British Association for Psychopharmacology consensus statement on evidence-based treatment of insomnia, parasomnias and circadian rhythm disorders.

Sleep disorders are common in the general population and even more so in clinical practice, yet are relatively poorly understood by doctors and other health care practitioners. These British Association for Psychopharmacology guidelines are designed to address this problem by providing an accessible up-to-date and evidence-based outline of the major issues, especially those relating to reliable diagnosis and appropriate treatment. A consensus meeting was held in London in May 2009. Those invited to attend included BAP members, representative clinicians with a strong interest in sleep disorders and recognized experts and advocates in the field, including a representative from mainland Europe and the USA. Presenters were asked to provide a review of the literature and identification of the standard of evidence in their area, with an emphasis on meta-analyses, systematic reviews and randomized controlled trials where available, plus updates on current clinical practice. Each presentation was followed by discussion, aimed to reach consensus where the evidence and/or clinical experience was considered adequate or otherwise to flag the area as a direction for future research. A draft of the proceedings was then circulated to all participants for comment. Key subsequent publications were added by the writer and speakers at draft stage. All comments were incorporated as far as possible in the final document, which represents the views of all participants although the authors take final responsibility for the document.

Modulation of TRPC5 cation channels by halothane, chloroform and propofol.

BACKGROUND AND PURPOSE: TRPC5 is a mammalian homologue of the Drosophila Transient Receptor Potential (TRP) channel and has expression and functions in the cardiovascular and nervous systems. It forms a calcium-permeable cation channel that can be activated by a variety of signals including carbachol (acting at muscarinic receptors), lanthanides (e.g. Gd3+) and phospholipids (e.g. lysophosphatidylcholine: LPC). Here we report the effects of inhalational (halothane and chloroform) and intravenous (propofol) general anaesthetics upon TRPC5. EXPERIMENTAL APPROACH: Human TRPC5 channels were expressed in HEK 293 cells and studied using fura-2 and patch-clamp recording to measure intracellular calcium and membrane currents respectively at room temperature. Human TRPM2 channels were studied for comparison. KEY RESULTS: TRPC5 activation by carbachol, Gd3+ or LPC was inhibited by halothane and chloroform at > or =0.1 and 0.2 mM respectively. Neither agent inhibited TRPM2. Propofol had an initial stimulatory effect on TRPC5 (evident in patch-clamp recordings only) and an inhibitory effect at > or =10 microM. TRPM2 was not affected by propofol. Propofol inhibited activation of TRPC5 by Gd3+ but not LPC, suggesting the effect was not directly on the channel. Propofol's anti-oxidant property was not necessary for its inhibitory effect because di-isopropyl benzene, a propofol analogue that lacks the hydroxyl group, also inhibited TRPC5. CONCLUSIONS AND IMPLICATIONS: The data show the sensitivity of TRPC5 channel to general anaesthetics and suggest that some of the effects could have clinical relevance. The effects may be explained in part by the sensitivity of the channel to biophysical properties of the lipid bilayer.

Basic pharmacologic mechanisms involved in benzodiazepine tolerance and withdrawal.

Benzodiazepines are widely prescribed for the treatment of anxiety and sleep disorders. Although safe, tolerance develops rapidly to their sedative activity and more slowly to their anticonvulsant activity. In animals anxiolytic tolerance has also been measured. Abrupt cessation of benzodiazepine treatment leads to symptoms of withdrawal. The mechanisms responsible for these phenomena are not known. Benzodiazepines act via GABA(A) receptors, but do not appear to produce tolerance and dependence by simple downregulation of receptor number. GABA(A) receptors are hetero-oligomers comprised of multiple subunits encoded by a multigene family. The molecular effects of long-term benzodiazepine exposure are reviewed and a model is presented that draws on results from a number of research groups working in this area.

Common effects of chronically administered antipanic drugs on brainstem GABA(A) receptor subunit gene expression.

