The neurophysiological basis of excessive daytime sleepiness: suggestions of an altered state of consciousness.

Excessive daytime sleepiness (EDS) is characterized by difficulty staying awake during daytime, though additional features may be present. EDS is a significant problem for clinical and non-clinical populations, being associated with a range of negative outcomes that also represent a burden for society. Extreme EDS is associated with sleep disorders, most notably the central hypersomnias such as narcolepsy, Kleine-Levin syndrome, and idiopathic hypersomnia (IH). Although investigation of these conditions indicates that EDS results from diminished sleep quality, the underlying cause for this impairment remains uncertain. One possibility could be that previous research has been too narrow in scope with insufficient attention paid to non-sleep-related aspects. Here, we offer a broader perspective in which findings concerning the impact of EDS on cortical functioning are interpreted in relation to current understanding about the neural basis of consciousness. Alterations in the spatial distribution of cortical activity, in particular reduced connectivity of frontal cortex, suggest that EDS is associated with an altered state of consciousness.

The effect of single and repeated tDCS sessions on motor symptoms in Parkinson's disease: a systematic review.

OBJECTIVES: to update understanding of the effectiveness of transcranial direct current stimulation (tDCS) on motor dysfunction in Parkinson's disease, since the last review was published in 2016. METHODS: in order to identify suitable publications for inclusion, an online search of the Pubmed, Scopus and Cochrane databases was carried out. Searches of relevant full-text articles were performed through specific keywords. The final database check was performed in July 2019. Papers were restricted to studies investigating motor rehabilitative effects of tDCS in adult patients with Parkinson's disease. Studies involving either single or repeated tDCS sessions with a sham or controlled trial type design (which incorporated outcomes on motor performance measures) were considered. As studies varied widely in terms of methodology, a qualitative analysis of the selected studies was performed using the Newcastle-Ottawa Quality Assessment Scale or the Delphi list (depending on the study design). RESULTS: twenty-nine studies were retained in this systematic review. Of the studies included, fifteen involved single tDCS session (patients = 256) and fourteen involved repeated tDCS sessions (patients = 294). Eight investigations of single tDCS and ten investigations of repeated tDCS demonstrated significant results. Studies involving multi- target stimulation demonstrated significant improvements on mobility (p=0.006), balance (by 50.9%), gait velocity (by 29%), fall reduction (p0.05) compared to mono-target stimulations. CONCLUSIONS: despite increasing evidence that tDCS may improve motor symptoms, the results showed that fully optimized tDCS protocols are not yet established.

Immunohistochemical assessment of mesotelencephalic dopamine activity during the acquisition and expression of Pavlovian versus instrumental behaviours.

Dopaminergic activity during Pavlovian or instrumental learning in key target regions of the mesotelencephalic dopamine system was investigated immunohistochemically using antibodies raised against glutaraldehyde-conjugated dopamine. Experiment 1 examined dopamine immunoreactivity during acquisition of a Pavlovian conditioned-approach response. Observations were taken at three stages of learning: initial, intermediate and asymptotic; each with a conditioned stimulus+ (CS+) group for whom visual or auditory stimuli immediately preceded an unconditioned stimulus (sucrose), and a conditioned stimulus- (CS-) group for whom stimuli and the unconditioned stimulus were unpaired. Animals learned to approach the alcove during CS+ presentations, whilst approach behaviour of the CS- group remained low. In general, target regions exhibiting a dopaminergic reaction responded maximally during the intermediate stage of acquisition, and were less responsive initially, and not responsive at all at asymptote. Specifically, the pattern of dopaminergic response was: shell more than core of the nucleus accumbens; prefrontal cortex, central and basolateral nuclei of the amygdala also significantly responsive. Mediodorsal and laterodorsal striatal regions were reactive only very early in training. Experiment 2 examined dopaminergic reaction following acquisition of a novel conditioned instrumental response. The conditioned response+ (CR+) group responded at a much higher rate on the lever for which unconditioned stimulus-associated stimuli were presented, than on the control lever. The conditioned response- (CR-) group responded at a low rate on both levers. In contrast with experiment 1, the most responsive regions were the core of the nucleus accumbens, medial prefrontal cortex and basolateral area of the amygdala. Thus, the acquisition, but not expression of Pavlovian associations activated dopamine within several key target regions of the mesotelencephalic dopamine system, and preferentially within the shell rather than core of the nucleus accumbens. By contrast, acquisition of a novel instrumental response preferentially activated the core of the nucleus accumbens, and basolateral area of the amygdala. These data carry significant implications for the potential role of these regions in learning and memory.

