisms have evolved for intercellular communication in metazoans, but some of the signaling molecules were already used in their predecessors. The social amoeba, Dictyostelium discoideum, is known to use peptides to trigger sporulation within fruiting bodies, but their sequences have not been defined. We found that the peptide signal spore differentiation factor 2 (SDF-2) is processed from acyl-CoA binding protein, AcbA. The mammalian homolog of AcbA is processed to diazepam binding inhibitor that binds to the GABA A receptor in the brain and to peripheral 1,4 benzodiazepine receptors. Although Dictyostelium has neither GABA A nor peripheral-type benzodiazepine receptors, we find that both a diazepam binding inhibitor peptide and diazepam (Valium) can mimic SDF-2 in a Dictyostelium bioassay. Mutants lacking AcbA sporulate well only when developed in chimeras with WT cells. Using a yeast system we show that ligand binding to the SDF-2 receptor histidine kinase, DhkA, inhibits phosphorelay, which can account for its ability to induce rapid sporulation.

α-Casozepine (α-CZP) is an anxiolytic-like bioactive decapeptide derived from bovine αs1-casein. The N-terminal peptide YLGYL was previously identified after proteolysis of the original peptide in an in vitro digestion model. Its putative anxiolytic-like properties were evaluated in a Swiss mice model using a light/dark box (LDB) after an intraperitoneal injection (0.5 mg/kg). The effect of YLGYL on c-Fos expression in brain regions linked to anxiety regulation was afterwards evaluated via immunofluorescence and compared to those of α-CZP and diazepam, a reference anxiolytic benzodiazepine. YLGYL elicited some anxiolytic-like properties in the LDB, similar to α-CZP and diazepam. The two peptides displayed some strong differences compared with diazepam in terms of c-Fos expression modulation in the prefontal cortex, the amygdala, the nucleus of the tractus solitarius, the periaqueductal grey, and the raphe magnus nucleus, implying a potentially different mode of action. Additionally, YLGYL modulated c-Fos expression in the amygdala and in one of the raphe nuclei, displaying a somewhat similar pattern of activation as α-CZP. Nevertheless, some differences were also spotted between the two peptides, making it possible to formulate the hypothesis that these peptides could act differently on anxiety regulation. Taken together, these results showed that YLGYL could contribute to the in vivo overall action of α-CZP.

Emerging wastewater treatment processes such as membrane bioreactors (MBRs) have attracted a significant amount of interest internationally due to their ability to produce high quality effluent suitable for water recycling. It is therefore important that their efficiency in removing hazardous trace organic contaminants be assessed. Accordingly, this study investigated the removal of trace organic chemical contaminants through a full-scale, package MBR in New South Wales, Australia. This study was unique in the context of MBR research because it characterised the removal of 48 trace organic chemical contaminants, which included steroidal hormones, xenoestrogens, pesticides, caffeine, pharmaceuticals and personal care products (PPCPs). Results showed that the removal of most trace organic chemical contaminants through the MBR was high (above 90%). However, amitriptyline, carbamazepine, diazepam, diclofenac, fluoxetine, gemfibrozil, omeprazole, sulphamethoxazole and trimethoprim were only partially removed through the MBR with the removal efficiencies of 24–68%. These are potential indicators for assessing MBR performance as these chemicals are usually sensitive to changes in the treatment systems. The trace organic chemical contaminants detected in the MBR permeate were 1 to 6 orders of magnitude lower than guideline values reported in the Australian Guidelines for Water Recycling. The outcomes of this study enhanced our understanding of the levels and removal of trace organic contaminants by MBRs.

Neuroinflammation is associated with the development of hypoactive delirium, which results in poor clinical outcomes. Drugs effective against hypoactive sur have not yet been established. Yokukansan has an anti-neuroinflammatory effect, making it potentially effective against hypoactive delirium. This study aimed to examine the effect of Yokukansan on the pentobarbital-induced loss of righting reflex duration extended with lipopolysaccharide (LPS)-induced neuroinflammation and diazepam-induced gamma-aminobutyric acid receptor stimulation in a mouse model. The active ingredients in Yokukansan and its anti-neuroinflammatory effect on the hippocampus were also investigated. Furthermore, we examined the in vitro anti-inflammatory effects of Yokukansan on LPS-stimulated BV2 cells, a murine microglial cell line. Findings revealed that treatment with Yokukansan significantly decreased the duration of pentobarbital-induced loss of righting reflex by attenuating the LPS-induced increase in interleukin-6 and tumor necrosis factor-alpha levels in the hippocampus. Moreover, treatment with Yokukansan significantly decreased the number of ionized calcium-binding adapter molecule-1-positive cells in the hippocampal dentate gyrus after 24 h of LPS administration. In addition, glycyrrhizic acid, an active ingredient in Yokukansan, partially decreased the duration of pentobarbital-induced loss of righting reflex. Treatment with Yokukansan also suppressed the expression of inducible nitric oxide, interleukin-6, and tumor necrosis factor mRNA in LPS-stimulated BV2 cells. Thus, these findings suggest that Yokukansan and glycyrrhizic acid may be effective therapeutic agents for treating neuroinflammation-induced hypoactive delirium. Graphical abstract

Benzodiazepines facilitate the inhibitory actions of GABA by binding to γ-aminobutyric acid type A receptors (GABAARs), GABA-gated chloride/bicarbonate channels, which are the key mediators of transmission at inhibitory synapses in the brain. This activity underpins potent anxiolytic, anticonvulsant and hypnotic effects of benzodiazepines in patients. However, extended benzodiazepine treatments lead to development of tolerance, a process which, despite its important therapeutic implications, remains poorly characterised. Here we report that prolonged exposure to diazepam, the most widely used benzodiazepine in clinic, leads to a gradual disruption of neuronal inhibitory GABAergic synapses. The loss of synapses and the preceding, time- and dose-dependent decrease in surface levels of GABAARs, mediated by dynamin-dependent internalisation, were blocked by Ro 15-1788, a competitive benzodiazepine antagonist, and bicuculline, a competitive GABA antagonist, indicating that prolonged enhancement of GABAAR activity by diazepam is integral to the underlying molecular mechanism. Characterisation of this mechanism has revealed a metabotropic-type signalling downstream of GABAARs, involving mobilisation of Ca2+ from the intracellular stores and activation of the Ca2+/calmodulin-dependent phosphatase calcineurin, which, in turn, dephosphorylates GABAARs and promotes their endocytosis, leading to disassembly of inhibitory synapses. Furthermore, functional coupling between GABAARs and Ca2+ stores was sensitive to phospholipase C (PLC) inhibition by U73122, and regulated by PLCδ, a PLC isoform found in direct association with GABAARs. Thus, a PLCδ/Ca2+/calcineurin signalling cascade converts the initial enhancement of GABAARs by benzodiazepines to a long-term downregulation of GABAergic synapses, this potentially underpinning the development of pharmacological and behavioural tolerance to these widely prescribed drugs.