WO2009016329A1

WO2009016329A1 - Use of gabaa receptor antagonists to treat cognitive impairment in patients with psychiatric conditions

Info

Publication number: WO2009016329A1
Application number: PCT/GB2007/002905
Authority: WO
Inventors: Edward Thomas Bullmore; Caroline Stephenson
Original assignee: Cambridge Enterprise Limited
Priority date: 2007-07-31
Filing date: 2007-07-31
Publication date: 2009-02-05

Abstract

The present invention relates to the treatment of impaired cognitive function in patients with psychiatric conditions such as schizophrenia with inhibitors of Ϝ-aminobutyric acid (GABA) neurotransmission, such as flumazenil. Methods of treatment and uses of blockers of inhibitory GABA neurotransmission are provided, along with screening methods for identifying further therapeutic agents.

Description

Use of GABA_A Receptor Antagonists to Treat Cognitive Impairment in Patients with Psychiatric Conditions

This invention relates to the treatment of cognitive impairment in patients with psychiatric conditions.

Impairments in executive function and memory are major impediments to social rehabilitation and predict poor clinical outcome in patients with psychiatric conditions such as schizophrenia^1"5

Cognitive deficits in such conditions are trait-like and heritable, but their neurobiological basis and rationale for targeted pharmacological intervention are not clear.^5"10

Despite the success of anti-psychotic drugs in treating positive symptoms, lack of effective pharmacotherapy for cognitive symptoms remains a major unmet need in the management of conditions such as schizophrenia. Previously published data indicates some therapeutic potential for benzodiazepines in the treatment of positive and negative psychotic symptoms³³'³⁴ but the effects of GABA modulating drugs on cognition in patients with psychiatric conditions have not been addressed previously.

The present inventors have discovered that psychiatric patients with impaired cognitive function who are treated with inhibitors of γ~ aminobutyric acid (GABA) neurotransmission, such as flumazenil, show improved cognitive performance, whereas healthy individuals treated in the same way show a slightly impaired performance (Menzies et al Arch Gen Psychiatry. 2007; 64 : 156-167) .

One aspect of the invention provides a method of treating cognitive impairment in an individual having a psychiatric condition comprising; administering a therapeutically effective amount of a compound which reduces or inhibits γ-aminobutyric acid (GABA) mediated neurotransmission to an individual in need thereof. The cognitive performance of the individual with the psychiatric condition may be improved by the administration of the GABA neurotransmission inhibitor as described herein.

A method described herein may further comprise assessing the cognitive function of the individual with the psychiatric condition to determine the level or extent of cognitive impairment. The assessment may be performed before administration of the-GABA neurotransmission inhibitor, for example to establish the necessity or suitability of the treatment for the individual. The assessment may be performed after administration, for example to establish the efficacy of the treatment in the individual .

Cognitive function relates to the mental abilities of the individual and may include understanding, reasoning and memory. Cognitive function may be assessed using standard diagnostic tests. For example, the working memory performance of an individual may be indicative of cognitive function. Working memory performance may be assessed using any convenient working memory test, for example the N-back test (Owen AM, et al Hum Brain Mapp. 2005 ; 25 : 46-59; Glahn DC, et al Hum Brain Mapp. 2005,-25: 60-69) .

In other embodiments, cognitive function may be assessed by assessing episodic or long term memory and/or executive function. Suitable tests for these parameters are available.

An individual suitable for treatment as described herein may have a psychiatric condition which is associated with impaired cognition. Impaired cognition may be caused by abnormalities of interneuronal structure or function. For example, an individual may have a psychiatric condition selected from the group consisting of schizophrenia, bipolar disorder, Huntington' s disease, dementias, developmental cognitive impairments, post traumatic/injury impairments or other causes of cognitive impairment .

The individual may be diagnosed with such a condition in accordance with standard neuropsychiatric diagnostic criteria. Neuropsychiatric diagnostic criteria for these conditions are set out, for example in the Diagnostic and Statistical Manual of Mental Disorders (text revision) , American Psychiatric Association (2000) American Psychiatric Publishing Inc (DSM-IV-TR) and the International Classification of Diseases 10 (ICD 10) .

Compounds which reduce or inhibit γ-aminobutyric acid (GABA) neurotransmission include GABA receptor antagonists. A GABA receptor antagonist may be specific for the target GABA receptor or may additionally act at other receptors. Suitable compounds include

GABA_A receptor antagonists. The GABA_A receptor is located at neuronal synapses and allows the passage of chloride ions across the membrane in response to GABA binding, contributing to the inhibitory postsynaptic potential. The GABA_A receptor may be composed of a combination of a, β, and Y subunits and is commonly a α₂β₂Y pentamer. GABA binds to the GABA_A receptor at the interface of α and β subunits (Hevers W et al (1998) MoI Neurobiol 18 (1) : 35-86; Sieghart W et al (2002) . Curr Top Med Chem 2 (8) : 795-816) .

A GABA_A receptor antagonist may, for example, reduce or block GABA stimulated conductance of chloride ions through the GABA_A receptor channel .

Benzodiazepines bind to a site at the interface of α and γ subunits of GABA_A receptor and increase or enhance GABA-mediated neurotransmission i.e. the conductance of chloride ions through the GABA_A receptor channel which is stimulated by GABA. Suitable GABA antagonists include benzodiazepine antagonists which block or inhibit the binding of benzodiazepines to the GABA receptor (for example by competitive inhibition) , thereby inhibiting or reducing benzodiazepine activity.

Suitable compounds include imidazobenzodiazepines, such as flumazenil (ethyl 8-fluoro-5, 6-dihydro-5-methyl-6-oxo-4H- imidazo [1,5-a] [1, 4] benzodiazepine-3 -carboxylate) , beta-carbolines such as ZK 93426, propyl-beta-carboline, and pyrazoloquinolines, such as CGS- 8216. In preferred embodiments, the compound is flumazenil (also known as (flumazepil, Ro 15-1788, Anexate^®, Lanexat^®, Mazicon^® and Romazicon^®) and isomers, salts, solvates, chemically protected forms , and prodrugs thereof .

A method of treating cognitive impairment in an individual having a psychiatric condition, such as schizophrenia, may comprise; administering a therapeutically effective amount of flumazenil or an isomer, salt, solvate, chemically protected form, or prodrug thereof to an individual in need thereof.