Panic disorder is an anxiety disorder that can be treated by long-term administration of tricyclic antidepressants such as imipramine, monoamine oxidase inhibitors such as phenelzine, or the selective serotonin reuptake inhibitor (SSRI) antidepressants. Clinical data also indicate that some benzodiazepines, such as alprazolam, are effective antipanic agents, and that their therapeutic onset is faster than that of antidepressants. Benzodiazepines are well known for their action at GABA(A) receptors, and preclinical data indicate that imipramine and phenelzine also interfere with the GABAergic system. In addition some clinical data lend support to decreased benzodiazepine-sensitive receptor function in panic disorder patients. Using imipramine, phenelzine and alprazolam, we investigated, in rats, the possibility that the therapeutic efficacy of antipanic agents stems from the remodeling of GABAergic transmission in the pons-medulla region. Of the 12 GABA(A) receptor subunit (alpha 1--6, beta 1--3, gamma 1--3) steady-state mRNA levels investigated, we observed an increase in the levels of the alpha 3-, beta 1- and gamma 2-subunit transcripts with all three antipanic agents tested. The effects of imipramine and phenelzine on these subunits occurred after 21 days of treatment, while alprazolam effects were observed after 3 days of administration. Histochemical data suggest that the alpha 3 beta 1 gamma 2 subunits comprise a receptor subtype in the pons-medulla region. Therefore, we conclude that these molecular events parallel the therapeutic profile of the drugs examined. We further propose that these events may correspond to a remodeling of the GABA(A) receptor population, and may be useful markers for investigation of the antipanic properties of drugs.

GABA(A) receptor gene expression in rat cortex: differential effects of two chronic diazepam treatment regimes.

Diazepam is widely prescribed as an anxiolytic but its therapeutic application is limited because with daily use tolerance develops to certain aspects of its pharmacological profile. We compared the effects of two dosing paradigms on GABA(A) receptor gene expression and benzodiazepine binding characteristics. Equivalent daily doses of 15 mg/kg/day diazepam were delivered either via constant infusion or daily subcutaneous injection for 14 days. The two distinct treatment regimes produced significantly different changes in GABA(A) receptor alpha4-, beta2-, beta3- and gamma1-subunit mRNA steady-state levels. Similar changes in the GABA enhancement of flunitrazepam binding and the BZ3/BZ2 subtype ratio determined ex vivo were produced, however, significant differences were found in [(3)H]-Ro 15-4513 binding between cortical tissue from diazepam injected animals compared with diazepam infused animals. Our data suggest that it is the diurnal fluctuations in receptor occupancy that are responsible for the different effects produced by these two dosing regimes.

The use of site-directed mutagenesis, transient transfection, and radioligand binding. A method for the characterization of receptor-ligand interactions.

Receptor-ligand interactions have traditionally been evaluated using a number of biochemical techniques including radioligand binding, photoaffinity labeling, crosslinking, and chemical modification. In modern biochemistry, these approaches have largely been superseded by site-directed mutagenesis in the study of protein function, owing in part to a better understanding of the chemical properties of oligonucleotides and to the ease with which mutant clones can now be generated. The Altered Sites II in vitro Mutagenesis System from the Promega Corporation employs oligonucleotides containing two mismatches to introduce specific nucleotide substitutions in the nucleic acid sequence of a target DNA. One of these mismatches will alter the primary sequence of a given protein, whereas the second will give rise to a silent restriction site that is used to screen for mutants. Transient transfection of tsA201 cells with mutant cDNA constructs using calcium phosphate as a carrier for plasmid DNA permits expression of recombinant receptors that can be characterized using radioligand binding assays. In this article, we focus on site-directed mutagenesis, heterologous expression in eukaryotic cells, and radioligand binding as a methodology to enable the characterization of receptor-ligand interactions.

Tyrosine 62 of the gamma-aminobutyric acid type A receptor beta 2 subunit is an important determinant of high affinity agonist binding.