An immunohistochemical examination of the effects of sensitisation on the mesotelencephalic dopaminergic response to d-amphetamine.

The dopaminergic response to d-amphetamine with or without prior repeated experience with the drug was investigated immunohistochemically in key target regions of the mesotelencephalic dopamine system using antibodies raised against glutaraldehyde-conjugated dopamine. This methodology permitted the unambiguous determination of dopaminergic activity within specific subregions of structures implicated in the behavioural effects of psychomotor stimulants drugs, and in the expression of behavioural sensitisation. Experiment 1 examined dopamine immunoreactivity in central or basolateral amygdala, shell or core of the nucleus accumbens, medial and lateral caudate-putamen and medial prefrontal cortex following the administration of various doses of d-amphetamine to drug-naïve rats. Whilst dose-related increases in dopaminergic activity were detected in all regions examined, a regional heterogeneity was clearly evident. For example, d-amphetamine enhanced dopaminergic activity preferentially within the shell subregion of the nucleus accumbens both with respect to the core subregion and to other striatal and non-striatal areas. Experiment 2 examined changes in dopaminergic activity following the administration of a low dose of d-amphetamine to d-amphetamine-sensitised rats and saline-pretreated control animals. Regional heterogeneity both between and within terminal areas was again detected. Thus, there was evidence of a preferential increase in dopaminergic activity within the shell of the nucleus accumbens of sensitised rats. Moreover, sensitisation to d-amphetamine increased the dopaminergic response to acute administration of d-amphetamine within all striatal and non-striatal areas examined. Comparison of this effect across subterritories of the areas under investigation revealed that in sensitised rats, acute administration of d-amphetamine elevated dopaminergic activity within the shell of the nucleus accumbens to a greater extent than within the core. These data therefore indicate that systemic administration of d-amphetamine is associated with regionally heterogeneous changes in dopaminergic activity within terminal regions of the mesotelencephalic dopamine system in both sensitised and unsensitised rats. Moreover, the present methodology permitted resolution of these changes at an anatomical level beyond that of conventional approaches.

Alpha-2 adrenergic receptor agonists block stress-induced reinstatement of cocaine seeking.

The alpha-2 adrenergic receptor agonists, clonidine, lofexidine and guanabenz, blocked stress- but not cocaine-induced reinstatement of cocaine seeking at doses that suppressed footshock-induced release of noradrenaline in prefrontal cortex and amygdala. Rats were trained to self-administer cocaine (0.5 mg/kg/infusion, i.v; 10-12 days) and, after a drug-free period (7-13 days), were returned to the self-administration chambers for daily extinction and reinstatement test sessions. Both intermittent footshock (15 min, 0.6 mA) and cocaine priming (20 mg/kg, i.p.) reinstated extinguished drug seeking. Pretreatment with either clonidine (20, or 40 microg/kg, i.p.) or lofexidine (50, 100, 150, or 200 microg/kg, i.p.) attenuated footshock- but not cocaine-induced reinstatement of cocaine seeking. Guanabenz (640 microg/kg, i.p.), an alpha-2 agonist with low affinity for imidazoline type-1 receptors, also attenuated footshock- but not cocaine-induced reinstatement of cocaine seeking. The results point to an important role for NE systems in the effects of footshock on relapse to cocaine seeking.

Effects of intra-amygdala R(+) 7-OH-DPAT on intra-accumbens d-amphetamine-associated learning. I. Pavlovian conditioning.