Other aspects of the invention provide a compound which reduces or inhibits γ-aminobutyric acid (GABA) -mediated neurotransmission for use in treating impaired cognitive function in an individual having a psychiatric condition, and the use of a compound which reduces or inhibits γ-aminobutyric acid (GABA) -mediated neurotransmission in the manufacture of a medicament for use treating impaired cognitive function in an individual having a psychiatric condition.

In some embodiments, a compound which reduces or inhibits γ~ aminobutyric acid (GABA) mediated neurotransmission may possess a selective activity on GABA neurotransmission and may show no other neurobiological activities. In other embodiments, a compound which reduces or inhibits γ-aminobutyric acid (GABA) mediated neurotransmission may act on multiple receptors and may display other neurobiological activities as well as inhibition of GABA neurotransmission. In some preferred embodiments, the compound may additionally reduce or inhibit dopamine mediated neurotransmission (i.e. the compound may be a dopamine antagonist), thereby ameliorating positive symptoms of schizophrenia as well as cognitive impairment .

While it is possible for an active compound to be administered alone, it is preferable to present it as a pharmaceutical composition (e.g., formulation) comprising at least one active compound, as defined above, together with one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilisers, preservatives, lubricants, or other materials well known to those skilled in the art and optionally other therapeutic or prophylactic agents.

Pharmaceutical compositions comprising a compound described herein admixed or formulated together with one or more pharmaceutically acceptable carriers, excipients, buffers, adjuvants, stabilisers, or other materials, as described herein, may be used in the methods described herein.

The term "pharmaceutically acceptable" as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well-known in the art of pharmacy. Such methods include the step of bringing the active compound into association with a carrier which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Formulations may be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, tablets, lozenges, granules, powders, capsules, cachets, pills, ampoules, suppositories, pessaries, ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses, electuaries, aerosols, patches or implants. The compound (s) or pharmaceutical composition comprising the compound (s) may be administered to a subject by any convenient route of administration, whether systemically/ peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion) or parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal ; by implant of a depot, for example, subcutaneously or intramuscularly.

In some embodiments, the compound (s) or pharmaceutical composition comprising the compound (s) may be administered by mechanical methods, for example by means of a patch or implant, such as a cutaneuous or sub-cutaneous patch.

Formulations suitable for oral administration (e.g., by ingestion) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus,- as an electuary; or as a paste .

Formulations suitable for parenteral administration (e.g. by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal) , include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the active compound in the solution is from about 1 ng/ml to about 10 μg/ml, for example, from about 10 ng/ml to about 1 μg/ml. The formulations may be presented in unit-dose or multi- dose sealed containers, for example, ampoules and vials, and may ^'be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.

Pharmaceutical formulations of flumazenil and other GABA antagonists are well-known in the art.

It will be appreciated that appropriate dosages of the active compounds, and compositions comprising the active compounds, can vary from patient to patient . Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side- effects .

Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment . Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.

In general, a suitable dose of the active compound is in the range of about 100 μg to about 250 mg per kilogram body weight of the subject per day. Where the active compound is a salt, an ester, prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.

Drug doses and timings of flumazenil may be selected on the basis of published pharmacokinetic data.53 conveniently, flumazenil may be administered as an intravenous bolus contained 0.9 mg of flumazenil, and the infusion delivered at a rate of 0.0102 mg/min, calculated to maintain steady plasma levels.

Other aspects of the invention relate to methods of identifying or screening for compounds which inhibit GABA_A receptor activity and may therefore by useful in ameliorating cognitive impairment in psychiatric patients. A method of screening for a compound useful in treating cognitive impairment in an individual having a psychiatric condition may comprise; contacting a GABA_A receptor with a GABA_A receptor agonist in the presence of a test compound and; determining the binding and/or activation of the GABA_A receptor by the agonist, wherein a decrease in binding and/or activation by the agonist in the presence relative to the absence of the test compound is indicative that the test compound is useful in treating cognitive impairment in an individual having a psychiatric condition.

The GABA_A receptor may be contacted with the GABA_A receptor agonist under conditions in which, in the absence of the test compound, the agonist binds to and/or activates the GABA_A receptor.

Binding of a GABA_A receptor agonist to a GABA_A receptor may be determined using any one of a range of binding assay techniques available in the art, including radioimmunoassay, scintillation proximetry assay and ELISA methods . Conveniently, the GABA_A receptor agonist may be labelled, for example with a fluorescent or radioactive group, and the amount of label which binds to immobilised GABA_A receptor measured.

Activation of the GABA_A receptor may be determined by measuring the ion conductance of receptor, preferably the chloride ion conductance. This may be achieved by conventional electrophysiological techniques.

The sequences of a, β, and Y subunits of the GABA_A receptor are well known in the art and publicly available on sequence databases.

GABA_A receptor agonists include natural ligands, such as GABA, and other molecules which bind to GABA_A receptors and increase or activate ion conductance, such as benzodiazepines.

Suitable benzodiazepines include alprazolam, bromazepam, diazepam , lorazepam, clonazepam, temazepam, oxazepam, flunitrazepam, riazolam, chlordiazepoxide, flurazepam, estazolam, and nitrazepam.

In some embodiments, the GABA_A receptor may be located in a membrane, preferably a cell membrane. A method of screening for a compound useful in treating cognitive impairment in an individual having a psychiatric condition may comprise; contacting a cell which expresses a GABA_A receptor with a GABA_A receptor agonist in the presence of a test compound and; determining the activation of the GABA_A receptor by the agonist, wherein a decrease in activation of the GABA_A receptor by the agonist in the presence relative to the absence of the test compound is indicative that the test compound is useful in treating cognitive impairment in an individual having a psychiatric condition.

Test compounds which may be screened using the methods described herein may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants, microbes or other organisms which contain several characterised or uncharacterised components may also be used.

Combinatorial library technology provides an efficient way of testing a potentially vast number of different compounds for ability to modulate an interaction. Such libraries and their use are known in the art, for all manner of natural products, small molecules, peptides, aptamers among others.

Suitable test compounds may include imidazobenzodiazepines, beta- carbolines and pyrazoloquinolines or other chemical groups.

The amount of test compound or compound which may be added to a method of the invention will normally be determined by serial dilution experiments. Typically, from about 0.001 nM to 1 mM or more of test compound may be used, for example from 0.01 nM to lOOμM, e.g. 0.1 to 50 μM, such as about 10 μM.

A method may comprise identifying the test compound as a compound which inhibits activation of GABA_A receptor which may be useful in the treatment or amelioration of impaired cognitive function in psychiatric patients.