The gamma-aminobutyric acid type A receptor (GABA(A)R) carries both high (K(D) = 10-30 nm) and low (K(D) = 0.1-1.0 microm) affinity binding sites for agonists. We have used site-directed mutagenesis to identify a specific residue in the rat beta2 subunit that is involved in high affinity agonist binding. Tyrosine residues at positions 62 and 74 were mutated to either phenylalanine or serine and the effects on ligand binding and ion channel activation were investigated after the expression of mutant subunits with wild-type alpha1 and gamma2 subunits in tsA201 cells or in Xenopus oocytes. None of the mutations affected [(3)H]Ro15-4513 binding or impaired allosteric interactions between the low affinity GABA and benzodiazepine sites. Although mutations at position 74 had little effect on [(3)H]muscimol binding, the Y62F mutation decreased the affinity of the high affinity [(3)H]muscimol binding sites by approximately 6-fold, and the Y62S mutation led to a loss of detectable high affinity binding sites. After expression in oocytes, the EC(50) values for both muscimol and GABA-induced activation of Y62F and Y62S receptors were increased by 2- and 6-fold compared with the wild-type. We conclude that Tyr-62 of the beta subunit is an important determinant for high affinity agonist binding to the GABA(A) receptor.

Determination of paroxetine levels in human plasma using gas chromatography with electron-capture detection.

A simple, rapid and sensitive procedure using gas chromatography with electron-capture detection to measure paroxetine levels in human plasma has been developed. The analyte was extracted from plasma with ethyl acetate after basification of the plasma and then derivatized with heptafluorobutyric anhydride before gas chromatographic separation. The calibration curves were linear, with typical r2 values >0.99. The assay was highly reproducible and gave peaks with excellent chromatographic properties.

The effect of treatment regimen on the development of tolerance to the sedative and anxiolytic effects of diazepam.

RATIONALE: Chronic treatment with benzodiazepines results in tolerance to their sedative and anxiolytic effects and there is considerable evidence that different mechanisms underlie the development of tolerance to different behavioural effects. OBJECTIVE: The purpose of the present experiment was to compare the behavioural effects of chronic treatment with diazepam (15 mg/kg per day) given as daily subcutaneous injections or by osmotic minipump. Both regimens resulted in continual receptor occupancy, but the daily injections also provided a period of higher brain concentrations. METHODS: Rats were tested in the holeboard, which provides measures of exploration and locomotor activity, and in the elevated plus-maze and social interaction tests of anxiety. For those in the subcutaneous injection group the tests were 2 h after injection, when brain concentrations were highest. RESULTS: Despite a higher brain concentration in the injected group, both groups showed tolerance to diazepam's sedative effects, after 7 days of treatment. In contrast, in the elevated plus-maze, there was tolerance to the anxiolytic effects in the pump group after 14 days, but a persisting anxiolytic effect in the injected group at 14 and 28 days. Whilst higher brain concentrations could explain this result in the plus-maze, they cannot account for the pattern observed in the social interaction test, where the injection group showed a significant anxiogenic effect at 28 days. CONCLUSIONS: Whereas the mechanism underlying tolerance to the sedative effects of diazepam was insensitive to the different treatment regimens, the results suggest that different adaptive mechanisms were triggered in the two tests of anxiety with a differential sensitivity to the treatment regimen. The adaptive mechanism predominating in the social interaction test was favoured by the injection regimen which produced intermittent peak concentrations. This mechanism seems to be an oppositional one, leading to an anxiogenic response, which was manifest despite high brain concentrations of diazepam at the time of testing.

Decreased GABA enhancement of benzodiazepine binding after a single dose of diazepam.

The GABA and benzodiazepine binding sites on GABA(A) receptors are allosterically coupled. The in vitro binding of 2 nM [3H]flunitrazepam to cortical and cerebellar membranes prepared from drug-naive rats was potentiated approximately 1.6-fold by 100 microM GABA. Potentiation in both regions was significantly reduced 4 or 12 but not 24 h after a single dose of 15 mg/kg diazepam. At 24 h after the last of 14 daily doses of diazepam, no differences in GABA potentiation were observed. Diazepam-induced changes in GABA(A) receptor gamma2-subunit gene transcription and alpha1-, beta2-, and gamma2-subunit steady-state mRNA levels did not appear to be temporally related to allosteric uncoupling.

Structural requirements for ligand interactions at the benzodiazepine recognition site of the GABA(A) receptor.

