Daniel Campbell-Meiklejohn

Dphil

Judi Wakeley Bsc

Vanessa Herbert Bsc

Jennifer Cook Bsc

Paolo Scollo Bsc

Manaan Kar

Ray Mbbs

M Sc Mrcpsych

Sudhakar Selvaraj

Dphil Mrcpsych

Richard E Passingham Dphil

MD FRCPsych Phillip Cowen

Robert D Rogers Ba

Msc Dphil

Robert D Rogers
email: robert.rogers@psych.ox.ac.uk

Serotonin and dopamine play complementary roles in gambling to recover losses

Keywords: serotonin, dopamine, loss-chasing, gambling, persistence, value Serotonin and dopamine in loss-chasing behaviour

Continued gambling to recover losses -'loss-chasing'is a prominent feature of social and pathological gambling. However, little is known about which neuromodulators influence this behaviour. In 3 separate experiments, we investigated the role of serotonin activity, D 2 /D 3 receptor activity, and beta-adrenoceptor activity on the loss-chasing of age and IQ-matched healthy adults randomised to treatment or an appropriate control/placebo. In Experiment 1, participants consumed amino acid drinks that did or did not contain the serotonin precursor, tryptophan. In Experiment 2, participants received a single 176µg dose of the D 2 /D 3 receptor agonist, pramipexole, or placebo. In Experiment 3, participants received a single 80mg dose of the beta-adrenoceptor blocker, propranolol, or placebo. Following treatment, participants completed a computerised loss-chasing game. Mood and heart rate was measured at baseline and following treatment. Tryptophan depletion significantly reduced the number of decisions made to chase losses, and the number of consecutive decisions to chase, in the absence of marked changes in mood. By contrast, pramipexole significantly increased the value of losses chased and diminished the value of losses surrendered. Propranolol markedly reduced heart rate but produced no significant changes in loss-chasing behaviour. Loss-chasing can be thought of as an aversively motivated escape behaviour controlled, in part, by the marginal value of continued gambling relative to the value of already accumulated losses. Serotonin and dopamine appear to play dissociable roles in the tendency of individuals to gamble to recover or to seek to 'escape' from previous losses. Serotonergic activity seems to promote the availability of loss-chasing as a behavioural option, while D 2 /D 3 receptor activity produces complex changes in the value of losses judged worth chasing. Sympathetic arousal, at least as mediated by beta-adrenoceptors, does not play a major role in loss-chasing choices.

Serotonin and dopamine in loss-chasing behaviour v3;

INTRODUCTION

Continued gambling to recover previous losses, or loss-chasing [START_REF] Lesieur | The Chase: career of the compuslive gambler, First Edition edn[END_REF], is a central feature of human decision-making [START_REF] Kahneman | Choices Values and Frames[END_REF]. However, in a clinical context, excessive loss-chasing is also a prominent indicator of impaired control in a significant proportion of those individuals who report problems with their gambling behaviour [START_REF] Corless | Gamblers' self-perceptions of the determinants of impaired control[END_REF][START_REF] Mcbride | A latent class analysis of DSM-IV pathological gambling criteria in a nationally representative British sample[END_REF][START_REF] Sacco | Differential Item Functioning of Pathological Gambling Criteria: An Examination of Gender, Race/Ethnicity, and Age[END_REF]. Left unchecked, loss-chasing can produce a dangerous spiral of gambling involvement, increasing financial liabilities but diminishing resources; and, potentially, the serious adverse family, social and occupational consequences of pathological gambling [START_REF] Lesieur | The compulsive gambler's spiral of options and involvement[END_REF].

At a psychological level, loss-chasing is complex and frequently involves conflicted motivational states, pitting the desire (or need) to keep playing against the dread of suffering even greater losses [START_REF] Lesieur | The Chase: career of the compuslive gambler, First Edition edn[END_REF]; powerful emotional states that are mediated by activity within dissociable neural circuits [START_REF] Campbell-Meiklejohn | Knowing when to stop: the brain mechanisms of chasing losses[END_REF]. Gambling to recover losses is also associated with heightened states of arousal (see below) and a preoccupation with gambling activities that is a prominent feature of the clinical presentation of gambling problems [START_REF] Dickerson | Chasing, arousal and sensation seeking in off-course gamblers[END_REF][START_REF] Mcbride | A latent class analysis of DSM-IV pathological gambling criteria in a nationally representative British sample[END_REF]. Consequently, loss-chasing may represent a salient target for the development of therapeutic interventions.

Despite its centrality to problem gambling, we know little about the way loss-chasing is influenced by the activity of neurochemical systems. A small amount of clinical evidence suggests that pathological gambling is associated with serotonergic dysfunction as exemplified by (inconsistent) reports of reduced concentrations of the serotonin metabolite, 5hydroxyindoleacetic acid in cerebrospinal fluid [START_REF] Bergh | Altered dopamine function in pathological gambling[END_REF][START_REF] Roy | Pathological gambling. A psychobiological study[END_REF] and by reports of increased prolactin release (and reports of a 'high') following acute challenge with Serotonin and dopamine in loss-chasing behaviour v3; 19 July 2010 4 the 5-HT 2c receptor agonist, meta-chlorophenylpiperazine [START_REF] Pallanti | Serotonin dysfunction in pathological gamblers: increased prolactin response to oral m-CPP versus placebo[END_REF]. Selective serotonin reuptake inhibitors have also shown promise as a treatment of pathological gambling via their anti-compulsive and anxiolytic effects (Grant andPotenza 2006, Pallesen et al 2007). Finally, serotonin exerts pronounced -albeit, complex -influences on impulsive behaviours [START_REF] Winstanley | Fractionating impulsivity: contrasting effects of central 5-HT depletion on different measures of impulsive behavior[END_REF], which both promote loss-chasing [START_REF] Breen | Chasing' in gambling behavior: Personality and cognitive determinants[END_REF] and are exaggerated in problem gamblers [START_REF] Blaszczynski | Impulsivity in pathological gambling: the antisocial impulsivist[END_REF].

The pathophysiology of problem-gambling is also highly likely to involve dysfunction of the dopaminergic midbrain, and its mesolimbic and prefrontal projections sites [START_REF] Hewig | Hypersensitivity to reward in problem gamblers[END_REF][START_REF] Potenza | Review. The neurobiology of pathological gambling and drug addiction: an overview and new findings[END_REF]). Compared to matched healthy control subjects, pathological gamblers show reduced neuronal responses within mesostriatal nuclei while engaging in a simulated gambling behaviour for monetary reward [START_REF] Reuter | Pathological gambling is linked to reduced activation of the mesolimbic reward system[END_REF]. Administration of the psychostimulant, amphetamine, to pathological gamblers can prime cognitions about gambling [START_REF] Zack | Amphetamine primes motivation to gamble and gambling-related semantic networks in problem gamblers[END_REF] while the D 2 receptor antagonist, haloperidol, can enhance the rewarding properties of such behaviour [START_REF] Zack | A D2 Antagonist Enhances the Rewarding and Priming Effects of a Gambling Episode in Pathological Gamblers[END_REF]. Finally, accumulating evidence indicates that dopaminergic treatments are associated with pathological gambling (and other impulse control problems) in a minority of patients with Parkinson's Disease [START_REF] Voon | Factors associated with dopaminergic drug-related pathological gambling in Parkinson disease[END_REF], presumably reflecting a disturbance of dopamine's wider role in reinforcement learning and the computation of action-value relationships [START_REF] Dagher | Personality, addiction, dopamine: insights from Parkinson's disease[END_REF]Robbins 2009, Voon et al 2010). Thus, the extant evidence suggests that both serotonin and dopamine dysfunction mediate aspects of problematic gambling [START_REF] Zeeb | Serotonergic and dopaminergic modulation of gambling behavior as assessed using a novel rat gambling task[END_REF]. However, to date, their role in the central feature of loss-chasing behaviour has not yet been explored experimentally.

One way to start to understand the neurochemical substrates of the excessive loss-chasing sometimes observed in problem gamblers is to investigate the roles of different neuromodulators in the chasing behaviour of healthy adults with limited gambling Serotonin and dopamine in loss-chasing behaviour v3; 19 July 2010 5 experiences. Information gained from such experiments will assist the formulation of hypotheses about how disturbances in the activity of neuromodulators mediate loss-chasing in the pathological state. Here, in 3 experiments, we used a behavioural model of loss-chasing developed in our laboratory and already validated with functional magnetic resonance imaging [START_REF] Campbell-Meiklejohn | Knowing when to stop: the brain mechanisms of chasing losses[END_REF] to compare the loss-chasing behaviour of nonclinical healthy adults (who reported only very limited gambling involvement) following manipulations of serotonergic, dopaminergic (D 2 /D 3 ) and beta-adrenoceptor activity.

In Experiment 1, we investigated the effects of tryptophan depletion on the tendency to continue gambling to recover losses and tested between 2 hypotheses with clearly divergent predictions. Serotonin is known to play a prominent role in the control of non-rewarded activity and the inhibition of behaviour following the occurrence of punishing or aversive events [START_REF] Soubrie | Serotonergic neurons and behavior[END_REF]. Furthermore, temporary reductions in central serotonin activity, achieved through tryptophan depletion, can diminish punishment-induced inhibition of ongoing behaviour in healthy adults [START_REF] Crockett | Reconciling the role of serotonin in behavioral inhibition and aversion: acute tryptophan depletion abolishes punishment-induced inhibition in humans[END_REF]. On this basis, we might expect that tryptophan depletion will increase the tendency to continue gambling in order to recover previous losses through a loss of serotonin-dependent behavioural inhibition.

On the other hand, serotonin also mediates important aspects of learning about negative events [START_REF] Bari | Serotonin Modulates Sensitivity to Reward and Negative Feedback in a Probabilistic Reversal Learning Task in Rats[END_REF][START_REF] Daw | Opponent interactions between serotonin and dopamine[END_REF][START_REF] Deakin | 5-HT and mechanisms of defence[END_REF][START_REF] Evers | Serotonergic modulation of prefrontal cortex during negative feedback in probabilistic reversal learning[END_REF]. [START_REF] Dayan | Serotonin, inhibition, and negative mood[END_REF] has proposed that failures of behavioural control following reductions in serotonin activity (experimental or clinical) can produce pervasive increases in the size of negative prediction errors that, in turn, engender negative affective states in vulnerable individuals [START_REF] Dayan | Serotonin, inhibition, and negative mood[END_REF]. Experimentally, tryptophan depletion can improve the accuracy of predictions of negative or punishing outcomes in healthy adults [START_REF] Cools | Acute tryptophan depletion in healthy volunteers enhances punishment prediction but does not affect reward prediction[END_REF]. Moreover, [START_REF] Evers | Serotonergic modulation of prefrontal cortex during negative feedback in probabilistic reversal learning[END_REF] showed that tryptophan depletion enhances neural activity in response to Serotonin and dopamine in loss-chasing behaviour v3; 19 July 2010 6 errors during reversal learning within the anterior cingulate region; an area that is activated while making decisions to stop chasing losses [START_REF] Campbell-Meiklejohn | Knowing when to stop: the brain mechanisms of chasing losses[END_REF]. Thus, we might also predict that tryptophan depletion will enhance the salience of bad outcomes during a run of losing gambles, and diminish subsequent loss-chasing behaviour.