A test compound identified using one or more initial screens as having ability to reduce or block activation of the GABA_A receptor, may be assessed further using one or more secondary screens. A secondary screen may, for example, involve testing for a biological function such as an effect on the activation of brain regions in an animal model. Typically, a compound may be administered to an individual and the individual subjected to cognitive testing and functional magnetic resonance imaging (fMRI) to assess the effect of the compound on brain function.

The test compound may be isolated and/or purified or alternatively, it may be synthesised using conventional techniques of recombinant expression or chemical synthesis. Furthermore, it may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals, either alone or in combination with other therapeutic agents, for the treatment of cognitive impairment in an individual having a psychiatric condition. Methods of the invention may thus comprise formulating the test compound in a pharmaceutical composition with a pharmaceutically acceptable excipient, vehicle or carrier for therapeutic application, as discussed further above.

Following identification of a compound which decrease in binding and/or activation of a GABA_A receptor by a GABA_A agonist , and which may therefore be useful in treating cognitive impairment in an individual having a psychiatric condition, a method may further comprise modifying the compound to optimise the pharmaceutical properties thereof.

The modification of a 'lead' compound identified as biologically active is a known approach to the development of pharmaceuticals and may be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, e.g. peptides are not well suited as active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal . Modification of a known active compound (for example, to produce a mimetic) may be used to avoid randomly screening large number of molecules for a target property.

Modification of a 'lead' compound to optimise its pharmaceutical properties commonly comprises several steps. Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. These parts or residues constituting the active region of the compound are known as its "pharmacophore". Once the pharmacophore has been found, its structure is modelled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR.

Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process.

In a variant of this approach, the three-dimensional structure of the compound is modelled. This can be especially useful where the compound changes conformation, allowing the model to take account of this in the optimisation of the lead compound.

A template molecule is then selected, onto which chemical groups that mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the modified compound is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.

The modified compounds found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Modified compounds include mimetics of the lead compound.

Further optimisation or modification can then be carried out to arrive at one or more final compounds for in vivo or clinical testing.

As described above, a compound identified and/or obtained using the present methods may be formulated into a pharmaceutical composition

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. All documents mentioned in this specification are incorporated herein by reference in their entirety. "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described above and tables described below.

Figure 1 shows maps of brain function during performance of the N- back working memory task. A, Systems of positive and negative brain load response in healthy volunteers after placebo administration (n=ll) , constituting a normal pattern of frontoparietal activation (red and yellow voxels) and temporocingulate deactivation (blue and purple voxels) . B, Systems of significant psychophysiological association between behavioral performance, area under the task performance curve (AUC [Pr] ) , and positive or negative brain load response across all participants and treatments (n=66) . The right side of each map represents the left side of the brain; the crosshairs indicate the origin of x and y dimensions, and the numbers denote the z dimension of each section in Talairach space.

Figure 2 shows the effects of schizophrenia and γ-aminobutyric acid-modulating drugs on N-back activation and deactivation systems. A, Scatterplot of individual mean positive and negative load responses showing a strong correlation between the magnitude of frontoparietal activation and temporocingulate deactivation across all participants and reduced group mean activation and deactivation in patients with schizophrenia. B, Mean positive and negative load response (averaged across both groups after each treatment) showing strongest activation and deactivation after placebo administration. C, Mean positive and negative load responses (averaged for each group after each treatment) showing reduced activation and deactivation after lorazepam and flumazenil administration in healthy volunteers and enhanced deactivation in patients with schizophrenia after flumazenil administration. Error bars represent standard error of the mean,- t, t statistic for the regional mean load-response .

Figure 3 shows the N-back working memory task performance in patients with schizophrenia and healthy volunteers after treatment with flumazenil, lorazepam, or placebo. A, Drug effects on behavioral performance (Pr) as a function of task difficulty for patients with schizophrenia (top) and healthy volunteers (bottom) . *Improved performance after flumazenil compared with placebo administration; at N=I, t43=-2.14; P=.04; at N=2, t43=-2.32; P=.03. tWorse performance after lorazepam compared with placebo administration (post hoc t43=3.1; P=.003) . B, Task performance accuracy summarized across all difficulty levels by the area under the task performance curve (AUC [Pr] ) . Error bars represent standard errors of means. Patients with schizophrenia showed a significant effect of drug on AUC(Pr) (post hoc analysis of variance [ANOVA] F2, 20=6.06; P=.009), but there was no significant effect of drug in healthy volunteers (post hoc ANOVA Fl .4, 13.5=1.00 ; P=.36).

Figure 4 shows the physiological correlates of working memory task performance in patients with schizophrenia and healthy volunteers after administration of lorazepam, flumazenil, or placebo. Scatterplot of the area under the task performance curve (AUC [Pr] ) vs positive and negative load responses averaged across all voxels in the systems shows significant association with task performance for all participants and treatments (n=66) . Superior performance is associated with greater frontoparietal activation and greater dorsal cingulate deactivation.

Figure 5 shows the effects of schizophrenia and γ-aminobutyric acid- modulating drugs on positive and negative load response in brain regions significantly associated with behavioral task performance. Plots of the mean area under the task performance curve (AUC [Pr] ) vs regional mean load response are shown separately by group and drug for 4 representative regions of significant psychophysiological association. A, Anterior cingulate cortex shows increased activation associated with superior performance in patients with schizophrenia after flumazenil administration. B and C, Bilateral parietal cortex shows attenuated activation associated with worse performance in patients with schizophrenia after lorazepam administration. D, Dorsal cingulate cortex shows attenuated deactivation after administration of both drugs in healthy volunteers and after lorazepam administration in patients with schizophrenia.

Table 1 shows demographic characteristics of schizophrenic patients and Matched Healthy Volunteers Participating in a Pharmacological fMRI Study of Working Memory. Abbreviations: fMRI, functional magnetic resonance imaging; NART, National Adult Reading Test; RBMT, Rivermead Behavioral Memory Test. *For age, df = 20; for the NART and RBMT scores and verbal fluency, df = 19 because results of the NART, RBMT, and verbal fluency were unavailable for 1 participant. tverbal fluency was assessed by (1) generation of exemplars within 1 minute from a semantic category and (2) generation of exemplars within 1 minute from a given starting letter.

Table 2 shows clinical characteristics of schizophrenic patients Abbreviations: BID, twice daily; BPRS, Brief Psychiatric Rating Scale; NA, not available; SANS, Scale for Assessment of Negative Symptoms; SAPS, Scale for Assessment of Positive Symptoms.