We have previously obtained evidence that the mesoamygdaloid dopamine projection modulates the acquisition of a conditioned response (CR) elicited by presentation of a conditioned stimulus (CS) predicting the availability of a natural (sucrose) reward. This property was found to be dependent upon D3, but not D1 or D2, dopamine receptor activation. The aim of the present study was to determine whether the mesoamygdaloid dopamine projection is similarly involved in the acquisition of a drug-associated CR. Thus, two groups of rats with guide cannulae aimed at the nucleus accumbens and amygdala were trained using a Pavlovian conditioning procedure in which an initially neutral CS was paired with a computer-controlled, bilateral intraaccumbens infusion of d-amphetamine (the unconditioned stimulus: US). Conditioning sessions were conducted in standard operant chambers, with each session consisting of a single CS-US trial. For one group of rats, CS presentation was positively correlated with the drug US (Paired group), while for the second group CS and US presentations were negatively correlated (Unpaired group). During training, locomotor activity was recorded and was utilised as the measure both of the unconditioned (UR) and conditioned response (CR). A within-subjects design was utilised to investigate the effect of post-session bilateral intraamygdala administration of R(+) 7-OH-DPAT on the development of the drug-associated CR. Hence, both Paired and Unpaired groups were exposed to two different CSs which were presented on alternate sessions. Post-session bilateral intra-amygdala administration of R(+) 7-OH-DPAT (10 nmol) followed sessions in which one CS was presented, while intra-amygdala vehicle followed sessions in which the alternate CS was presented. The development of a CR occurred only in the presence of a CS that had been positively correlated with presentation of the drug US. Post-session, intra-amygdala administration of R(+) 7-OH-DPAT enhanced the acquisition of this CR. However, R(+) 7-OH-DPAT was without effect upon the unconditioned response to intra-accumbens d-amphetamine. Our previous data indicate a comparable effect of R(+) 7-OH-DPAT on conditioning to a CS associated with a non-drug, natural reward. Therefore, taken together, these findings suggest that D3 dopamine receptors within the amygdala modulate specifically the acquisition of Pavlovian conditioned responses, regardless of whether drug or natural rewards constitute the US.

Effects of intra-amygdala R(+) 7-OH-DPAT on intra-accumbens d-amphetamine-associated learning. II. Instrumental conditioning.

Rats were trained to associate an initially neutral conditioned stimulus (CS) with a response-independent, intra-accumbens infusion of d-amphetamine (the unconditioned stimulus; US). Elsewhere, we have reported that as a result of this training, presentations of the CS alone elicited a conditioned response consisting of increased locomotor activity and that acquisition of this conditioned response was enhanced by post-session, intra-amygdala infusion of the dopamine D3 receptor preferring agonist, R(+) 7-OH-DPAT. Here, in this same group of animals, we have examined the conditioned rewarding properties of the drug-associated CS by determining its ability to support the acquisition of a novel instrumental response in the absence of drug reward. Thus, rats were presented with two novel levers. Presentation of the drug-associated CS was made contingent upon depression of one of the levers (CR lever), while responding upon the other lever (NCR lever) had no programmed consequences. Preferential responding upon the lever delivering the drug-associated CS was observed despite a 6-week interval between CS-US training and the conditioned reward test. Intra-accumbens administration of d-amphetamine (0-20 microg) increased the control over behaviour exerted by the CS, increasing CR. but not NCR lever responding. In contrast, rats that received three pairings of an intra-accumbens infusion of d-amphetamine in combination with intra-amygdala infusion of R(+) 7-OH-DPAT, 3 weeks prior to testing, displayed similar rates of response upon both levers and were insensitive to the potentiation of responding for conditioned reward following intra-accumbens d-amphetamine. However, intra-accumbens d-amphetamine stimulated locomotor activity in a similar, dose-related manner in both groups. In this way, rats that had received intra-accumbens infusion of d-amphetamine in combination with intra-amygdala infusion of R(+) 7-OH-DPAT appeared exactly like control group rats, for which the CS had been paired with intra-accumbens d-amphetamine on a negative basis only. A locomotor activity test indicated that one behavioural consequence of intra-amygdala administration of R(+) 7-OH-DPAT was the reduction of the unconditioned locomotor response resulting from intra-accumbens administration of d-amphetamine. Hence, the present data demonstrate that the conditioned rewarding properties of a drug-associated CS are specific to the CS-US association and are relatively insensitive to decay over time. However, the rewarding properties of a drug-associated CS were selectively abolished following activation of amygdala D3 receptors during presentation of the drug reward. Potential explanations for this effect are discussed, including the possibility that intra-amygdala R(+) 7-OH-DPAT reduced the incentive value of the US.

Double dissociation of the behavioural effects of R(+) 7-OH-DPAT infusions in the central and basolateral amygdala nuclei upon Pavlovian and instrumental conditioned appetitive behaviours.