In Experiment 2, we investigated the effects of a single dose of the non-ergoline D2/D3 receptor agonist, pramipexole. Alongside other dopaminergic treatments, treatment with pramipexole has been associated with gambling problems in a subset of Parkinson's Disease patients [START_REF] Voon | Factors associated with dopaminergic drug-related pathological gambling in Parkinson disease[END_REF]. However, there has been no test of whether D2/D3 receptors agonists alter chasing behaviour during a run of losing gambles.

Pramipexole is significantly more selective for D 3 than D 2 receptors and binds to dopamine (autoreceptor and post-synaptic) receptors in mesolimbic reward pathways (Camacho-Ochoa et al 1995) (see Supplemental Information). Single low doses of pramipexole (e.g. 0.5mg) can impair reinforcement learning in healthy adults [START_REF] Pizzagalli | Single dose of a dopamine agonist impairs reinforcement learning in humans: behavioral evidence from a laboratory-based measure of reward responsiveness[END_REF], and increase risky choices in lottery-type games [START_REF] Riba | Dopamine agonist increases risk taking but blunts reward-related brain activity[END_REF], possibly through blunted reward signalling of mesolimbic pathways [START_REF] Riba | Dopamine agonist increases risk taking but blunts reward-related brain activity[END_REF][START_REF] Santesso | Single dose of a dopamine agonist impairs reinforcement learning in humans: evidence from eventrelated potentials and computational modeling of striatal-cortical function[END_REF]. In the light of this, and evidence that low doses of pramipexole, and other agents acting upon D2 receptors impair the signalling of bad outcomes ('negative prediction errors') [START_REF] Frank | A mechanistic account of striatal dopamine function in human cognition: psychopharmacological studies with cabergoline and haloperidol[END_REF][START_REF] Santesso | Single dose of a dopamine agonist impairs reinforcement learning in humans: evidence from eventrelated potentials and computational modeling of striatal-cortical function[END_REF][START_REF] Van Eimeren | Dopamine agonists diminish value sensitivity of the orbitofrontal cortex: a trigger for pathological gambling in Parkinson's disease?[END_REF], we tested the hypothesis that single doses of pramipexole increase loss-chasing behaviour and, perhaps, influence the value of losses that individuals are prepared to chase.

Although it is unlikely that the findings we report in Experiments 1 and 2 reflect gross changes in subjective states associated with either tryptophan depletion or treatment with Serotonin and dopamine in loss-chasing behaviour v3; 19 July 2010 7 pramipexole, it is possible that our observations relate to changes in alerting or arousal, perhaps reflecting the relatively prolonged protocols of pharmacological experiments. For example, while tryptophan depletion typically does not modify state affect in adults who have been screened for affective disorders, it may attenuate physiological (cardiac) responses to negative performance feedback [START_REF] Van Der Veen | Acute tryptophan depletion in healthy males attenuates phasic cardiac slowing but does not affect electrocortical response to negative feedback[END_REF]. Moreover, field studies indicate that commercial gambling is associated increased in sympathetic arousal [START_REF] Anderson | Real and laboratory gambling, sensation-seeking and arousal[END_REF]Brown 1984, Meyer et al 2000). However, it is unclear whether changes in arousal might increase or decrease the tendency to keep gambling to recover losses. Previously, we have found that single doses of the beta-adrenoceptor antagonist, propranolol, reduced decisionmakers' attention towards punishment-related cues [START_REF] Rogers | Effects of beta-adrenoceptor blockade on components of human decision-making[END_REF], potentially releasing loss-chasing behaviour. Therefore, in Experiment 3, we tested whether changes in arousal, as reflected in the kind of reduced heart rate produced in healthy adults by a single dose of the beta-adrenoceptor antagonist propranolol would influence loss-chasing behaviour.

Materials and Methods

Participants and Designs

All participants were given a clinical examination by an experienced psychiatrist, including a semi-structured SCID-I interview to ensure that none of the following exclusion criteria were met: (i) major physical illness, (ii) current or previous DSM-IV major mood or psychotic disorder (iii) current or previous DSM-IV substance abuse disorder. Participants were assessed with the South Oaks Gambling Screen [START_REF] Lesieur | The South Oaks Gambling Screen (SOGS): a new instrument for the identification of pathological gamblers[END_REF]; all scores were either 0 or 1, indicating no evidence of gambling problems or pathological gambling.

Experiment 1. Thirty four healthy adults participated. None had any history of mood disorder; there was no restriction on the phase of menstrual cycle in female participants.

Serotonin and dopamine in loss-chasing behaviour v3; 19 July 2010 8 Seventeen participants (8 males) ingested an amino-acid drink that did not contain tryptophan (T-) and 17 participants (8 males) ingested an amino-acid drink that did contain tryptophan (T+). The T+ participants and T-participants were matched in terms of their gender (see Table S1), age (F< 1.00), and cognitive ability (Raven et al 1998) (F(1,30)< 2.08).

Participants followed a low-protein diet (<2g) the day before the study, and fasted overnight before attending the laboratory at 8.30 am on the day of the experiment. Measures of state positive and negative affect [START_REF] Watson | Development and validation of brief measures of positive and negative affect: the PANAS scales[END_REF] were taken at this time along with 15ml blood samples to obtain total plasma tryptophan concentrations. Participants then drank an amino-acid drink over a 60min period. None of the participants reported side effects beyond transitory nausea. Participants were given a low-protein (<2g) lunch at midday. Repeat state positive and negative affect measurements, and a second blood sample, were collected +5h after consumption of the amino acid drink, before completing the loss-chasing game.

Experiment 2. Thirty healthy adults were randomly assigned to receive 176µg of pramipexole (PPX) or placebo (Placebo-PPX). Each group contained 7 males. There were no significant differences between those participants who received placebo and those who received pramipexole in terms of their age or cognitive ability (Table S2) (both Fs< 1.00).

The 176µg dose of pramipexole used in Experiment 2 is comparable to dosages shown to be clinically effective for restless leg syndrome [START_REF] Manconi | First night efficacy of pramipexole in restless legs syndrome and periodic leg movements[END_REF]. There are good reasons to suppose that the subjective [START_REF] Hamidovic | Effects of low to moderate acute doses of pramipexole on impulsivity and cognition in healthy volunteers[END_REF] and behavioural [START_REF] Pizzagalli | Single dose of a dopamine agonist impairs reinforcement learning in humans: behavioral evidence from a laboratory-based measure of reward responsiveness[END_REF][START_REF] Riba | Dopamine agonist increases risk taking but blunts reward-related brain activity[END_REF][START_REF] Santesso | Single dose of a dopamine agonist impairs reinforcement learning in humans: evidence from eventrelated potentials and computational modeling of striatal-cortical function[END_REF] effects of low doses of dopaminergic agents reflect pre-synaptic actions at the auto-receptors that regulate the activity of mid-brain dopaminergic neurones [START_REF] Frank | Hold your horses: a dynamic computational role for the subthalamic nucleus in decision making[END_REF]O'Reilly 2006, Grace 1995). As Serotonin and dopamine in loss-chasing behaviour v3; 19 July 2010 9 described below, we replicate findings that single (1mg) low doses of pramipexole reduce psychometric measurements of state positive affect in healthy adults and that have been taken to suggest a pre-synaptic mode of action [START_REF] Hamidovic | Effects of low to moderate acute doses of pramipexole on impulsivity and cognition in healthy volunteers[END_REF]. However, our 176µg dose is also comparable to those shown to reduce serum prolactin over 2hr [START_REF] Schilling | Neuroendocrine and side effect profile of pramipexole, a new dopamine receptor agonist, in humans[END_REF], at least raising the possibility that our results also involve some post-synaptic receptor activity [START_REF] Ben-Jonathan | Dopamine: a prolactin-inhibiting hormone[END_REF].

Participants attended the laboratory at 8.30am and completed baseline assessments of state positive and negative affect [START_REF] Watson | Development and validation of brief measures of positive and negative affect: the PANAS scales[END_REF]. Baseline measures of systolic/diastolic blood pressure (BP) and heart rate (HR) were collected. Following this, participants received a single 176µg dose of pramipexole or a gelatine capsule containing lactose. Two hours later (+2h), further measurements of systolic/diastolic BP and HR were taken. State positive and negative affect were also collected at this time, before completion of the loss-chasing game.

Experiment 3. Fourteen (7 males) participants were randomly assigned to receive 80mg propranolol (Placebo-PPL) and 14 participants (8 males) were randomly assigned to receive a lactose placebo (PLA-PPL). The 2 groups of participants were well-matched in terms of their age (see Table S4), F<1, and their cognitive ability (F(1, 24)= 1.87).

Participants attended the laboratory in the mornings having fasted for 2h and without caffeine intake. State positive and negative affect (PANAS) [START_REF] Watson | Development and validation of brief measures of positive and negative affect: the PANAS scales[END_REF], systolic BP, diastolic BP and HR was assessed at baseline and then every 30min thereafter. Participants completed the computerised loss-chasing game +75min following treatment.

Serotonin and dopamine in loss-chasing behaviour v3; 19 July 2010 10

Loss-chasing Game

A version of our loss-chasing game suitable for functional magnetic resonance imaging has been described in detail elsewhere [START_REF] Campbell-Meiklejohn | Knowing when to stop: the brain mechanisms of chasing losses[END_REF]. On each play, participants were required to choose between gambling to recover a loss (at the risk of doubling its size) or quitting (and sustaining a certain loss). Such dilemmas induce risky choices in a variety of social and economic contexts [START_REF] Shafir | Decision Making[END_REF]. Descriptive theories of choice (under uncertainty) attribute this behaviour to the fact that losses fall on the convex part of a psychophysical function relating nominal value (e.g. monetary outcomes) to subjective value or utility, such that the decreases in utility associated with chasing and suffering larger losses are proportionately smaller than the decreases in utility associated with certain but smaller losses [START_REF] Kahneman | Choices Values and Frames[END_REF]. Previously, we found that gambling to recover losses during our game is positively associated with psychometric measures of the tendency to chase losses in other gambling activities [START_REF] Campbell-Meiklejohn | Knowing when to stop: the brain mechanisms of chasing losses[END_REF].