Table 3 shows behavioral performance on a verbal N-Back paradigm as a function of working memory load, in schizophrenic patients and healthy volunteers after administration of placebo, Lorazepam, or Flumazenil Abbreviation: AUC(Pr), area under the task performance curve. *Calculated as a discrimination index (Pr) by subtracting the proportion of nontarget items incorrectly identified as targets (false alarms) from the proportion of items correctly identified as targets (hits) . tTotal working memory performance AUC(Pr) was summarized by calculating the area under each subject's curve of the discrimination index (Pr) vs task difficulty.

Experiments The following experiments are described in Menzies et al Arch Gen Psychiatry. 2007; 64 : 156-167.

Methods Sample Twelve male right-handed patients with an operational DSM-IV47 diagnosis of schizophrenia and 12 male right-handed healthy volunteers matched with patients for age and premorbid IQ (measured using the National Adult Reading Test)⁴⁸ participated in the study (Table 1) . Healthy volunteers were recruited by advertisement from the local population. Schizophrenic patients were recruited by a consultant psychiatrist from a clinical rehabilitation service for patients with chronic psychosis in Cambridge, England. Additional clinical data are given in Table 2. All participants were screened to exclude psychiatric or neurological disorders (other than schizophrenia in the patients) , head injury, history of substance abuse, current medication (in particular, benzodiazepines or anticholinergic drugs) with the exception of antipsychotics in the patient group, and contraindications to MRI. All participants had negative urinalysis findings for illicit drug metabolites when recruited and were screened for episodic memory and executive function using the Rivermead Behavioral Memory Test⁴⁹ and a verbal fluency test (Table 1) .

One patient was excluded after failure to perform the task during MRI. To maintain a balanced design for factorial analysis (important in ensuring that interaction effects remain orthogonal) and to allow permutation testing of the imaging data,^50"52 1 volunteer was also excluded at random, leaving 11 patients and 11 control subjects for behavioral and imaging analysis. A secondary analysis of the behavioral data, including the 12th volunteer, is also reported in the supplemental material (available at http://www- bmu.psychiatry.cam.ac.uk/data/menzies) . The study was approved by the local research ethics committee of Addenbrooke ' s Hospital, Cambridge, and the Medicines Control Agency, London, England. After a complete description of the study, written informed consent was obtained from all participants. The participants were paid an honorarium and travel expenses.

Study Design and Drug Treatment

We used a placebo-controlled, randomized, double-blind, balanced factorial design with, drug treatment as a within-subject factor and diagnostic group as a between-subjects factor. Each participant underwent MRI on 4 occasions at 2 -week intervals. Structural MR images were acquired during the first session, which also acclimated participants to the scanner and experimental task. In the remaining 3 sessions, participants were assigned to 1 of 3 treatments: placebo, intravenous (IV) flumazen.il, or oral lorazepam. To control for task practice effects entailed by the repeated-measures design, the order of drug administration was counterbalanced across participants. One patient's data after placebo administration only were unavailable; mean values were imputed where appropriate. Lorazepam and flumazenil are widely used clinically and were well tolerated by our study participants. Drug doses and timings were selected on the basis of published pharmacokinetic data.53 To control for different modes of drug administration, we used the following regimen: 120 minutes before all MRI sessions, participants received an oral capsule, and 10 minutes be- fore MRI they were administered a 10-mL IV bolus followed by an IV infusion until the MRI session was finished (approximately 90 minutes later) . In the placebo condition, the oral tablet contained lactose, and the IV bolus and infusion contained isotonic sodium chloride solution. In the lorazepam condition, the oral capsule contained 2 mg of lorazepam, and the IV bolus and infusion contained isotonic sodium chloride solution.

In the flumazenil condition, the oral capsule contained lactose, but the IV bolus contained 0.9 mg of flumazenil, and the infusion delivered flumazenil at a rate of 0.0102 mg/min, calculated to maintain steady plasma levels. N-back Working Memory Task

The N-back test of verbal working memory was presented at 3 levels of difficulty in a blocked periodic paradigm.³⁸'³⁹ At the easiest level, N=I, subjects were asked to indicate when the current letter in a series of visually presented letters was the same as the immediately preceding one; at more difficult levels, N=2 or N=3 , subjects were asked to indicate when the current letter was the same as that presented 2 or 3 trials earlier. Subjects were also asked to perform a sensorimotor control task, N=O, in which they had to indicate whether the current letter was X. The interstimulus interval (stimulus duration) was 2200 milliseconds; 13 stimuli were presented in each of 4 blocks at each level of difficulty (52 stimuli at each level of difficulty; 16 blocks in total) . There were 3 or 4 target stimuli per block; that is, the probability of a target trial was 23% or 31%. Each block was preceded by a brief (2000-millisecond) visual cue to perform the task at a certain level of difficulty; the order of the blocks of trials at different difficulty levels was counterbalanced to control for nonspecific practice effects during the course of each experiment lasting 8 minutes and 10 seconds.

Performance was monitored behaviourally by a right handed button press, with one button for targets and another for non targets. A program for stimulus presentation and behavioural monitoring was written in DMDX.54

FMRJ Data Acquisition

Gradient-echo echoplanar imaging data depicting blood oxygen level- dependent contrast were acquired using a commercially available 3T MRI scanner (Bruker Medspec S300 System; Bruker, Etlinger, Germany) at the Wolfson Brain Imaging Centre of Addenbrooke ' s Hospital, Cambridge. During each acquisition, 454 images consisting of 21 near-axial slices were collected with the following settings: repetition time, 1100 milliseconds; echo time, 27.5 milliseconds; flip angle, 65°; slice thickness, 4 mm with an interslice gap of 1 mm; matrix size, 64x64; and in-plane resolution, 3.75 mm. The first 6 images, acquired before starting the working memory paradigm, were discarded to allow for Tl equilibrium, leaving 448 images available for analysis. To minimize problems of image-acquisition at 3T, for example, in the orbitofrontal and inferior temporal regions, all tests on imaging data were performed only at voxels where all subjects had a non zero activation statistic, thus avoiding spurious results due to signal dropout.