His101 of the GABA(A) receptor alpha1 subunit is an important determinant of benzodiazepine recognition and a major site of photolabeling by [3H]flunitrazepam. To investigate further the chemical specificity of the residue in this position, we substituted it with phenylalanine, tyrosine, lysine, glutamate, glutamine, or cysteine. The mutant alpha subunits were coexpressed with the rat beta2 and gamma2 subunits in TSA201 cells, and the effects of the substitutions on the binding of benzodiazepine site ligands were examined. [3H]Ro 15-4513 bound to all mutant receptors with equal or greater affinity than to the wild-type receptor. However, flunitrazepam and ZK93423 recognition was adversely affected by substitutions of the amino acid in this position. The binding of the antagonists, Ro 15-1788 and ZK93426, was also sensitive to the mutations, with the largest decreases in affinity occurring with the tyrosine, lysine, and glutamate substitutions. In all mutants that recognized flunitrazepam, GABA potentiated the binding of this ligand to a similar extent, suggesting that it is a full agonist at these receptors. The effects of GABA on the binding of Ro 15-1788 and Ro 15-4513 suggest that their efficacies may have been changed by some of the substitutions. This study further emphasizes the importance of the residue at position 101 in both ligand recognition and pharmacological effect.

Effects of phenelzine and imipramine on the steady-state levels of mRNAs that encode glutamic acid decarboxylase (GAD67 and GAD65), the GABA transporter GAT-1 and GABA transaminase in rat cortex.

There is an increasing body of evidence suggesting that GABA plays an important role in the therapeutic effects of antidepressant/antipanic drugs. Phenelzine and imipramine are efficacious in the treatment of depression and panic disorder and phenelzine has been reported to elevate GABA levels while imipramine enhances GABA release in rat brains. In the present study, using a multiprobe quantitative solution hybridization assay, we measured the steady-state levels of mRNAs that encode glutamic acid decarboxylase (GAD67 and GAD65), the GABA transporter GAT-1 and GABA transaminase (GABA-T) in rat cortex after treatment with constant infusion (via osmotic minipumps) of phenelzine or imipramine for a short-term (3 days) or long-term (21 days) period. We found that none of the treatments gave rise to significant changes in the steady-state levels of mRNAs encoding GAD67, GAD65 or GABA-T at any time point. The steady-state levels of GAT-1 mRNA were increased significantly (23%) after long-term, but not by short-term, treatment with phenelzine. Imipramine treatment, short- or long-term, did not alter the steady-state levels of GAT-1 mRNA. These results suggest that the GABA enhancing effects of phenelzine or imipramine in rat cortex do not affect the steady-state levels of mRNAs that encode GAD67, GAD65 and GABA-T. Further, the previously observed increases in GABA levels or GABA release induced by these drugs are probably not a consequence of changes in the expression of these genes.

Chronic diazepam exposure decreases transcription of the rat GABA(A) receptor gamma2-subunit gene.

The rate of transcription of the GABA(A) receptor gamma2-subunit gene in rat cortex has been measured using the nuclear run-off transcriptional assay. Exposure of rats to diazepam (15 mg/kg/day for 14 days) caused a significant reduction in the level of nascent GABA(A) receptor gamma2-subunit transcripts. Therefore, a component of the cellular response to chronic benzodiazepine exposure includes events which take place at the level of transcription of a GABA(A) receptor gene.

Chronic zolpidem treatment alters GABA(A) receptor mRNA levels in the rat cortex.

The effect of chronic zolpidem treatment on the steady-state levels of gamma-aminobutyric acidA alpha1-6, beta1-3 and gamma1-3 subunit mRNAs in rat cortex has been investigated. Male Sprague-Dawley rats were injected once daily, for 7 or 14 days, with 15 mg/kg of zolpidem in sesame oil vehicle. The levels of the alpha4 and beta1 subunit mRNAs were significantly increased after 7 days of treatment and the level of alpha1 subunit mRNA was significantly decreased after 14 days of treatment, as determined by solution hybridization. These results are compared to the previously determined effects of an equivalent schedule of treatment with diazepam.

The quantitative analysis of multiple mRNA species using oligonucleotide probes in an S1 nuclease protection assay.