Figure 1 about here At the start of the game, participants were told that they had a fictional £20,000 to play with, but that the participant with the most points at the end of the experiment would win a real prize of £70. On each 'round' of the game, an initial £10, £20, £40, £80 or £160 was subtracted from their game total. This amount appeared below the choices: 'Quit' and 'Play' (Figure 1). At this point, participants could choose to 'Quit', sustaining this loss and ending the round immediately ('quit-loss' outcome), or they could choose to 'Play', that is, chase the loss. Thus, they could gamble on recovering an amount equal to the loss but at the risk of increasing their losses by the same amount. If the outcome of a decision to gamble were positive ('chase-win' outcome), the loss was recovered and the round ended. If the outcome Serotonin and dopamine in loss-chasing behaviour v3; 19 July 2010 11 were negative ('chase-loss' outcome), the loss was doubled and participants given another chance to quit or to chase in the next choice of the round. The options for each choice -'Play' or 'Quit'-appeared equally often on the left and right sides of the displays.

Outcome displays (see Figure 1), indicated whether participants had won a gamble and that no money was lost ('chase-win'); whether they had lost a gamble and the amount lost ('chaseloss'); or the amount lost if participants chose to quit the round ('quit-loss'). At the end of each round, participants were also informed of their final losses in a 'round-loss' display. This display indicated the total cumulative losses for that round, in red text if the losses were greater than 0 but in green text if 0. Rounds of the loss-chasing game began with losses of £10, £20, £40, £80 or £160. If participants continued losing, losses kept doubling until they reached £640, at which point the round ended, having incurred the maximum loss.

All participants played 20 rounds of the loss-chasing game. Chase-win outcomes were positioned randomly within each round such that winning outcomes occurred equally often after any number of (between 0 and 5) consecutive losses. The outcomes of the loss-chasing game were distributed such that 14 rounds returned all losses if participants decided to play on every choice of the game. However, 6 rounds resulted in the maximum loss of £640.

Participants were not told anything about the probabilities of good versus bad outcomes so that their decisions were made under conditions of 'ambiguity' [START_REF] Camerer | Recent developments in modeling preferences: Uncertainty and ambiguity[END_REF]. In order to discourage participants from adopting conservative strategies by which they quit early to preserve as much of their play money as possible, no information was provided about their cumulative game total of play money during the game. Participants were also informed that they would not achieve the best possible score by exclusively playing or quitting. 12 To summarise, participants were confronted with a series of dilemmas involving a choice between gambling to recover a loss of at the risk of doubling its size, or sustaining the loss and ending the chase, while at the same time preserving as much as possible the resources that allowed play to continue [START_REF] Campbell-Meiklejohn | Knowing when to stop: the brain mechanisms of chasing losses[END_REF]. The value of these resources (experimenter-defined points) was provided by the context of an inter-participant competition requiring participants to retain as many points as possible. This mixture of nominal and actual rewards have been used in economics to demonstrate behaviour qualitatively and quantitatively similar to that observed outside the laboratory [START_REF] Cubitt | On the validity of the random lottery incentive system[END_REF].

Statistical Analysis

Dependent measures included the proportion of choices to gamble (or chase) out of all choices made during the game, and the mean number of losses chased per round. We analysed the magnitude (or value) of losses chased and the magnitude (or value) of losses surrendered during the game. These values were expressed as ratios to the mean values of all losses encountered during the game; see Supplementary Information for more details.

Demographic, subjective and loss-chasing measures for the 3 experiments were tested using one-way analysis of variance (ANOVA) with the between-subjects factors of treatment (T-vs T-, pramipexole vs placebo, or propranolol vs placebo) and gender.

RESULTS Experiment 1: Tryptophan Depletion

Physiological and subjective effects. Consumption of the amino acid drink without tryptophan (in the T-treatment) produced a significant reduction in total plasma concentration Serotonin and dopamine in loss-chasing behaviour v3; 19 July 2010 13 5 hours later compared to the control drink (in the T+ treatment) (see Table S1). However, the T-treatment did not produce any marked changes in either state positive or negative affect compared to the T+ treatment (Table S1) (all F(1, 30)s< 2.29). In contrast to the effects on the proportion of gambles to recover losses, there was no significant change in the value of losses that the tryptophan-depleted participants decided to chase (expressed as ratios to the mean values of all losses encountered during the game; Supplementary Information) (Figure 3a) compared to the participants who received the control procedure (Fs< 1). Neither was there any significant change in the value of losses surrendered when deciding to quit (Figure 3b) (Fs< 1).

Experiment 2: Pramipexole

Physiological and subjective effects. Systolic BP, diastolic BP and heart rate were not significantly altered following treatment with pramipexole compared to treatment with placebo (see Supplementary Information and Table S3) (all F(1, 25)s< 1.86).

Serotonin and dopamine in loss-chasing behaviour v3; 19 July 2010 14 Treatment with pramipexole significantly reduced state positive affect in comparison to placebo across the +2h following treatment, F(1, 26)= 10.05, p< .005 (Table S2). Specifically, while positive affect tended to increase following treatment with placebo (F(1, 13)= 3.53, p= .08), it was significantly decreased following treatment with pramipexole (F(1, 13)= 6.84, p< .05). At +2h, when completing the loss-chasing game, participants who received pramipexole reported lower positive affect than those who received placebo (F(1, 26)= 8.34, p< .01).

Pramipexole did not alter state negative affect compared to placebo (all Fs< 1).

Figure 3 about here

Loss-chasing.

Pramipexole slightly reduced the number of decisions to chase, and the number of consecutive decisions to chase, during a run of losing gambles compared to placebo (Figure 2); however, neither of these effects were statistically significant (Fs< 1). However, by contrast, pramipexole significantly increased the value of losses that participants decided to gamble to recover (Figure 3a) (F(1, 26)= 4.94, p< .05), and also significantly reduced the value of losses participants surrendered (Figure 3b) (F(1, 26)= 5.87, p< .05). These changes in the value of losses chased and surrendered remained significant when positive affect at +2hr was entered as a covariate, (F1, 25)= 4.48, p< .05 and F(1, 25)= 4.39, p< .05, respectively).

They were also broadly unaltered when the statistical analysis was performed on the unadjusted value of losses chased or values surrendered (see Supplementary Information).

Experiment 3: Propranolol

Physiological and subjective effects. Propranolol did not produce significantly different larger or smaller changes in systolic or diastolic BP compared to placebo, (all Fs< 1). HR diminished over the 75min following treatment (73.64±10.82bpm vs 62.04±7.68bpm), F(1, Serotonin and dopamine in loss-chasing behaviour v3; 19 July 2010 15 24)= 60.30, p< .0001. However, this reduction was significantly greater following propranolol compared to placebo (Table S5) (F(1, 24)= 4.98, p< .05). Since baseline HR tended to be greater in participants treated with propranolol compared to participants treated with placebo (F(1, 24)= 2.64), we also examined the treatment effects on the proportionate change in participants' HR. This confirmed that propranolol produced a significantly larger reduction in heart rate compared to placebo (18.64±8.45% vs 11.08±11.38%) (F(1, 24)= 4.64, p< .05).

State positive and state negative affect were not substantially different following treatment with propranolol compared to treatment with placebo (see Supplementary Information and Table S4) (F< 1.00 and F(1, 24)= 1.61, respectively). There were no significant group differences in either measure at +75min when the loss-chasing game was completed.

Loss-chasing. There were no significant differences between propranolol and placebo in terms of the number of decisions to chase, number of consecutive decisions to chase (Figure 2), or the value of losses chased and the value of losses surrendered (Figure 3), all Fs< 1.

DISCUSSION

Our findings suggest that serotonin and dopamine play complementary roles in the tendency to keep gambling to recover losses. Serotonin activity appears to play a role in sustaining losschasing behaviour while dopamine activity, involving at least the D 2 /D 3 receptor system, appears to regulate the magnitude of losses chased or surrendered at that point when players decide to quit gambling. By contrast, both these aspects of loss-chasing are broadly independent of changes in sympathetic arousal, at least as mediated by beta-adrenoceptor activity. Our data highlight novel hypotheses about the monoaminergic mechanisms that promote the expression of this central, but poorly understood, aspect of gambling behaviour.

Serotonin and dopamine in loss-chasing behaviour v3; 19 July 2010 16

In Experiment 1, we investigated the effects of tryptophan depletion to test whether central serotonin activity mediates loss-chasing behaviour. This might have been manifested in at least 2 ways. First, several lines of evidence suggest that serotonin mediates the inhibition of non-rewarded or punished behaviour [START_REF] Crockett | Reconciling the role of serotonin in behavioral inhibition and aversion: acute tryptophan depletion abolishes punishment-induced inhibition in humans[END_REF][START_REF] Dayan | Serotonin, inhibition, and negative mood[END_REF][START_REF] Soubrie | Serotonergic neurons and behavior[END_REF]). So, tryptophan depletion, leading to a reduction in serotonin activity, might have been expected to increase gambling to recover losses in our healthy adult participants. By contrast, serotonin activity also plays a significant role in learning from, and coping with, aversive events [START_REF] Bari | Serotonin Modulates Sensitivity to Reward and Negative Feedback in a Probabilistic Reversal Learning Task in Rats[END_REF][START_REF] Daw | Opponent interactions between serotonin and dopamine[END_REF][START_REF] Deakin | 5-HT and mechanisms of defence[END_REF][START_REF] Evers | Serotonergic modulation of prefrontal cortex during negative feedback in probabilistic reversal learning[END_REF]. Given that tryptophan depletion can also improve the prediction of punishing outcomes [START_REF] Cools | Acute tryptophan depletion in healthy volunteers enhances punishment prediction but does not affect reward prediction[END_REF], and enhance neural responses to punishing outcomes within the anterior cingulate cortex [START_REF] Evers | Serotonergic modulation of prefrontal cortex during negative feedback in probabilistic reversal learning[END_REF], we also anticipated that tryptophan depletion might increase the salience of bad outcomes and diminish loss-chasing behaviour. In fact, while producing no marked changes in healthy adults' state affect, tryptophan depletion significantly reduced the proportion of decisions participants made to chase losses, and also reduced the number of consecutive decisions to chase, during a run of losing gambles. This suggests that, in this instance at least, serotonin activity helps to sustain loss-chasing rather than inhibit it.

Descriptive theories of choice under uncertainty attribute loss-chasing behaviour to the idea that the prospective reductions in subjective value or utility associated with chasing and suffering larger losses still are proportionately smaller than the reductions in utility associated with the smaller losses already incurred [START_REF] Kahneman | Choices Values and Frames[END_REF]. Under these conditions, it makes sense for gamblers to continue to play, so long as the necessary resources are available. From this perspective, loss-chasing can be viewed as an aversively-motivated escape behaviour but one controlled, at least in part, by the marginal utility of continued play relative to its cessation. Our finding that tryptophan depletion reduced our behavioural model of loss-chasing suggests that, in this instance at least, diminished central serotonin activity Serotonin and dopamine in loss-chasing behaviour v3; 19 July 2010 17 reduced the marginal utility of continued play, perhaps by increasing the salience of future bad outcomes across their range of values (Cools et al 2008, Deakin and[START_REF] Deakin | 5-HT and mechanisms of defence[END_REF].