Behavioural Data Analysis

We used a repeated-measures analysis of variance (ANOVA) to model within-subject effects of block repetition, task difficulty, drug treatment, and session order and the between subjects effect of diagnostic group on task performance. Performance was measured using response latency and the discrimination index (Pr) , a standard accuracy metric derived from signal detection theory. We calculated Pr=P/T-FP/N as the hit rate minus the false-positive rate, ie, the number of positive hits (correct identifications of a target [P] ) divided by the total number of target trials (T) minus the number of false-positive trials (FP) divided by the total number of non-target trials (N) . To summarize overall accuracy of task performance at all levels of difficulty in the paradigm, we calculated the area under the curve (AUC) of Pr vs task difficulty. Greater AUC(Pr) indicates better task performance with consideration of all difficulty levels.

Non-responses were excluded from calculation of the Pr index and response latency. Where necessary, the Huynh-Feldt epsilon algorithm was applied to correct for nonsphericity. Homogeneity of variance was checked by the Levene test, and we used post hoc t tests where appropriate .

fMRI Data Analysis

Each individual data set was preprocessed to correct for head motion effects,⁵⁵ detrended, and smoothed by a 2-dimensional Gaussian filter with a standard deviation of 1.875 mm. Positive or negative brain functional load response, defined as an increase ^■ or a decrease in brain activation linearly related to the 4 levels of increasing task difficulty, was estimated by regressing a hemodynamically convolved contrast on the movement corrected time series at each voxel . For each participant, the resulting brain maps of the load response, normalized by the standard error of the load-response estimation at each voxel, were co-registered in the space of the Montreal Neurological Institute-echoplanar imaging template image using affine transformation ⁵⁶'⁵⁷.

Considering first only data acquired from the healthy volunteers after placebo administration, we used a permutation test to identify voxels where the median load response was significantly greater (more strongly negative or positive) than under the null hypothesis that the median load response was not related to task performance . This analysis produced a map of the brain systems normally activated or deactivated by the Nback working memory task (Figure IA) .

We then explored group and drug effects on the load response in these systems in 2 ways. First, we used the normal activation and deactivation systems as a mask to define the mean load response across all voxels constituting these systems in data acquired from all participants after all treatments. This allowed us to describe the group and drug effects on load response at a systems level (Figure 2) . Second, to localize the group and drug effects with greater anatomical resolution, we fitted a 2 -way ANOVA model to the load-response statistics estimated at each voxel of the normal activation and deactivation systems and again used permutation testing to identify spatial clusters of voxels where there were significant group and drug effects.

Finally, we investigated the relationship between the cognitive and brain functional effects of drug treatment in 2 ways. First, we simply estimated the correlations between the summary performance metric, AUC(Pr), and the sum of the absolute mean load-response statistics in the activation and deactivation systems. Second, we regressed AUC(Pr) on the load-response statistics estimated at each voxel in standard space and identified, by permutation testing, voxels where there was significant psychophysiological association between the task performance and brain function (Figure IB) . All statistical brain mapping was conducted non-parametrically using previously validated methods for permutation testing.⁵¹'⁵⁷ To control for type I (false-positive) error rates in the context of the statistical tests potentially entailed for brain mapping in fMRI, we used the following strategies:

(1) 2-way ANOVA of group and drug effects was restricted to a reduced search volume defined by the normal activation and deactivation systems;

(2) a cluster-level statistic (the sum of spatially contiguous voxel statistics exceeding a preliminary threshold) that we have previously shown increases sensitivity and reduces search volume was used for all analyses⁵¹'⁵⁸; and

(3) the P value for all maps was chosen such that the expected number of false-positive clusters was less than 1 cluster per map.

Results

Demographic, Psychological, and Clinical Characteristics

The 2 groups were well-matched by age and premorbid IQ; however, the schizophrenic patients had significant impairments on the memory and verbal fluency tests (Table 1) .

Clinically, the schizophrenic patients had a chronic disorder and were moderately symptomatic despite receiving antipsychotic drugs (most frequently, clozapine) (Table 2) .

Working Memory Task Performance

Discrimination Index

Descriptive statistics on the discrimination index (Pr) stratified by level of difficulty, drug treatment, and diagnostic group are summarized in Table3. The repeated measures ANOVA demonstrated a main effect of task difficulty (F3, 60=7.58; P_.001) and a main effect of drug (F2, 40=3.44; P=.04). Across all of the participants, we found the expected decline in task performance as task difficulty increased and that the 2 drugs generally had opposing effects, with a tendency for improved performance after flumazenil administration and impaired performance after lorazepam administration. The post- hoc t tests of drug effects showed a significant performance impairment across all subjects after lorazepam compared with placebo administration (t351=3.29; P=.001) but non-significant effects of flumazenil compared with placebo administration (t351=-l.12; P=.26).

However, the behavioral effects of the drugs were clearly modulated by task difficulty and diagnostic status. There was a significant drug_difficulty interaction (F_5-5, ₁₀₉.₂=2.24; P=.05), with the effects of the drug being particularly strong at the N=I and N=2 levels of difficulty. Importantly, there was a group_drug interaction (Fa,₄₀=3.96; P=.03) owing to greater, significant effects of lorazepam and flumazenil in schizophrenic patients compared with the non-significant effects of both drugs in healthy volunteers (see Figure 3 for post hoc t tests) . There was also a significant group_drug_difficulty interaction (F_5-5,_109-2=2.42 ; P=.04), indicating that GABA modulating drugs had differential effects on the cognitive load response in the schizophrenic patients compared with the healthy volunteers (Figure 3A) .

There was no significant main effect of task repetition (practice) (F₂.₃,₄₆.₃=0.05;P= .37) or session order (F_L7134-7=I .36 ;

P=.27),nor was the order_group interaction (F_1-7, _34-7=0.07; P=.91) significant, providing indication that pharmacological effects were not confounded by the order of drug administration and that counterbalancing of drug order between groups successfully controlled this potential bias.

Area Under the Task Performance Curve

To summarize each subject's behavioural response, their total working memory performance across all levels of task difficulty was calculated as the area under the task performance curve (AUC[Pr])

(Figure 3 Band Table 3) . For all of the drug treatments, performance was approximately 10% impaired in the schizophrenic patients. Within the healthy volunteer group, performance decrements were due to both lorazepam (-2.5%compared with placebo) and flumazenil (-3.1%).

Within the patient group, the performance-degrading effect of lorazepam was more marked(-7.4%) , and flumazenil had a performance- enhancing effect (+6.7%) . The ANOVA results, with drug treatment as a within subject factor and diagnostic group as a between subjects factor, showed a significant main effect of drug (F₂,₄₀=4.25; P=.02) and a drug_group interaction (F₂,₄₀= 4.85; P=.01) on the AUC(Pr). Again, the drug x group interaction was due to an increased susceptibility of the patients to improved performance after flumazenil administration and impaired performance after lorazepam administration.