Dopaminergic cell bodies located within the ventral mesencephalon innervate the amygdaloid complex, a region critically involved in the attribution of affective significance to environmental stimuli. Recently, we have shown that post-session intra-amygdala administration of a D3 dopamine receptor agonist enhances selectively the acquisition of an appetitive conditioned response. In the present study, we have investigated the potential involvement of the central nucleus and the basolateral nuclei of the amygdala in mediating this effect. Thus, rats were trained to associate an arbitrary stimulus (CS+) with the availability of 10% sucrose reward. Post-session infusions of the D3 receptor-preferring agonist, R(+) 7-OH-DPAT, were made into either the central nucleus or basolateral nuclei. Acquisition of a conditioned approach response was enhanced by R(+) 7-OH-DPAT infusions within the central nucleus, but not within the basolateral nuclei. Drug infusions into either region failed to affect approach behaviour elicited by presentation of a control stimulus (CS-), explicitly unpaired with sucrose reward. The effects of pre-test infusions of R(+) 7-OH-DPAT on the instrumental properties of the stimuli were then determined. Rats were presented with two novel levers, depression of one lever resulted in presentation of the CS+, while presentation of the CS- was contingent upon depression of the other lever. Rates of response upon each lever as well as the ability of the conditioned stimuli subsequently to elicit conditioned approach behaviour were recorded. Data revealed a double dissociation of the effects of R(+) 7-OH-DPAT on the expression of the Pavlovian and instrumental properties of the reward-related stimulus. Thus, within the central nucleus R(+) 7-OH-DPAT dose-dependently attenuated expression of the conditioned approach response, but had no effect upon instrumental responding maintained by the conditioned reward. In contrast, within the basolateral nuclei, R(+) 7-OH-DPAT had no effect upon expression of conditioned approach behaviour, but abolished selectively the ability of the reward-associated stimulus to support the acquisition of a novel instrumental response. Hence, these data indicate that distinct regions of the amygdaloid complex process distinct aspects of conditioned appetitive behaviours.

Enhanced stimulus-reward learning by intra-amygdala administration of a D3 dopamine receptor agonist.

The amygdala is considered to be a critical neural substrate underlying the formation of stimulus-reward associations, and is known to receive substantial innervation from dopaminergic neurons located within the ventral mesencephalon. However, relatively little is known about the function of the mesoamygdaloid dopamine projection in stimulus-reward learning. Recently, we have found post-session intra-amygdala microinjections of d-amphetamine to enhance appetitive Pavlovian conditioning as assessed in a discriminative approach task. In the present study, we have examined the effects of dopamine receptor agonists possessing relative selectivity for the D1, D2 and D3 receptor subtypes in order to examine more fully the role of the mesoamygdaloid dopamine projection in stimulus-reward learning. Thus, subjects were trained to associated an initially neutral stimulus (CS+) with 10% sucrose reward (US). A second, control stimulus (CS-) was also presented but never paired with sucrose reward. In order to measure specifically the conditioned response to CS+/CS- presentation, responding during CS and US presentations was measured separately. Immediately following each training session, subjects received bilateral intra-amygdala infusion of 0.1, 1 or 10 nmol/side of SKF-38393, quinpirole or 7-OH-DPAT. Infusions of SKF-38393 or quinpirole were without effect on CS+ approach. However, post-session intra-amygdala infusions of 7-OH-DPAT enhanced selectively CS+ approach in a dose-dependent fashion. No dose of any drug affected CS- approach, US behaviours, or measures of extraneous behaviour. Subsequent acquisition of a novel conditioned instrumental response was also unaffected. Thus, the present data indicate a selective involvement of the D3 dopamine receptor subtype in the modulation of stimulus-reward learning by the mesoamygdaloid dopamine projection.

Enhanced acquisition of discriminative approach following intra-amygdala d-amphetamine.