Further experiments will be needed to establish the relationship between serotonin activity and gambling to recover losses. However, given serotonin's complex contribution to impulse control, we should not assume that this relationship will be linear [START_REF] Winstanley | Fractionating impulsivity: contrasting effects of central 5-HT depletion on different measures of impulsive behavior[END_REF].

Our finding that tryptophan depletion reduced loss-chasing is in line with other observations, obtained using simple elicitation procedures to measure risk attitudes, that carriers of the 10repeat allele of the STin2 gene (that results in higher serotonin tone) show increased riskseeking choices for losses [START_REF] Zhong | A neurochemical approach to valuation sensitivity over gains and losses[END_REF]. By contrast, our present data are apparently inconsistent with findings that 2 weeks treatment with tryptophan, as a dietary substrate reduced shifts between risk-averse choices when making single decisions between certain gains and uncertain larger or smaller gains, and risk-seeking choices when making single decisions between certain losses and uncertain larger or smaller losses [START_REF] Murphy | The role of serotonin in non-normative risky choice: the effects of tryptophan supplements on the 'reflection effect' in healthy adult volunteers[END_REF].

Collectively, these data indicate that serotonin's influence upon gambling to recover losses may vary depending upon a number of psychological and pharmacological factors, including whether the experimental situation involves single or multiple consecutive choices and whether there is a context of other choices that involve positive expected values.

The effects of a single 176µg dose of pramipexole were quite different. This treatment did not increase the proportion of decisions to chase losses or the number of consecutive decisions to chase during a run of losing gambles; however, pramipexole did significantly increase the value of losses that participants were willing to chase and, at the same time, reduce the value of losses that participants were willing to surrender when quitting. Thus, a single dose of pramipexole induced a preference for chasing larger losses at the expense of smaller losses.

Serotonin and dopamine in loss-chasing behaviour v3; 19 July 2010 18

We acknowledge that the mode of action of the single 176µg dose of pramipexole used in Experiment 2 remains uncertain. While the behavioural effects of low doses of dopaminergic drugs may reflect pre-synaptic action at the auto-receptors of dopamine neurones within the mid-brain [START_REF] Frank | Hold your horses: a dynamic computational role for the subthalamic nucleus in decision making[END_REF]O'Reilly 2006, Santesso et al 2009), single doses of 100µg and 200µg pramipexole may also reduce serum prolactin, suggesting a post-synaptic action of the drug at dopamine receptors in the anterior pituitary [START_REF] Schilling | Neuroendocrine and side effect profile of pramipexole, a new dopamine receptor agonist, in humans[END_REF]. Here, replicating previous findings, we note that our dose of 176µg pramipexole also significantly reduced participants' positive state affect [START_REF] Hamidovic | Effects of low to moderate acute doses of pramipexole on impulsivity and cognition in healthy volunteers[END_REF]. This suggests that, in this experiment at least, doses of pramipexole influenced the performance of our loss-chasing game via activity at D2/D3 dopamine auto-receptors D 2 and D 3 receptors are predominantly expressed within reinforcement pathways in the nucleus accumbens and amygdala [START_REF] Camacho-Ochoa | Rat brain binding sites for pramipexole, a clinically useful D3-preferring dopamine agonist[END_REF] where both appear to influence the reinforcement value of drugs of stimulants such a cocaine [START_REF] Caine | D3 receptor test in vitro predicts decreased cocaine self-administration in rats[END_REF][START_REF] Thiel | Stimulation of dopamine D2/D3 but not D1 receptors in the central amygdala decreases cocaine-seeking behavior[END_REF]. At the current time, we have no way of knowing which of these receptor subtypes makes the larger contribution to the loss-chasing behaviour observed. Previous experiments have suggested that activity at D 2 receptors can impair learning from the bad outcomes of risky decisions ('no-go learning') by impairing the expression of dips in midbrain dopamine activity that signal negative prediction errors [START_REF] Frank | A mechanistic account of striatal dopamine function in human cognition: psychopharmacological studies with cabergoline and haloperidol[END_REF], Frank et al 2007a, Frank et al 2007b, Frank et al 2009). However, our data suggest that this insensitivity to losing outcomes associated with D 2 /D 3 receptor activity produces more complex changes in risky choices than a simple failure to learn from negative events. Rather, we speculate that impairments in the detection of dips in dopamine activity following bad outcomes produced a straight failure to register small losses; thus increasing the number of pramipexole-treated participants' decisions to quit for small stakes. However, the reduced Serotonin and dopamine in loss-chasing behaviour v3; 19 July 2010 19 sensitivity to losing outcomes associated with D 2 /D 3 activity also further diminished the reduction in subjective value associated with chasing unsuccessfully and suffering larger losses still relative to the reduction in subjective value associated with already certain losses; thus promoting decisions to chase for larger value losses compared to placebo.

Changes in reinforcement learning following treatment with pramipexole [START_REF] Pizzagalli | Single dose of a dopamine agonist impairs reinforcement learning in humans: behavioral evidence from a laboratory-based measure of reward responsiveness[END_REF] are associated with altered signalling within the anterior cingulate region following bad outcomes [START_REF] Santesso | Single dose of a dopamine agonist impairs reinforcement learning in humans: evidence from eventrelated potentials and computational modeling of striatal-cortical function[END_REF] and blunted signalling within striatum following good outcomes [START_REF] Riba | Dopamine agonist increases risk taking but blunts reward-related brain activity[END_REF]. Previously, we have observed that attenuated neural responses to bad gambling outcomes within the anterior cingulate sulcus is also associated with continued chasing behaviour during performance of our loss-chasing game [START_REF] Campbell-Meiklejohn | Knowing when to stop: the brain mechanisms of chasing losses[END_REF]. This is consistent with recent electrophysiological evidence that the reward-related functions of the anterior cingulate and mid-line structures may be disrupted in pathological gamblers [START_REF] Hewig | Hypersensitivity to reward in problem gamblers[END_REF]. Therefore, the findings of Experiment 2 raise the possibility that single doses of pramipexole increase the value of losses judged worth chasing via altered reinforcement signalling within a distributed neural circuit encompassing the anterior cingulate region and its afferent ventral striatal targets [START_REF] Nakano | Neural circuits and functional organization of the striatum[END_REF].

Finally, the results of Experiment 3 indicate that while a single dose of 80mg propranolol significantly reduced heart rate compared to placebo, it did not significantly alter the number of decisions to chase losses, the value of losses chased, or the value of losses surrendered. These findings suggest that the cognitive and emotional aspects of losschasing modelled by our game -though obviously not the excitement associated with commercial gaming activities [START_REF] Anderson | Real and laboratory gambling, sensation-seeking and arousal[END_REF] -are not influenced by manipulations of beta-adrenoceptor activity. They also provide some reassurance that Serotonin and dopamine in loss-chasing behaviour v3; 19 July 2010 20 the effects of tryptophan depletion and pramipexole we observed in Experiment 1 and 2 cannot be attributed to undetected changes in sympathetic and/or peripheral arousal.

However, loss-chasing behaviour might well be influenced by other aspects of noradrenaline function including activity of α2-adrenoceptors that influence activity of the ascending innervation of the locus coeruleus and modulate the processing of negative decision outcomes in the cingulate area [START_REF] Riba | Noradrenergic stimulation enhances human action monitoring[END_REF].

Several limitations to our findings need to be addressed in future investigations. First, while our loss-chasing game captures the essential feature of continued play that brings mounting losses, this feature limits our ability to isolate the specific psychological mechanisms that might be influenced by serotonin and D2/D3 activity to influence gambling to recover losses. Tryptophan depletion and single low doses of pramipexole produced distinct behavioural changes in gambling to recover losses, but additional experiments are needed to establish how these changes relate to what we already know about serotonin's role in avoidance or punishment-induced inhibition [START_REF] Crockett | Reconciling the role of serotonin in behavioral inhibition and aversion: acute tryptophan depletion abolishes punishment-induced inhibition in humans[END_REF][START_REF] Soubrie | Serotonergic neurons and behavior[END_REF]) and what we know about the role of D 2 receptors in learning from negative outcomes [START_REF] Frank | Hold your horses: a dynamic computational role for the subthalamic nucleus in decision making[END_REF]. Second, the clinical implications of these findings need to be explored by examining the effects of serotonergic and dopaminergic treatments on performance of our loss-chasing game in samples of pathological gamblers; as well as testing loss-chasing as a model of impaired control in other addictions (Rogers et al in press). We might also examine the role of other neurotransmitters, such as the opiate and glutamate systems, which may sustain gambling problems [START_REF] Grant | N-acetyl cysteine, a glutamate-modulating agent, in the treatment of pathological gambling: a pilot study[END_REF][START_REF] Grant | Predicting response to opiate antagonists and placebo in the treatment of pathological gambling[END_REF].

Pathological gambling is a source of enormous personal and family distress and represents a significant public health issue [START_REF] Shaffer | Gambling and related mental disorders: a public health analysis[END_REF]. Yet, we know very These findings suggest that the general persistence of gamblers in playing to recover losses is modulated by serotonin activity while the evaluation of losses that gamblers judge worth chasing is mediated by activity of the D2/D3 receptor system.

Figure 2

 2 Figure 2 about here

  biological factors that confer vulnerability for gambling problems, with no licensed pharmacological treatments currently available to clinicians. The experiments represented here represent one way to start to tackle these issues empirically; namely, by investigating the neural and pharmacological basis of the cognitive and behavioural biases evident in the individuals who present at the clinic.

Figure 1

 1 Figure1Display sequences for the loss-chasing game. At the beginning of each round of the game, a loss was imposed and a decision made either to play (gamble further) or quit (to accept the loss) and end the round. Consecutive losses and decisions occurred until a maximum round loss of £640 was incurred, participants won a gamble and cleared their losses or participants chose to quit, at which point the round ended.

Figure 2

 2 Figure 2 Persistence of loss-chasing behaviour in 3 samples of healthy, non-clinical adult participants following tryptophan depletion (vs a control amino acid drink), a single 176µg of the D 2 /D 3 receptor agonist, pramipexole (vs placebo) and a single 80mg dose of the betaadrenoceptor antagonist, propranolol (vs placebo). (a) Mean proportion of decisions to chase losses during the loss-chasing game. (b) Mean consecutive number of decisions to chase losses per round of the loss-chasing game. * p< .05.

Figure 3

 3 Figure 3 The use of value information in the loss-chasing behaviour in 3 samples of healthy, non-clinical participants following tryptophan depletion (vs a control amino acid drink), a single 176µg of the D 2 /D 3 receptor agonist, pramipexole (vs placebo) and a single 80mg dose of the beta-adrenoceptor antagonist, propranolol (vs placebo). (a) Mean value of losses chased (adjusted to the value of all losses encountered). (b) Mean value of losses surrendered (adjusted to the value of all losses encountered). * p< .05.

ACKNOWLEDGEMENTS

This research was funded by a Medical Research Council studentship to Daniel Campbell-Meiklejohn and by an independent award from the Biotechnology and Biological Sciences Research Council (BBSRC) to Robert Rogers. We would also like to thank Michael Frank for helpful suggestions about an earlier version of this manuscript.