Response Latency

Results of the ANOVA of response latency showed a main effect of load (F₂.₅,so.₂=36.1; P_.001), with an expected increase in latency as task difficulty increased. In addition, we found a group_difficulty interaction (F₂.₅,₅₀.₂=6.03 ; P=.002) because latency was not greatly prolonged by increased task difficulty in the patient group. There was no effect of drug or a group_drug interaction on latency, implying that non-specific sedative or slowing effects of drug treatment were not prominent .

N-Back Activation And Deactivation

To map brain areas normally activated or deactivated during the N- back working memory task, we first estimated the load response in fMRI data acquired only from healthy volunteers after placebo administration (Figure 1 and supplemental material [available at http://www-bmu.psychiatry.cam.ac.uk/data/menzies]). This demonstrated that a large-scale system of regions had a positive load response (increased activation as the task difficulty increased) , including the cerebellum and the bilateral prefrontal and premotor (approximate Brodmann areas [BAs] 6, 8, 9, 10, 44, 45, 46, and 47), parietal (BAs 7 and 39), and anterior cingulate (BA 32) cortices . There was also a system of distributed regions with the opposite pattern of a negative load response (increased deactivation as the task difficulty increased) , including the cerebellum, the bilateral temporal (BAs 20, 21, 28, 36, 38, 41, and 42), posterior cingulate and parietooccipital (BAs 2, 18, 19, 23, 24, 31, and 40), and medial frontal and anterior cingulate (BAs 6, 8, 9, 11, 24, 25, and 32) cortices, and the striatum. For the sake of brevity, we will subsequently describe these 2 complementary systems as the N-back activation and deactivation networks, respectively.

Considering the load-response statistics estimated in these regions for all participants, we found that the magnitude of activation and deactivation were significantly correlated (r=0.66; n=66; P_.001), that is, participants who showed the strongest positive load response in the N-back activation network tended to show the strongest negative load response in the N-back deactivation network (Figure 2A) . The schizophrenic patients showed reduced activation and reduced deactivation compared with the healthy volunteers (Figure 2C) (ANOVA: main effect of group on activation and deactivation [as summarized by absolute sum of mean load- response statistics across both networks]; F_li2o=5.29; P=.03). On average across all subjects, lorazepam and flumazenil also tended to reduce activation and deactivation (Figure 2B) . (ANOVA: main effect of drug on activation and deactivation; F_x.₄₉,₂₉.₇=3.63; P=.05.) However, consideration of drug effects separately by group showed that, whereas both GABA-modulating drugs reduced activation and deactivation in healthy volunteers and lorazepam also reduced activation in schizophrenic patients, flumazenil tended to enhance deactivation in the patient group (Figure 2C) . (ANOVA: group_drug interaction on deactivation; Fl.68, 33.5=8.93; P=.001.)

Neurophysiological Correlates Of Task Performance

The extent of activation and deactivation (as summarized by the absolute sum of the mean load-response statistics across the 2 networks) was correlated with behavioral performance as summarized by the AUC(Pr) (r=0.48; n=66; P<.001). This indicates that participants who performed the task better tended to activate the frontoparietal cortex and deactivate the temporocingulate cortex more strongly than did those with poor task performance .

To specifically address the relationship between task performance and brain function, we performed a second analysis in which we regressed the summary performance measure AUC(Pr) on the load response at each voxel of fMRI data. We found 2 main classes of 2007/002905

brain regions where variable task performance significantly predicted physiological load response (Figure IB) . Superior task performance predicted a greater positive load response in the anterior cingulated and medial frontal cortex (BAs 24 and 32) , the bilateral posterior parietal cortex (BAs 7, 39, and 40) , the thalamus, and the caudate nucleus. In these areas, better performing individuals showed increased brain activation with increasing task difficulty. In addition, superior task performance predicted a greater negative load response in the posterior and dorsal cingulate cortices (Bas 23 and 24) . In these areas, better-performing individuals showed decreased deactivation with increasing task difficulty. These opposing changes in activation as a function of increased task difficulty were associated across all subjects (mixed-effects regression, t₄₃=-7.29; P<.001), meaning that the subjects who performed well had marked increases in frontoparietal activation and marked decreases in posterior cingulate activation at difficult levels of the task (Figure 4) .

Exploratory plots of the AUC(Pr) vs load response in each of these regions considered separately (Figure 5) confirmed that (1) schizophrenic patients generally performed poorly and had a reduced positive or negative load response compared with healthy volunteers;

(2) the effect of lorazepam markedly reduced the load response in schizophrenic patients; and (3) improved performance by schizophrenic patients after flumazenil administration was associated with an increased positive load response in the anterior cingulate cortex.

We found that the GABA-modulating drugs had differential effects on behavioural measures of working memory performance in schizophrenic patients compared with healthy volunteers . We also found that variation in cognitive performance was generally correlated with variation in brain function and that schizophrenic patients demonstrated an abnormal pattern of drug effects on frontoparietal activation and temporocingulate deactivation. Behavioral Effects of Gaba-Modulating Drugs On Task Performance After placebo administration, the schizophrenic patients had a relatively impaired working memory performance compared with healthy volunteers . As shown in Figure 3 and Table 3 , lorazepam impaired performance in all participants, but the degree of impairment was greater in the patient group. The effects of flumazenil were also different between groups: a tendency for impaired performance in healthy volunteers contrasted with a significant enhancement of performance in schizophrenia patients (for post-hoc t tests, see Figure 3) . These observations cannot be explained readily in terms of differential drug effects on processing speed or general sedative drug effects (e.g. lorazepam) because there was no significant group-drug interaction for response latency.