This study examined the role of the mesoamygdaloid dopamine projection in stimulus-reward learning. Bilateral post-session intra-amygdala microinjections of d-amphetamine were carried out in rats during training in a discriminative approach task known to be sensitive to experimental manipulations of the amygdala. The experiment consisted of two phases: discriminative approach training, and a subsequent assessment of instrumental conditioned reward efficacy. During discriminative approach training, subjects were trained to associate a neutral stimulus with 10% w/v sucrose reward. Each trial consisted of a 1-s light stimulus followed by a 5-s presentation of the sucrose reward. Approach behaviour into the recess housing sucrose reward was measured during each trial. Inappropriate approach behaviour (approach outside of the trial periods) was punished by delaying the next trial. Intra-amygdala d-amphetamine (10 microg/side) enhanced the rate of acquisition of discriminative approach behaviour. This effect was most evident early during training (sessions 2-4) and by the tenth session both groups had reached similar asymptotic performance. Horizontal and vertical activity increased slightly across sessions, but there was no indication of a differential effect of d-amphetamine. Thus, intra-amygdala microinjections of d-amphetamine enhanced selectively the acquisition of the stimulus-reward association. During a subsequent test of instrumental conditioned reward, presentation of the conditioned light stimulus was made contingent upon performance of a novel lever-pressing response (probability 0.5). Responding on a second, control lever was without programmed consequences. Sucrose reward was not available at any point, and subjects were tested drug-free. In both groups the conditioned stimulus was found to possess significant conditioned rewarding efficacy. Extraneous behaviour was increased in the d-amphetamine group but the rewarding properties of the conditioned stimulus were unaltered. These findings demonstrate that the mesoamygdaloid dopamine projection modulates the acquisition of a stimulus-reward association, but is apparently without subsequent effect on the rewarding efficacy of a conditioned stimulus.

Repeated d-amphetamine enhances stimulated mesoamygdaloid dopamine transmission.

The mesoaccumbens dopamine pathway exhibits an enhanced dopaminergic response to a challenge injection of d-amphetamine or cocaine after repeated intermittent exposure to that drug. Much research has focused on the potential role of this sensitised response in the enhanced propensity of drug-associated stimuli to elicit relapse. However, the amygdala is acknowledged to play a critical role in stimulus-reward learning, and recent work suggests that the mesoamygdaloid dopamine pathway exerts a significant influence upon amygdala function. In the present study, rats were administered d-amphetamine (1 mg/kg, I.P.) or vehicle once per day, for 14 days. After 11 untreated days, a locomotor assay showed that prior repeated administration of d-amphetamine led to a markedly enhanced locomotor response to 0.5 mg/kg d-amphetamine. There was no effect of d-amphetamine pretreatment upon the response to a novel environment, or to injection with vehicle. Following a total of 14 days in the home cage, subjects were implanted with microdialysis probes within the amygdala, and for comparison also within the nucleus accumbens. Baseline and d-amphetamine-stimulated (0.5 mg/kg) levels of extracellular dopamine were assessed for each brain region. Results showed that baseline levels of dopamine were very similar in sensitised and control animals. By contrast, prior treatment with d-amphetamine enhanced dopamine overflow in response to a challenge with d-amphetamine both in the nucleus accumbens and amygdala. These results indicate that changes in the pattern of dopamine transmission both in the nucleus accumbens, and the amygdala, accompany the behavioural sensitisation observed after repeated exposure to d-amphetamine. Hence, an enhanced propensity of drug-associated stimuli to elicit relapse may not depend solely upon changes relating to the mesoaccumbens dopamine projection.

Amygdala and hippocampus control dissociable aspects of drug-associated conditioned rewards.

Limbic innervation of the nucleus accumbens via the ventral subiculum/hippocampus and basolateral area of the amygdala has been shown to determine dissociable aspects of behaviour controlled by stimuli associated with natural rewards. However, the respective contributions of the ventral subiculum and amygdala to behaviour governed by drug-associated stimuli remain to be determined. Experiments consisted of two phases: drug-stimulus training, and subsequent stimulus-only testing. Initial training sessions were of two alternating forms. During drug sessions, responding upon one lever resulted in an infusion of 1 microgram d-amphetamine into the nucleus accumbens, whilst during saline sessions d-amphetamine was replaced with saline. Each infusion (drug or saline) was preceded with either a light, or tone. Responding upon a control lever had no programmed consequences. Following training, the levers were retracted, and instead two novel vertical bars were extended from the chamber ceiling. Movement of one bar produced the drug-associated stimulus, whilst the alternative bar produced the saline-associated stimulus. Infusions of the AMPA receptor antagonist CNQX into the ventral subiculum or basolateral area of the amygdala (0, 0.2, 2.0 nmol) were made immediately before the start of each session. Intra-basolateral area of the amygdala CNQX reduced responding upon the drug-associated stimulus bar, but at the same time increased responding upon the saline-associated stimulus bar. By contrast, intra-ventral subiculum CNQX reduced drug-associated stimulus responding selectively. Neither manipulation affected levels of activity within the operant chamber extraneous to the bar-pushing response. Hence, the basolateral area of the amygdala appeared to have determined the degree of discriminative control exerted over behaviour by the drug-associated stimulus, whilst the ventral subiculum is suggested to have determined the efficacy of the conditioned reward.