DISCLOSURE

We report no biomedical financial interests or potential conflicts of interest.


Himashi Anver

Andor Peter D Ward

Martin Magony

Vreugdenhil

Andor Magony

PhD Martin Vreugdenhil
email: m.vreugdenhil@bham.ac.uk

NMDA receptor hypofunction phase-couples independent gamma oscillations in the rat visual cortex

Keywords: Hallucinogenic gamma oscillation phase-coupling Gamma oscillation, ketamine, phencyclidine, NMDA receptor, hallucinogenic, psychotomimetic

NMDA receptor hypofunction phase-couples independent gamma oscillations in the rat visual cortex.

Introduction

Hallucinations are perceptions in the absence of a stimulus. Where hallucinations are often auditory in schizophrenia, hallucinations associated with neurodegenerative dementias like Lewy body Dementia, and Parkinson's disease with dementia are normally visual [START_REF] Metzler-Baddeley | A review of cognitive impairments in dementia with Lewy bodies relative to Alzheimer's disease and Parkinson's disease with dementia[END_REF]. Hallucinations, and distortions of visual perception [START_REF] Muetzelfeldt | Journey through the K-hole: phenomenological aspects of ketamine use[END_REF]) are also a desired action of dissociative drugs like the N-methyl-D-aspartic acid (NMDA) receptor antagonists ketamine and phencyclidine (PCP). Because a single sub-anaesthetic dose of ketamine or PCP is psychotomimetic [START_REF] Farber | The NMDA receptor hypofunction model of psychosis[END_REF] and agents that enhance NMDA receptor function are antipsychotic [START_REF] Kantrowitz | High dose D-serine in the treatment of schizophrenia[END_REF][START_REF] Schlumberger | Effects of a positive allosteric modulator of mGluR5 ADX47273 on conditioned avoidance response and PCP-induced hyperlocomotion in the rat as models for schizophrenia[END_REF][START_REF] Marek | Glutamatergic (Nmethyl-D-aspartate receptor) hypofrontality in schizophrenia: too little juice or a miswired brain?[END_REF], NMDA receptor hypofunction is regarded a shared process in both psychotic pathologies and substance abuse drugs [START_REF] Coyle | Substance use disorders and Schizophrenia: a question of shared glutamatergic mechanisms[END_REF]). Understanding the mechanism by which NMDA receptor hypofunction causes aberrant perceptions may lead to better antipsychotic treatment.

Perceptions may arise from matching of data-driven (bottom-up) information processing in the sensory cortices with (top-down) information driven by previous experiences stored in higher cortical areas. This process requires neuronal synchronization with millisecond-precision provided by network oscillations at gamma frequencies (30-120 Hz) [START_REF] Herrmann | Cognitive functions of gamma-band activity: memory match and utilization[END_REF][START_REF] Engel | Dynamic predictions: oscillations and synchrony in topdown processing[END_REF]. Reduced NMDA receptor-mediated activation of interneurons in cortical networks leads to pyramidal neuron hyperexcitability [START_REF] Homayoun | NMDA receptor hypofunction produces opposite effects on prefrontal cortex interneurons and pyramidal neurons[END_REF], which was hypothesized to cause aberrant perceptions by disruption of gamma synchronization [START_REF] Marek | Glutamatergic (Nmethyl-D-aspartate receptor) hypofrontality in schizophrenia: too little juice or a miswired brain?[END_REF]). However, psychosis and hallucinations have rather been associated with gamma hypersynchronization [START_REF] Hakami | NMDA receptor hypofunction leads to generalized and persistent aberrant gamma oscillations independent of hyperlocomotion and the state of consciousness[END_REF][START_REF] Spencer | Left auditory cortex gamma synchronization and auditory hallucination symptoms in schizophrenia[END_REF][START_REF] Flynn | Increased absolute magnitude of gamma synchrony in first-episode psychosis[END_REF][START_REF] Spencer | Left auditory cortex gamma synchronization and auditory hallucination symptoms in schizophrenia[END_REF][START_REF] Herrmann | Human EEG gamma oscillations in neuropsychiatric disorders[END_REF].

In the neocortex high-frequency gamma oscillations (HFγ; > 70 Hz) coexist with lowfrequency gamma oscillations (LFγ; < 70 Hz) and the two can operate independently and serve different aspects of information processing processing [START_REF] Wyart | Neural dissociation between visual awareness and spatial attention[END_REF][START_REF] Vidal | Visual grouping and the focusing of attention induce gamma-band oscillations at different frequencies in human magnetoencephalogram signals[END_REF] [START_REF] Crone | High-frequency gamma oscillations and human brain mapping with electrocorticography[END_REF]). Recently we demonstrated that HFγ and LFγ can coexist in the same column of the visual cortex in vitro and operate independently [START_REF] Oke | High-frequency gamma oscillations coexist with low-frequency gamma oscillations in the rat visual cortex in vitro[END_REF].

Interestingly the NMDA receptor antagonist APV selectively reduced the dominant frequency of HFγ so that it approached the frequency of the unaltered LFγ [START_REF] Oke | High-frequency gamma oscillations coexist with low-frequency gamma oscillations in the rat visual cortex in vitro[END_REF].

We therefore propose that NMDA receptor hypofunction causes phase-coupling of two normally phase-independent gamma generating networks, causing hypersynchrony and a distortion of perception due to aberrant matching of top-down information with bottom-up information. Here we test the effect of NMDA receptor hypofunction on HFγ and LFγ recorded in the rat visual cortex in vitro and demonstrate that NMDA receptor hypofunction phasecouples the two gamma generating networks.

Materials and methods

Preparation

Male adult Sprague Dawley rats (200-300 g, Charles-River, Margate, UK) were anaesthetized with by intraperitoneal injection of a ketamine (75 mg.kg -1 ) / medetomidine (1 mg.kg -1 ) mixture and killed by cardiac perfusion with a chilled sucrose-based solution containing 189 mM sucrose, 2.5 mM KCl, 26 mM NaHCO 3 , 1.2 mM NaH 2 PO 4 , 0.1 mM CaCl 2 , 5 mM MgCl 2 , 10 mM D-glucose; pH 7.4. All procedures conformed to the UK Animals (Scientific Procedures) Act 1986. The brain was removed and coronal slices (400 μm thick) were cut around 7-6 mm caudal from bregma. The dorsal neocortex containing the visual cortex areas V1 and V2 was isolated from the rest of the slice by scalpel cuts in chilled sucrose-based solution. Slices were either immediately placed at the interface between artificial cerebrospinal fluid (aCSF, at 7 ml/min) and moist carbogen (95% O 2 and 5 % CO 2 at 300 ml/min) at 32 ºC, or stored in a static interface type chamber at 23 ºC for later use. aCSF contained 125 mM NaCl, 3 mM KCl, 26 mM NaHCO 3 , 1.25 mM NaH 2 PO 4 , 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM D-glucose; pH was set at 7.4. Slices were allowed to recover for one hour before recordings started.

Electrophysiological recordings

Gamma oscillations were evoked by adding kainate (400 nM) and the muscarinic acetylcholine receptor agonist carbachol (20 µM) [START_REF] Buhl | Cholinergic activation and tonic excitation induce persistent gamma oscillations in mouse somatosensory cortex in vitro[END_REF][START_REF] Oke | High-frequency gamma oscillations coexist with low-frequency gamma oscillations in the rat visual cortex in vitro[END_REF], and left to develop for one hour before oscillation characteristics were further explored.

Field potentials were recorded with aCSF-filled glass pipette recording electrodes (4-5 MΩ), amplified with Neurolog NL104 AC-coupled amplifiers (Digitimer, Welwyn Garden City, UK), band-pass filtered at 1-200 Hz with Neurolog NL125 filters (Digitimer), and any noise locked to the mains supply (50 Hz) was removed with Humbug noise eliminators (Digitimer). The signal was then digitized and sampled at 2 kHz using a CED-1401 (Cambridge Electronic Design, Cambridge, UK) and Spike-2 software (Cambridge Electronic Design). Slices were scanned for sites where high-frequency gamma oscillations and low-frequency gamma oscillations were found in the same column, which was usually in the medial part of the secondary visual cortex.

Intracellular current-clamp recordings were made using sharp (60-80 MΩ) pipettes, filled with 3 M potassium methylsulphate. Using a MX1641 motorized microdrive (Siskyou, Grants Pass, OR, USA) the electrode was driven (at 5 µm steps) into the tissue parallel to the layers up to a depth of 250 µm in search for neurons. The membrane potential was amplified using an Axoclamp-2A amplifier (Axon Instruments, Burlingham, CA, USA), low-pass filtered at 2 kHz and sampled at 10 kHz. Impaled cells were first inspected and accepted for recording if the input resistance was greater than 30 MΩ, the resting membrane potential was stable and more polarized than -55 mV in the presence of kainate and carbachol, and action potentials were overshooting.

Drugs were applied at about two hours after application of kainate and carbachol when the oscillation power and frequency were stable. Drug-induced changes after 25 minutes were compared with baseline values. Drugs were used diluted from the following frozen stock solutions. Ketamine; 50 mM in H 2 O, PCP; 2 mM in H 2 O (5S,10R)-(+)-5- d]cyclohepten-5,10-imine maleate (MK-801); 10 mM in H 2 O, ZnCl 2 ; 30 µM in Ketamine and PCP were washed out for 30 minutes and if the drug effect was reversible a second higher concentration of the same drug was applied.

Data analysis

The oscillation power was calculated from 60 s recording epochs by fast Fourier transforms (1 Hz bin size, Hanning window), using Spike-2 software. The gamma frequency band was set at 20-80 Hz for recording at 32 °C, taking into account the temperature dependence of gamma oscillations observed in the visual cortex [START_REF] Oke | High-frequency gamma oscillations coexist with low-frequency gamma oscillations in the rat visual cortex in vitro[END_REF]. The dominant oscillation frequency was determined as the frequency of peak power in the power spectrum smoothed by a 5-point simple moving average. To take into account possible changes in dominant oscillation frequency the power was averaged over -5 Hz to 5 Hz of the dominant frequency. Waveform crosscorrelograms were calculated over 60 s digitally high-pass filtered (FIR at 10 Hz) epochs.

To obtain membrane waveform averages field potential recordings were band-pass filtered (with a phase-true FIR filter) from 10 to 200 Hz. The maximum amplitude for gamma cycles was determined for this signal so that on average one cycle per second exceeded this value. Gamma cycles of medium (0.4-0.6 times the maximum) amplitude were marked at their trough minimum and waveform averages of gamma cycles (> 300 cycles, time-zeroed at the sorted marks) were then calculated from the 10 Hz high-pass (FIR) filtered membrane potential.