However, they are compatible with the hypothesis that cognitive deficits in schizophrenia are related to inhibitory neuronal abnormalities. There is now considerable histopathological evidence indicating cellular abnormalities of inhibitory interneurons in schizophrenic patients.¹¹'¹²'²⁵ The parvalbumin expressing, chandelier class of cortical interneurons is most clearly implicated in the pathophysiology of schizophrenia. ³⁰'⁵⁹ These cells, concentrated in neocortex layers III through V, form clustered inhibitory synapses on axon initial segments and somas of pyramidal cells and are thus powerfully positioned to coordinate pyramidal firing. Cellular abnormalities of GABA interneurons have been reported, especially in the anterior cingulate and prefrontal cortices, in postmortem studies of schizophrenia.²⁷'²⁸'⁶⁰ There is also evidence of reduced prefrontal expression of messenger RNA coding the GABA-synthesizing enzyme glutamic acid decarboxylase and the trophic protein reelin, which is released presynaptically from some classes of interneurons and is involved in sustaining the plasticity of dendritic spines on pyramidal cells .³⁰'³¹'^61"65

These presynaptic changes would be expected to attenuate inhibitory neurotransmission by cortical interneurons. However, there is also evidence of reduced prefrontal expression of messenger RNA coding the GABA membrane transporter ^ll4e which would tend to increase synaptic availability of GABA, and increased postsynaptic density of GABAA receptors,^27"29 which would tend to enhance postsynaptic inhibitory GABA effects. It has been persuasively argued that the primary patho-genetic event is presynaptic down-regulation of GABA synthesis, which triggers compensatory changes in postsynaptic receptor density.¹¹'¹²'³² However, it is currently unclear whether the net effect of presynaptic down-regulation of GABA synthesis and release, combined with postsynaptic compensatory changes in GABAA receptor density, is to increase or to decrease the inhibitory tone prevailing on pyramidal cells.

Our behavioral data, indicating that working memory deficits in schizophrenia can be exacerbated by lorazepam (a positive allosteric modulator of GABA transmission) and ameliorated by flumazenil (an antagonist or a partial inverse agonist at the GABAA receptor) , are compatible with the view that cognitive impairment may reflect a pathological state of overinhibition in schizophrenia. These data imply that postsynaptic changes supposed to compensate for presynaptic down-regulation of GABA synthesis must in some sense be over-compensatory.

N-Back Activation And Deactivation Networks

In healthy volunteers undergoing MRI after placebo administration, the performance of the N-back working memory task at increasing levels of difficulty predictably engendered increasing activation in a large-scale system consisting of the prefrontal, premotor, antdrior cingulate, and parietal cortices and the cerebellum. A similar activation pattern was reported in a meta-analysis of 24 functional neuroimaging studies of the N-back paradigm³⁸ and positive load-response properties of the prefrontal, premotor, and anterior cingulate cortices have been reported across a diverse range of cognitive tasks.⁶⁶ In addition, we found in healthy volunteers after placebo administration that increasing task difficulty caused increasing deactivation in a large-scale system consisting of the lateral temporal, dorsal, and posterior cingulate cortices. Deactivation of similar regions has previously been reported in some working memory studies; posterior cingulate 05 deactivation, in particular, has been reported across a wide range of cognitive tasks.^57"71 Theoretical interest in fMRI deactivation has focused recently on the idea that common patterns of deactivation across multiple experimental tasks may represent reallocation of activation resource from a resting or "default mode" network, including the posterior cingulate cortex, to other brain regions specialized for processing task demands.^72"75 More generally, we can think of the brain as more or less efficient in rationing a finite activation resource between functionally competitive large-scale neurocognitive systems.⁷⁰'⁷²'⁷³'⁷⁶ In keeping with this economic model, it has been shown previously, as well as in these data, that a greater magnitude of activation in task-related regions is associated with a greater magnitude of deactivation in default regions.⁷⁰'⁷⁶ We have further shown, for the first time, that the strength of correlate activation/deactivation or efficiency of competition—between task-related and default systems is associated with superior cognitive performance. In this theoretical context, we interpret the fMRI data in schizophrenic patients as indicating relative inefficiency of resource reallocation between competitive systems. Similar patterns of reduced frontal activation and/or reduced lateral temporal deactivation have been reported in functional neuroimaging studies of schizophrenia and discussed in terms of hypofrontality or frontotemporal dysconnectivity.^77"81 Neurocognitive inefficiency has also been invoked conceptually to describe abnormal associations between cognitive performance and the magnitude of frontal activation in schizophrenia'⁸²'⁸³ However, neurotransmitter mechanisms for these functional neuroimaging phenomena have not previously been investigated.

Our observation that lorazepam and flumazenil reduced both frontoparietal activation and temporocingulate deactivation in healthy volunteers, provides indication that normal inhibitory interneuronal function is critical for efficient systems competition. The more salient effects of lorazepam on brain function in schizophrenic patients, which were essentially to exaggerate the disease-related profile of competitive inefficiency, suggest that abnormalities of GABA neurons may also subtend abnormalities of large-scale correlated brain activation in schizophrenia.

Therapeutic Implications The main clinical inference from these data is that cognitive adverse effects of benzodiazepines should be monitored closely in schizophrenia. Few studies have examined the effects of benzodiazepine on cognition in schizophrenia, with the exception of a single photon emission tomography study suggesting a differential relationship between benzodiazepine receptor binding and cognitive performance in schizophrenic patients compared with healthy controls.⁸⁴ Our data indicate that a single dose of lorazepam administered to schizophrenic patients can cause deterioration of working memory to a much greater extent than the same dose administered to healthy controls.

Few published reports have examined pharmacotherapies for cognition in schizophrenia. These include discussion of the benefits of atypical vs typical antipsychotics⁸⁵'⁸⁶ and, motivated by the cholinergic model of Alzheimer disease, an interest in cholinergic agonists and acetylcholinesterase inhibitors .⁸⁷ Most recently, Spence et al⁸⁸ reported increased activation of the anterior cingulated correlated with N-back performance, in schizophrenic patients after modafinil administration. Based on data indicating a primary presynaptic deficit in GABA synthesis, there is interest in the use of alpha2- selective GABAA agonists,¹²'³² but there are no empirical data yet available to support this . Our results alternatively suggest that further efforts to discover GABA- modulating drugs for cognition might justifiably include compounds with antagonist properties at the benzodiazepine site, like flumazenil but more conveniently administered.

Methodological Issues

Our sample size is small and there are attendant risks of type II (false-negative) error. The schizophrenic patients were receiving medication and were chronically ill (Table 2) . It is difficult to predict the interactions that may occur between antipsychotics and the GABA modulating drugs used herein, and thus it is unclear how generalizable these results would be to first-episode or medication- naive patients (although Perault et al⁸⁹ report a lack of interaction between the effects of lorazepam and an atypical antipsychotic on memory) . In addition, we assumed a linear relationship between task, difficulty and physiological load response; this may be a simplistic assumption necessitated by the limited number of levels of task difficulty available for modeling nonlinear load response.⁸² Pharmacological MRI is increasingly used for measuring the functional effects of psychoactive drugs on large-scale neurocognitive systems. ⁹⁰

However, interpretation of pharmacological MRI findings is complex. It is possible that GABA modulating drugs could cause changes in task-related blood oxygen level-dependent contrast by direct vascular effects, although the correlation between fMRI findings and behavioral outcome measures reduces the plausibility of this interpretation of our findings. We have reported evidence of differential effects of GABA-tnodulating drugs on cognition and related brain function in schizophrenic patients compared with healthy volunteers. We argue that these results are compatible with a pathological state of over inhibition in schizophrenia, leading to inefficient reallocation of activation resources between task-related and default brain systems.