The quantitative measurement of steady-state mRNA levels is fundamental to the analysis of gene expression. A variety of techniques are widely used to achieve this including Northern blotting, RNase protection, and S1 nuclease protection. We describe here in detail a relatively recent extension of the S1 nuclease protection technique (1) in which radiolabeled oligonucleotides are used as probes in a solution hybridization assay (2). The principle advantage of this technique is that it allows, in a single RNA sample, the simultaneous measurement of the relative levels of at least six mRNA species, including that of a control mRNA. Further, a large number of RNA samples can be analyzed at one time.

Rapid, sensitive procedure to determine buspirone levels in rat brains using gas chromatography with nitrogen-phosphorus detection.

Reported here is a rapid, sensitive and relatively inexpensive procedure using gas chromatography with nitrogen-phosphorus detection (GC-NPD) to quantify buspirone levels in brains of rats. The analyte was directly extracted from brain homogenate with toluene after basification and then subjected to GC-NPD analysis using a capillary column. The calibration curves were linear over the range of 10 to 320 ng per 2 ml of brain homogenate, with typical r2 values >0.99. The assay was highly reproducible and gave peaks with excellent chromatographic properties.

Dimethyl sulfoxide/propylene glycol is a suitable solvent for the delivery of diazepam from osmotic minipumps.

The functional integrity of Alzet osmotic minipumps was assessed using two organic solvents (50% (v/v) dimethyl sulfoxide (DMSO)/50% (v/v) propylene glycol (PG) and 100% tetraglycol) which dissolve diazepam, an aqueous insoluble benzodiazepine. Both solvents showed a significant decrease in output rate over time: the decline with tetraglycol was, however, more marked and variable. Further, the DMSO/PG vehicle demonstrated a comparable decline in rate (1.45%) to that of the control vehicles saline and water (1.12%). DMSO/PG is therefore a suitable solvent for the chronic delivery of diazepam from osmotic minipumps.

Molecular biology of the GABA(A) receptor: functional domains implicated by mutational analysis.

GABA(A) receptors are the major inhibitory neurotransmitter receptors in mammalian brain. They belong to a family of ligand-gated ion channels that also includes the nicotinic acetylcholine receptors, glycine receptors and 5HT(3) receptors. Each receptor in the family is believed to be a pentamer of homologous subunits that assemble to form a central transmembrane ion pore which, in the case of the GABA(A) receptor, is anion-selective. For almost twenty years, there has been tremendous interest in the structure and function of GABA(A) receptors, not only because of their importance in regulating brain excitability but also because these proteins are the specific targets for a wide variety of therapeutic agents including the anxiolytic benzodiazepines and barbiturates. Molecular cloning has revealed that GABA(A) receptors are heterogeneous, being formed by combinations of different isoforms of several subunit classes (alpha, beta, gamma, delta). The physiological and pharmacological properties of individual GABAA receptor subtypes appear to depend on their precise subunit complement. In this review, we focus on the application of modern techniques in molecular biology, particularly mutational analysis, to identify structural domains of these receptors that are important for ligand recognition and receptor function.

A novel G protein-coupled P2 purinoceptor (P2Y3) activated preferentially by nucleoside diphosphates.

A partial cDNA was isolated by hybridization screening of an embryonic chick brain library for P2Y purinoceptors. After extension to full length, it revealed an open reading frame that encoded a protein, P2Y3, of 328 amino acids that is nearest in sequence identity to the G protein-coupled P2 purinoceptors obtained by DNA cloning. Expression of P2Y3 in cRNA-injected Xenopus oocytes confirmed that this cDNA encodes a member of the metabotropic purinoceptor family, with a novel order for the relative activities of nucleotides. At 100 microM concentrations, ADP gave the highest activity, and UTP and UDP were also strongly active. When expressed in the human T cell line Jurkat, P2Y3 mediated transient increases in intracellular Ca2+ in response to various nucleotides. Again, an unusual agonist rank order was revealed, with uridine nucleotides being more potent than adenosine nucleotides and UDP being the most potent agonist tested (half-maximal concentration, 0.13 microM) and 10-fold more potent than UTP. 2-Methylthlo-ATP was of relatively low activity in both systems. The receptor transcript is expressed in brain, spinal cord, kidney, and lung and is highly abundant in the spleen but not in other peripheral tissues that we tested. The results indicated that P2Y3 is a previously unknown P2 purinoceptor subtype with a preference for nucleoside diphosphates.