To determine the relationship between different events peri-event interval distributions (between -50 ms and 50 ms) were calculated from the timings of the different events and a distribution deviating significantly from a uniform distribution was considered to indicate a relationship between the timing of the two events. To determine the phase relationship between oscillations in recordings from different layers, peri-event intervals were calculated using MATLAB software (The MathWorks, Natick, USA). Simultaneous 100 s recordings were bandpass filtered (with a phase-true FIR filter) at±5 Hz of the dominant frequency of LFγ (determined in layer V) and HFγ (determined in layer III). A Hilbert transform provided a discrete-time analytic signal that was converted to phase angles in radians. From the Hilbert-peak times from both recordings peri-event interval distributions were calculated.

Statistics

Data are expressed as means ± standard error of the mean (SEM), n indicates the number of slices or cells tested. The effect of drugs was tested by unpaired comparison with matched controls, using a two-tailed Student's t-test with and alpha level of 0.05. Deviation from a uniform distribution was determined with the Kolmogorov-Smirnov (K-S) Z test with and alpha level of 0.05.

Results

High-frequency gamma oscillations coexists with low-frequency gamma oscillations

We induced rhythmic field potential oscillations in cortex slices by bath application of kainate (400 nM) and carbachol (20 µM) that substitute for the glutamatergic and cholinergic inputs lost due to the slicing [START_REF] Oke | High-frequency gamma oscillations coexist with low-frequency gamma oscillations in the rat visual cortex in vitro[END_REF][START_REF] Buhl | Cholinergic activation and tonic excitation induce persistent gamma oscillations in mouse somatosensory cortex in vitro[END_REF]. Oscillations had substantial power in the gamma frequency range (example in Figs. 1Ai/Bi), which was set at 20-80 Hz for recording at 32 °C, taking into account the temperature dependence of gamma oscillations [START_REF] Oke | High-frequency gamma oscillations coexist with low-frequency gamma oscillations in the rat visual cortex in vitro[END_REF].

After one hour the visual cortex in each slice was scanned along layer IV to select a cortical column with oscillatory activity with power peaks in both the low-frequency gamma oscillation (LFγ) frequency band (20-45 Hz) and the high-frequency gamma oscillation (HFγ) frequency band (46-80 Hz) range [START_REF] Oke | High-frequency gamma oscillations coexist with low-frequency gamma oscillations in the rat visual cortex in vitro[END_REF], which was usually found in area V2M (Figure S1).

Simultaneous field potential recordings were then made from layer III (0.5 mm from pial surface) and from layer V (1.2 mm from pial surface, Figure S1) of the selected column. The oscillatory activity in layer III (example in Figure 1Ai, grey trace) was invariably faster than the oscillatory activity in layer V (example in Figure 1Ai). The power spectrum of the recording from layer III peaked at frequencies in the HFγ range (Table 1, example in Figure 1Bi, grey line), whereas the power spectrum of the recording from layer V peaked at frequencies in the LFγ range (Table 1, example in Figure 1Bi, black line).

Ketamine selectively decelerates HFγ

Application of the psychotomimetic drug ketamine (10 µM) reduced the dominant frequency of HFγ in layer III, without affecting the dominant frequency of LFγ in layer V (Table 1, example in Figure 1Aii and1Bii). We determined the change in dominant frequency of HFγ in layer III and LFγ in layer V at 25 minutes after application of different ketamine concentrations and compared it with that after 25 minutes of continued perfusion with control aCSF (Table 1).

Ketamine caused a significant reduction in dominant frequency of HFγ at concentrations of 1 µM and up (Figure 1C). The ketamine-induced deceleration of HFγ in layer III, defined as the ketamine-induced reduction in dominant frequency minus the change with control aCSF, as function of ketamine concentration. (Figure 1C, grey symbols) was fitted with a sigmoid function of the form: effect = maximum effect/(1 + ([drug]/EC 50 ) -h )), with a maximum effect of 22.1±0.7 Hz, a concentration of half maximum effect (EC 50 ) of 2.7±0.3 µM and a slope (h) of 1.1±0.1. This EC 50 is well within the range of concentrations obtained with sub-anaesthetic doses used recreationally (shaded area in Figure 1C). The ketamine-induced deceleration of LFγ in layer V was only significant at very high concentrations (Figure 1C, black symbols). To allow for the frequency shift, the effect of ketamine on dominant gamma oscillation power was assessed by taking the average power over +5 Hz and -5 Hz from the dominant frequency. The dominant HFγ power in layer III increased at lower concentrations and decreased at higher concentrations of ketamine (Figure 1D, grey symbols). The increase in dominant LFγ power in layer V at concentrations from 10 µM (Figure 1D, black symbols) may well reflect the contribution of decelerated HFγ in layer III. Effects of ketamine were completely reversible up to a concentration of 3 µM (not shown).

Deceleration of HFγ causes hypersynchrony

Under control conditions HFγ in layer III was independent of LFγ in layer V [START_REF] Oke | High-frequency gamma oscillations coexist with low-frequency gamma oscillations in the rat visual cortex in vitro[END_REF]). In the presence of ketamine the difference between the dominant frequency of LFγ in layer V and that of the decelerated HFγ in layer III became so small that there was a possibility that the two gamma oscillation generating networks could become phase-locked. We tested the prediction that ketamine would increase the phase-link between the two oscillators, by determining the phase relationship between HFγ in layer III and LFγ in layer V from the perievent intervals, calculated from a Hilbert transform of band-pass (± 5 Hz around dominant frequency) filtered recordings. Before ketamine, the peri-event interval distribution was not different from uniform in 6 out of 7 slices (K-S test P< 0.05, example in Figure 2Ai), indicating the absence of a relationship between the phase of HFγ in layer III and the phase of LFγ in layer V. After ketamine (10 µM) the peaks in the inter-event distribution showed a significant relationship between the oscillation phase in layer III and that in layer V (example in Figure 2Aii). The K-S test Z increased from 1.11±0.11 in control to 4.23±0.21 in ketamine (Paired Student's t-test P< 0.001, n= 7, Figure 2Aiii). On average layer III gamma troughs were 4.3±0.5 ms ahead of layer V gamma troughs. This indicates that, whereas in control the two oscillators operate independently, the ketamine-induced deceleration of HFγ in layer III causes phasecoupling of the gamma generating networks in layer III and layer V.

Under control conditions LFγ in layer V is spatially coherent, unlike HFγ in layer III [START_REF] Oke | High-frequency gamma oscillations coexist with low-frequency gamma oscillations in the rat visual cortex in vitro[END_REF]). To test whether the phase-coupling of the decelerated HFγ in layer III to LFγ would increase the spatial coherence of the gamma oscillation in layer III, we determined the cross correlation maximum between two electrodes placed 500 µm apart in layer III of the same slice.

Cross correlation was small in control (example in Figure 2Bi), but increased in the presence of ketamine (10 µM, example in Figure 2Bii). For five slices the cross correlation in layer III increased from 0.22±0.02 in control to 0.50±0.05 in ketamine (paired Student's t-test P< 0.01, Figure 2Biii). For the same slices the cross correlation maximum between two electrodes placed 500 µm apart in layer V was not affected by ketamine (0.54±0.05 in control versus 0.58±0.06 in ketamine, n.s.). This suggests that the increase in spatial coherence of gamma oscillations in layer III is due to the phase coupling of the decelerated HFγ in layer III to LFγ in layer V.

Ketamine causes entrainment of layer III pyramidal neurons by LFγ

Like other cortical gamma oscillations [START_REF] Buhl | Cholinergic activation and tonic excitation induce persistent gamma oscillations in mouse somatosensory cortex in vitro[END_REF][START_REF] Cunningham | Gamma oscillations induced by kainate receptor activation in the entorhinal cortex in vitro[END_REF], HFγ in layer III is dependent on recurrent inhibition [START_REF] Oke | High-frequency gamma oscillations coexist with low-frequency gamma oscillations in the rat visual cortex in vitro[END_REF]) and layer III pyramidal neuron firing is entrained by rhythmic IPSCs and EPSCs phase-locked to HFγ in layer III (unpublished observations MV). To test whether ketamine affects the relationship between the activity of layer III pyramidal neurons and the gamma oscillations in the different layers, we recorded cells in layer III under sharp electrode current-clamp conditions. Six cells identified as pyramidal neurons (see Figure S2) were recorded before and after ketamine (10 µM).

To test the relationship between action potential firing times and gamma oscillation phase, neurons were made to fire at ~ 2 Hz by manually adjusting the holding current. The distribution of intervals between action potential peaks (500 per neuron) and HFγ troughs in layer III (example in Figure 3Ai) was tested for uniformity. For 5 out of 6 neurons the spike timing distribution showed a significant relationship (K-S test for uniformity, P< 0.05) between firing and HFγ troughs in layer III, with the peak firing probability in these neurons 4.3±0.7 ms ahead of the HFγ trough. In contrast to the entrainment of firing by HFγ, the distribution of intervals between action potential peaks and LFγ troughs in layer V showed no entrainment of the same six layer III pyramidal neurons by LFγ in layer V (example in Figure 3Bi).

In the presence of ketamine (10 µM) the firing of the same layer III pyramidal neurons was still entrained by the now decelerated HFγ in layer III, but the relation was less tight (example in Figure 3Aii). The K-S test Z was reduced from 2.96±0.26 in control to 2.47±0.31 in ketamine (paired Student's t-test P< 0.05, n= 6, Figure 3Aiii). However, for 4 out of 6 layer III pyramidal neurons the firing was now entrained by LFγ in layer V (example in Figure 3Bii), with the peak firing probability in these neurons 0.5±1.0 ms after the HFγ trough. The K-S test Z increased from 0.95±0.14 in control to 1.87±0.19 in ketamine (paired Student's t-test P< 0.01, n= 6, Figure 3Biii). These observations demonstrate that layer III pyramidal neurons that under normal conditions ignore the LFγ in layer III, become entrained by it in the presence of ketamine.

Ketamine increases rhythmic synaptic inputs.

All pyramidal neurons recorded received a constant barrage of IPSPs and EPSP. To determine how synaptic inputs are related to the different gamma oscillations, we held the same neurons manually just below firing threshold and constructed the membrane potential waveform averages, time-zeroed at the trough of medium-amplitude HFγ gamma cycles in layer III (example in Figure 3Ci). The membrane potential gamma cycle had its peak amplitude 3.4±0.6 ms ahead of the HFγ trough in layer III. The waveform averages of the membrane potential timezeroed to the LFγ troughs in layer V (example in Figure 3Di) show that the membrane oscillation was only weakly coupled to LFγ in layer V, peaking 1.3±0.8 ms ahead of the LFγ trough.

In the presence of ketamine the membrane potential waveform average time-zeroed at the decelerated HFγ troughs in layer III was larger (example in Figure 3Cii) and peaked later (1.0±0.7 ms, paired Student's t-test P< 0.01, n= 6,). The maximum (peak-to-trough) amplitude was 0.23±0.06 mV in ketamine versus 0.18±0.04 mV in control (paired Student's t-test P< 0.05, n= 6, Figure 3Ciii). Ketamine strongly increased the maximum amplitude of the membrane potential waveform average for LFγ troughs in layer V (example in Figure 3Dii), without affecting the peak time (0.4±0.7 ms after the LFγ trough, n.s.). The maximum amplitude was 0.26±0.06 mV versus 0.10±0.03 mV in control (paired Student's t-test P< 0.01, n= 6, Figure 3Diii). These observations demonstrate that ketamine increases the LFγ-related rhythmic synaptic inputs to layer III pyramidal neurons.