Table 1

Table 2

Table 3

References :

1. Elvevag B, Goldberg TE. Crit Rev Neurobiol. 2000; 14:1-21.

2. Barnett JH et al Psychol Med. 2005 ; 35 : 1031-1041.

3. Green MF. Am J Psychiatry. 1996; 153 : 321-330. 4. Harvey PD, et al Am J Psychiatry. 1998 ; 155 : 1080-1086.

5. Kuperberg G, et al Curr Opin Neurobiol .2000; 10 : 205-210.

6. Bartok E, et al Prog Neuropsychopharmacol Biol Psychiatry. 2005;29:621-625.

7. Brewer WJ, et al .AmJ Psychiatry. 2005 ; 162 : 71-78. 8. Keefe RS et al Psychiatry Res. 1994 ; 53 : 1-12.

9. Kremen WS et al Schizophr Bull. 1994; 20 : 103-119.

10. Egan MF, et al Biol Psychiatry. 2001; 50 : 98-107.

11. Lewis DA, et al Nat Rev Neurosci . 2005 ; 6 : 312-324.

12. Guidotti A et al Psychopharmacology (Berl) . 2005 ; 180 : 191-205. 13. Coyle JT. Biochem Pharmacol. 2004 ; 68 : 1507-1514.

14. Buzsaki G et al Science. 2004;304 : 1926-1929.

15. Buzsaki G et al Trends Neurosci. 2004 ; 27 : 186-193.

16. Cobb SR et al Nature. 1995 ; 378 : 75-78.

17. Engel AK et al . Nat Rev Neurosci. 2001; 2 : 704-716. 18. Howard MW et al . Cereb Cortex. 2003 ; 13 : 1369-1374.

19. Sederberg PB et al . J Neurosci. 2003 ; 23 : 10809-10814.

20. McBain CJ et al . Nat Rev Neurosci. 2001;2 : 11-23.

21. Jones EG. et al Cereb Cortex. 1993 ;3 : 361-372.

22. Tamas G, et al Nat Neurosci. 2000; 3:366-371. 23. Spencer KM et al J Neurosci. 2003; 23: 7407-7411.

24. Spencer KM et al Proc Natl Acad Sci U S A. 2004; 101 : 17288-17293.

25. Benes FM. et al Res Brain Res Rev. 2000 ; 31 : 251-269.

26. Benes FM, et al Neuropsychopharmacology. 2001; 25:1-27.

27. Benes FM, et al J Neurosci. 1992 ; 12 : 924-929. 28. Benes FM, et al . Neuroscience . 1996; 75 : 1021-1031.

29. VoIk DW, et al . Cereb Cortex. 2002 ; 12 : 1063-1070.

30. Hashimoto T et al J Neurosci. 2003 ; 23 : 6315-6326.

31. Akbarian S, et al Arch Gen Psychiatry. 1995; 52:258-266.

32. Lewis DA, et al . Psychopharmacology (Berl). 2004; 174 : 143-150. 33. Wolkowitz OM et al . Am J Psychiatry. 1991; 148 : 714-726.

34. Carpenter WT Jr, et al . Am J Psychiatry. 1999; 156: 299-303.

35. Park S et al . Arch Gen Psychiatry. 1992 ; 49 : 975-982. 36. Lencz T et al Arch Gen Psychiatry. 2003 ; 60 : 238-243.

37. Gold JM, et al Arch Gen Psychiatry. 1997; 54 : 159-165.

38. Owen AM, et al Hum Brain Mapp. 2005 ;25 : 46-59.

39. Glahn DC, et al Hum Brain Mapp. 2005; 25: 60-69. 40. Doble A. et al 1999; 13 (suppl 1):S11-S2O.

41. O'Boyle C, et al . Br J Anaesth. 1983 ; 55 : 349-356.

42. File SE et al Psychopharmacology (Berl) . 1986; 88 : 1-11.

43. Neave N et al Br Dent J. 2000 ; 189 : 668-674.

44. LaI H et al FASEB J. 1988;2 : 2707-2711. 45. DeLorey TM, et al Eur J Pharmacol. 2001 ; 426 : 45-54.

46. VoIk D et al Am J Psychiatry. 2001; 158 : 256-265.

47. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) . Washington, DC: American Psychiatric Association; 1994. 48. Nelson H. National Adult Reading Test (NART) Manual. Windsor, England: NFER Publishing Co; 1982.

49. Wilson B et al J Clin Exp Neuropsychol . 1989; 11 : 855-870.

50. Howell D. Statistical Methods for Psychology. Belmont, Calif: Duxbury Press; 2002

Claims

1. A method of treating cognitive impairment in an individual having a psychiatric condition comprising; administering a therapeutically effective amount of a compound which reduces or inhibits γ-aminobutyric acid (GABA) -mediated neurotransmission to an individual in need thereof.

2. A method according to claim 1 wherein the compound is a GABA_A antagonist.

3. A method according to claim 2 wherein the compound is a benzodiazepine antagonist .

4. A method according to claim 3 wherein the compound is an imidazobenzodiazepine, beta-carboline or pyrazoloquinoline.

5. A method according to any one of the preceding claims wherein the compound is flumazenil .

6. A method according to any one of the preceding claims wherein the psychiatric condition is schizophrenia or bipolar disorder.

7. Use of a compound which reduces or inhibits γ-aminobutyric acid (GABA) -mediated neurotransmission in the manufacture of a medicament for use in a method of treating cognitive impairment in an individual having a psychiatric condition.

8. A method of screening for a compound useful in treating cognitive impairment in an individual having a psychiatric condition comprising; contacting a GABA_A receptor with a GABA_A receptor agonist in the presence of a test compound and; determining the binding and/or activation of the GABA_A receptor by the agonist, wherein a decrease in binding and/or activation by the agonist in the presence relative to the absence of the test compound is indicative that the test compound is useful in treating cognitive impairment in an individual having a psychiatric condition.