PCP decelerates HFγ and causes hypersynchrony

The psychotomimetic drug PCP has a similar NMDA receptor-antagonistic action to ketamine and we predicted that PCP would have a ketamine-like effect on gamma oscillations in the visual cortex. Like with ketamine, application of 1 µM PCP reversibly decelerated HFγ in layer III, but did not affect the dominant frequency of LFγ in layer V (Table 1, example in Figure S3A/B). Like ketamine, 1 µM PCP phase-locked the gamma-generating networks in layer III and layer V that were independent in control (Figure S3C). The deceleration of HFγ in layer III as function of PCP concentration (Figure S3D) was fitted with a sigmoid function with a maximum effect of 21.7±0.9 Hz, an EC 50 of 0.16±0.02 µM and a slope of 1.7±0.3. PCP suppressed the dominant HFγ power at higher concentrations and increased the dominant LFγ power at intermediate concentrations (Figure S3E), possibly due to the contribution from the decelerated HFγ in layer III. At concentrations obtained with doses used to induce hallucinations PCP has the same network effects as ketamine.

NMDA receptor modulating drug effects

Ketamine and PCP are not selective NMDA receptor antagonists and may exert their effect through inhibition of I h [START_REF] Tanabe | Inhibition of hyperpolarization-activated cation currents by phencyclidine and some sigma ligands in rat hippocampal CA1 pyramidal neurons in vitro[END_REF]) and/or dopamine D 2 receptor stimulation [START_REF] Seeman | Dopamine D2High receptors stimulated by phencyclidines, lysergic acid diethylamide, salvinorin A, and modafinil[END_REF]). Therefore we tested the effect of the selective non-competitive NMDA receptor antagonist MK-801. MK-801 (10 µM) decelerated HFγ, but left the dominant frequency of LFγ unchanged (Table 1). MK-801 increased the dominant power of both HFγ and LFγ (Table 1).

NMDA receptor activation also requires occupation of the glycine site by its endogenous ligand D-serine [START_REF] Oliet | Regulation of N-methyl-D-aspartate receptors by astrocytic Dserine[END_REF]. We reduced endogenous D-serine levels by increasing Dserine metabolism by applying the enzyme DAAO. DAAO (0.17 U/ml, 60 minutes) selectively decelerated HFγ (Table 1, example in Figure 4A). These observations, together with the increase in HFγ power observed with the competitive NMDA receptor antagonist APV [START_REF] Oke | High-frequency gamma oscillations coexist with low-frequency gamma oscillations in the rat visual cortex in vitro[END_REF], confirm that the HFγ-decelerating effect of ketamine and PCP is mediated through NMDA receptor antagonism.

In order to determine which NMDA receptor type is responsible we tested the effect of Ro 25-6981 (10 µM), a selective antagonist of NR2B subunit-containing NMDA receptors [START_REF] Fischer | Ro 25-6981, a highly potent and selective blocker of N-methyl-D-aspartate receptors containing the NR2B subunit. Characterization in vitro[END_REF]) and of nanomolar zinc, which selectively blocks NR2A subunit-containing NMDA receptors [START_REF] Paoletti | High-affinity zinc inhibition of NMDA NR1-NR2A receptors[END_REF]. Ro 25-6981 selectively decelerated HFγ, increased dominant LFγ power and reduced dominant HFγ power (Table 1, example in Figure 4B). However, zinc (30 nM ZnCl 2 ) had no effect on the dominant frequency of HFγ or LFγ or on their dominant gamma power (Table 1). These observations indicate that the deceleration of HFγ by NMDA receptor antagonists is mediated by antagonism of NR2B subunit-containing NMDA receptors.

Based on the observation that NMDA receptor hypofunction can cause phase-coupling of the gamma oscillators by selectively decelerating HFγ, we predicted that the opposite effect would be obtained by increasing NMDA receptor activation. To test this we applied NMDA directly [START_REF] Mann | Control of hippocampal gamma oscillation frequency by tonic inhibition and excitation of interneurons[END_REF] to slices with HFγ and LFγ. NMDA (3 µM) selectively accelerated HFγ and suppressed the gamma power of both HFγ and LFγ (Table 1). NMDA receptor activity depends on the activation of the glycine site of the receptor. Increasing glycine site occupation by applying a high concentration of D-serine (100 µM) selectively accelerated HFγ without affecting oscillation power (Table 1, example in Figure 4C). NMDA receptor activation can also be increased by activation of metabotropic glutamate group I receptors [START_REF] Pisani | Enhancement of NMDA responses by group I metabotropic glutamate receptor activation in striatal neurones[END_REF]. In line with this the metabotropic glutamate group I receptor agonist DHPG (10 µM) selectively accelerated HFγ, without affecting the oscillation power (Table 1, example in Figure 4D). These observations confirm that, whatever the method, increasing NMDA receptor activity selectively accelerates HFγ.

Discussion

We investigated the effect of psychotomimetic NMDA receptor antagonists ketamine and PCP and demonstrated that in the visual cortex NMDA receptor hypofunction causes phasecoupling of both HFγ in layer III and layer III pyramidal neuron activity to LFγ in layer V, by selectively decelerating HFγ. This effect is mediated through D-serine-dependent NR2B subunitcontaining receptors, and can be reversed by increasing NMDA receptor activity.

Ketamine and PCP selectively decelerate HFγ in layer III with an EC 50 within the subanaesthetic concentration range. At sub-anaesthetic doses ketamine strongly increases cortical LFγ power in the behaving rat [START_REF] Hakami | NMDA receptor hypofunction leads to generalized and persistent aberrant gamma oscillations independent of hyperlocomotion and the state of consciousness[END_REF]) and in healthy humans [START_REF] Hong | Gamma and delta neural oscillations and association with clinical symptoms under subanesthetic ketamine[END_REF].

This increase can be explained by the increase in HFγ power in layer III, and by the deceleration and increased spatial coherence of HFγ. Reducing the NMDA receptor-mediated drive of cortical interneurons increases pyramidal neuron firing rate [START_REF] Homayoun | NMDA receptor hypofunction produces opposite effects on prefrontal cortex interneurons and pyramidal neurons[END_REF] which, according to computational modelling, would increase in cortical gamma power [START_REF] Spencer | The functional consequences of cortical circuit abnormalities on gamma oscillations in schizophrenia: insights from computational modeling[END_REF].

Because the spatial coherence of HFγ is relatively low in control conditions [START_REF] Oke | High-frequency gamma oscillations coexist with low-frequency gamma oscillations in the rat visual cortex in vitro[END_REF], recordings taken from the cortical surface are likely to miss the HFγ that can be recorded with indwelling electrodes and magnetoencephalography [START_REF] Tallon-Baudry | Oscillatory synchrony and human visual cognition[END_REF]. The deceleration of HFγ and subsequent phase-coupling to the spatially more-coherent LFγ will increase the spatial coherence of HFγ and the rhythmic IPSC amplitude in layer III pyramidal neurons, and hence will increase the power of the oscillation recorded with surface electrodes (illustrated in Figure 5).

Why does NMDA receptor modulation selectively affect HFγ frequency? The relatively high frequency of HFγ could be caused by 1) faster IPSC decay kinetics [START_REF] Whittington | Synchronized oscillations in interneuron networks driven by metabotropic glutamate receptor activation[END_REF][START_REF] Heistek | GABAergic synapse properties may explain genetic variation in hippocampal network oscillations in mice[END_REF], 2) smaller IPSC amplitude [START_REF] Atallah | Instantaneous modulation of gamma oscillation frequency by balancing excitation with inhibition[END_REF], but see [START_REF] Oke | High-frequency gamma oscillations coexist with low-frequency gamma oscillations in the rat visual cortex in vitro[END_REF], 3) increased interneuron firing rate due to increased NMDA receptor-mediated depolarization and/or reduced tonic GABA A ergic inhibition of interneurons [START_REF] Mann | Control of hippocampal gamma oscillation frequency by tonic inhibition and excitation of interneurons[END_REF], or 4) the selective NMDA receptor-mediated activation of fast-firing parvalbuminexpressing interneurons [START_REF] Middleton | NMDA receptor-dependent switching between different gamma rhythm-generating microcircuits in entorhinal cortex[END_REF]. The consistent, bi-directional effects of modulating NMDA receptor activation on HFγ frequency suggest that NMDA receptor hypofunction decelerates HFγ by reduced activation of fast-firing, probably parvalbuminexpressing interneurons in the superficial layers that drive HFγ. Interneurons involved in LFγ in layer V may not be dependent on NMDA receptor-mediated activation. Indeed, GABA A ergic IPSCs in layer V neurons are NMDA receptor activation-independent [START_REF] Ling | Recruitment of GABAA inhibition in rat neocortex is limited and not NMDA dependent[END_REF].

Because NMDA receptor-mediated modulation of interneuron activity can be presynaptic, NMDA receptor antagonists may selectively reduce glutamate release to interneurons involved in HFγ. Indeed, NR2B-containing (and not NR2A-containing) NMDA auto-receptors modulate glutamate release in visual cortex and are located in layer II/III [START_REF] Li | Presynaptic NR2B-containing NMDA autoreceptors mediate gluta-matergic synaptic transmission in the rat visual cortex[END_REF]. This is consistent with the differential effects of zinc and Ro 25-6981 in decelerating HFγ.

Psychosis and hallucinations have been associated with increased cortical LFγ [START_REF] Flynn | Increased absolute magnitude of gamma synchrony in first-episode psychosis[END_REF][START_REF] Spencer | Left auditory cortex gamma synchronization and auditory hallucination symptoms in schizophrenia[END_REF][START_REF] Becker | Electrophysiological correlates of flickerinduced color hallucinations[END_REF]) and the gestalt-induced gamma oscillation is decelerated in the visual cortex of schizophrenics [START_REF] Spencer | Neural synchrony indexes disordered perception and cognition in schizophrenia[END_REF]). Because NMDA receptor hypofunction is a common factor in the action of substance abuse drugs and schizophreniaassociated psychosis [START_REF] Coyle | Substance use disorders and Schizophrenia: a question of shared glutamatergic mechanisms[END_REF][START_REF] Farber | The NMDA receptor hypofunction model of psychosis[END_REF], the latter could be due to an increased propensity of a cortical network to hypersynchronize [START_REF] Spencer | Left auditory cortex gamma synchronization and auditory hallucination symptoms in schizophrenia[END_REF], due to network changes that can decelerate HFγ.

Firstly, parvalbumin-expressing fast-firing interneurons may be functionally impaired. In the visual cortex of schizophrenics parvalbumin-immunoreactivity is selectively reduced [START_REF] Hashimoto | Conserved regional patterns of GABA-related transcript expression in the neocortex of subjects with schizophrenia[END_REF]. GABA synthesis is reduced in parvalbumin-expressing cortical interneurons [START_REF] Hashimoto | Gene expression deficits in a subclass of GABA neurons in the prefrontal cortex of subjects with schizophrenia[END_REF], possibly due to reduced activity of TrkB receptors, that are normally predominantly expressed on parvalbumin-expressing interneurons [START_REF] Lewis | Cortical inhibitory neurons and schizophrenia[END_REF]), but are reduced in schizophrenics [START_REF] Hashimoto | Relationship of brain-derived neurotrophic factor and its receptor TrkB to altered inhibitory prefrontal circuitry in schizophrenia[END_REF]. Increased activation of the remaining TrkB receptors may therefore have antipsychotic potential.

Secondly, the NMDA receptor-dependent activation of interneurons may be reduced.

Although the total cellular expression of NR2 proteins is not consistently altered in the cortex of schizophrenics, the trafficking of NR2B subunit-containing NMDA receptors to their pre-and postsynaptic locations is impaired [START_REF] Kristiansen | Expression of the NR2B-NMDA receptor trafficking complex in prefrontal cortex from a group of elderly patients with schizophrenia[END_REF]). In the chronic low-dose MK-801treated rat model the parvalbumin-expressing interneurons have a reduced NMDA receptor expression [START_REF] Xi | Dizocilpine (MK-801) induces distinct changes of N-methyl-D-aspartic acid receptor subunits in parvalbumin-containing interneurons in young adult rat prefrontal cortex[END_REF]. As increased activation of NR2B subunit-containing NMDA receptors accelerated HFγ, selective facilitation of these receptors may therefore have antipsychotic potential.

NMDA receptor activation requires co-activation of the glycine site by its endogenous ligand D-serine [START_REF] Oliet | Regulation of N-methyl-D-aspartate receptors by astrocytic Dserine[END_REF]. Schizophrenia has been associated with aberrant NMDA receptor D-serine/glycine site function [START_REF] Labrie | The involvement of the NMDA receptor D-serine/glycine site in the pathophysiology and treatment of schizophrenia[END_REF] and increasing D-serine levels has an antipsychotic-like profile [START_REF] Kantrowitz | High dose D-serine in the treatment of schizophrenia[END_REF][START_REF] Marek | Glutamatergic (Nmethyl-D-aspartate receptor) hypofrontality in schizophrenia: too little juice or a miswired brain?[END_REF]. Interestingly, in the rat visual cortex glycine site agonists facilitate glutamate release in layer III by acting on presynaptic NR2B subunit-containing NMDA auto-receptors [START_REF] Li | Tonic facilitation of glutamate release by glycine binding sites on presynaptic NR2B-containing NMDA autoreceptors in the rat visual cortex[END_REF]. The deceleration of HFγ with reducing endogenous D-serine levels and the acceleration with exogenous D-serine confirm that the deceleration of HFγ is involved in the psychotic action of NMDA receptor hypofunction.

NMDA receptor activation is facilitated by metabotropic glutamate receptor group 1 activation [START_REF] Pisani | Enhancement of NMDA responses by group I metabotropic glutamate receptor activation in striatal neurones[END_REF]) through mGluR5 receptors. Although the mGluR5 expression is not altered in the cortex of schizophrenics [START_REF] Gupta | Metabotropic glutamate receptor protein expression in the prefrontal cortex and striatum in schizophrenia[END_REF], increasing mGluR5 receptor activity has promising antipsychotic effects [START_REF] Schlumberger | Effects of a positive allosteric modulator of mGluR5 ADX47273 on conditioned avoidance response and PCP-induced hyperlocomotion in the rat as models for schizophrenia[END_REF], possibly due to the acceleration of HFγ observed with DHPG.

Thirdly, non-NMDA receptor-dependent activation of fast-spiking interneurons may be impaired. Metabotropic glutamate receptor GluR2-selective agonists selectively activate fastspiking interneurons in the cortex [START_REF] Sun | Differential metabotropic glutamate receptor expression and modulation in two neocortical inhibitory networks[END_REF] and have an antipsychotic action [START_REF] Marek | Glutamatergic (Nmethyl-D-aspartate receptor) hypofrontality in schizophrenia: too little juice or a miswired brain?[END_REF]. The expression of kainate receptor subunit GluR5 (not mGluR6) mRNA was reduced in cortical interneurons of schizophrenics [START_REF] Woo | Differential alterations of kainate receptor subunits in inhibitory interneurons in the anterior cingulate cortex in schizophrenia and bipolar disorder[END_REF]. Because the GluR5 subunitselective kainate receptor agonist ATPA accelerated HFγ frequency [START_REF] Oke | High-frequency gamma oscillations coexist with low-frequency gamma oscillations in the rat visual cortex in vitro[END_REF]), selective GluR5 potentiators may have an antipsychotic potential [START_REF] Marek | Glutamatergic (Nmethyl-D-aspartate receptor) hypofrontality in schizophrenia: too little juice or a miswired brain?[END_REF].

At a cellular level the ketamine-induced deceleration of HFγ caused the firing of layer III pyramidal neurons to be entrained by LFγ in layer V. Whereas in control conditions the firing of pyramidal neurons in the superficial layers is independent of that in the deep layers, NMDA receptor hypofunction aligns the firing of the two populations, increasing the interactions between the layers (illustrated in Figure 5). Because the decelerated HFγ in layer III phase leads LFγ in layer V, as in the somatosensory cortex under control conditions [START_REF] Buhl | Cholinergic activation and tonic excitation induce persistent gamma oscillations in mouse somatosensory cortex in vitro[END_REF], the EPSPs from layer III pyramidal neurons may arrive just before the IPSCs in layer V pyramidal neurons. This is likely to increase the information transfer between the layers during gamma oscillations. In addition it may increase the synaptic strength of layer III to layer V pyramidal neuron connections through Hebbian plasticity and change information transfer long-term. In addition the phase-coupling of neuronal firing across layers is likely to increase burst firing in layer V pyramidal neurons that stretch through all layers [START_REF] Larkum | A new cellular mechanism for coupling inputs arriving at different cortical layers[END_REF][START_REF] Larkum | Top-down dendritic input increases the gain of layer 5 pyramidal neurons[END_REF]).

Functionally it is tempting to speculate how hallucinations can arise from the increased coupling of processes in the superficial layers and the deep layers. Co-existence of HFγ and LFγ is found throughout the cortex, including the auditory cortex and prefrontal cortex [START_REF] Crone | High-frequency gamma oscillations and human brain mapping with electrocorticography[END_REF]). In the visual cortex HFγ and LFγ are associated with different aspects of visual information processing [START_REF] Wyart | Neural dissociation between visual awareness and spatial attention[END_REF][START_REF] Vidal | Visual grouping and the focusing of attention induce gamma-band oscillations at different frequencies in human magnetoencephalogram signals[END_REF][START_REF] Chaumon | Unconscious learning versus visual perception: Dissociable roles for gamma oscillations revealed in MEG[END_REF] and relate to the degree of top-down or bottom-up processing (Tallon-Baudry, personal communication). Conscious perception of complex visual stimuli may need the matching of stimulus-driven bottom-up information with memory-based top-down predictions [START_REF] Wyart | Neural dissociation between visual awareness and spatial attention[END_REF][START_REF] Herrmann | Cognitive functions of gamma-band activity: memory match and utilization[END_REF][START_REF] Engel | Dynamic predictions: oscillations and synchrony in topdown processing[END_REF]. Top-down processes may modulate the temporal structure of bottom-up processes [START_REF] Engel | Dynamic predictions: oscillations and synchrony in topdown processing[END_REF], and/or boost the response of layer V neurons when bottom-up and top-down information arrives simultaneously at basal and apical dendrites respectively [START_REF] Siegel | A functional gamma-band defined by stimulus-dependent synchronization in area 18 of awake behaving cats[END_REF][START_REF] Larkum | Top-down dendritic input increases the gain of layer 5 pyramidal neurons[END_REF]). The

  H 2 O, (aR,bS)-a-(4-Hydroxyphenyl)-b-methyl-4-(phenylmethyl)-1 -piperidinepropanol maleate (Ro 25-6981); 10 mM in H 2 O, (S)-3,5-Dihydroxyphenylglycine (DHPG); 10 mM in H 2 O. Dserine was directly added to the aCSF. The D-serine metabolising enzyme D-amino acid oxidase (DAAO); 800 U/ml was derived from rhodotorula gracilis and kindly donated by Professor Dale (University of Warwick). Ketamine, Ro 25-6981 and MK-801 were purchased from Tocris (Bristol, UK). All other drugs and aCSF salts were purchased from Sigma (Poole, UK).

Figure 1 .

 1 Figure 1. Ketamine decelerates selectively HFγ. A. Example of oscillatory activity

Figure 2 .

 2 Figure 2. Ketamine increases phase-coupling. A. Peri-event interval distribution of the

Figure 3 .

 3 Figure 3. Ketamine effect on layer III pyramidal neurons. A. Peri-event interval distribution

Figure 4 .

 4 Figure 4. Effect of NMDA receptor modulation on HFγ. A. Power spectrum of a one-minute

Figure 5 .

 5 Figure 5. Illustration of the effect of hypersynchrony within and across layers. Recordings of

Acknowledgments

We thank the Medical Research Council (UK) for support. We thank Professor Jefferys (University of Birmingham) for discussion and use of equipment. We thank B. Shakila and Professor Dale (University of Warwick) for the production and kind donation of DOOA.

The negative symptoms in schizophrenics are associated with gamma band hyposynchronisation [START_REF] Spencer | Left auditory cortex gamma synchronization and auditory hallucination symptoms in schizophrenia[END_REF][START_REF] Herrmann | Human EEG gamma oscillations in neuropsychiatric disorders[END_REF], which poses the question how gamma band hypersynchronisation can arise from the same distorted network?

Computational modelling has demonstrated that different kinds of network abnormalities associated with schizophrenia can produce gamma oscillation deficits as well as increases [START_REF] Spencer | The functional consequences of cortical circuit abnormalities on gamma oscillations in schizophrenia: insights from computational modeling[END_REF]. We propose that even in a network with reduced LFγ, the coincidence of several of the modulatory factors that each can cause selective deceleration of HFγ in the superficial layers, may cause a temporary cross-layer gamma band hypersynchronization and consequent distortion of perception, due to aberrant matching of top-down information with bottom-up information. Our study suggests that a combination of therapies to counteract several of these modulating factors may provide the most effective antipsychotic therapy.

Disclosure/Conflict of Interest

The authors declare that, except for income received from the primary employer, or stipends unrelated to pharmaceutical corporations, no financial support or compensation has been received from any individual or corporation over the last three years of research and there are no personal financial holdings that could be perceived as constituting a potential conflict of interest.