Concurrent treatment with verapamil prevents diazepam withdrawal-induced anxiety, in the absence of altered calcium flux in cortical synaptosomes.

Rats, chronically treated with diazepam (4 mg/kg/day) for 28 days, displayed increased anxiety when tested in the elevated plus-maze, 42 hr after the last dose. This anxiogenic withdrawal response was entirely prevented by the concurrent administration of the calcium channel antagonist, verapamil. No anxiolytic effect of chronic administration of verapamil was observed in vehicle-treated rats. To investigate the possibility that increased calcium function in nerve terminals might underlie diazepam withdrawal-induced anxiety, the uptake by cortical synaptosomes of 45Ca2+ was studied. Both fast (3-sec) and slow (60-sec) phase uptake were measured. No changes in basal (5 mM), potassium-stimulated (55 mM) or net uptake were observed during either fast or slow phase uptake. It is concluded that increased calcium influx in nerve terminals in the cortex does not underlie the anxiogenic effect of withdrawal of the benzodiazepine but that further studies must be carried out in other regions of the brain.

Effects of nitrendipine, chlordiazepoxide, flumazenil and baclofen on the increased anxiety resulting from alcohol withdrawal.

1. Male hooded Lister rats were fed a liquid diet containing 10% absolute ethanol for 4-5 weeks. Control rats received the liquid diet in amounts controlled to produce equal weight gain. 2. The rats were tested 7.5 h after withdrawal of ethanol and 30 min after i.p. injection with nitrendipine, chlordiazepoxide or baclofen or 20 min after i.p. injection with flumazenil. 3. Nitrendipine (25-100 mg/kg) was unable to reverse the anxiogenic responses detected on withdrawal from ethanol, but the highest dose did reduce withdrawal tremor. 4. Chlordiazepoxide (10 mg/kg), flumazenil (4 mg/kg) and baclofen (1.25 mg/kg) significantly reversed the anxiogenic response detected on withdrawal from ethanol. 5. These reversals of ethanol withdrawal responses are similar to the reversal of the increased anxiety detected on withdrawal from chronic treatment with benzodiazepines. 6. The mechanisms and clinical implications of these drug-induced reversals are discussed.

Chronic diazepam treatment in rats causes long-lasting changes in central [3H]-5-hydroxytryptamine and [14C]-gamma-aminobutyric acid release.

The effects of chronic diazepam administration to rats on the central release of [3H]-5-hydroxytryptamine ([3H]-5-HT) and [14C]-gamma-aminobutyric acid ([14C]-GABA, ex vivo) were examined. Chronic (5 and 21 days) administration of diazepam (4 mg kg-1 i.p. daily for 21 days) reduced the K-evoked (20 mM KCl) release of [3H]-5-HT from frontal cortex by approximately 50%. Remarkably, this decrease was still present 1 week after diazepam withdrawal. Chronic diazepam treatment did not significantly affect hippocampal [3H]-5-HT release but after 21 days the K-evoked release of [14C]-GABA was more than doubled and remained elevated 30 h after withdrawal; it returned to control levels after 1 week, and decreased below control levels after 2 weeks. This study indicates that chronic diazepam treatment produces striking changes in transmitter release in rats that persist long after treatment has ceased.

A theory of benzodiazepine dependence that can explain whether flumazenil will enhance or reverse the phenomena.

Repeated administration of benzodiazepines (BDZs) produces dependence in man and animals and this is reflected in the phenomena of tolerance and withdrawal responses. In BDZ-dependent animals the BDZ-receptor antagonist flumazenil (Ro 15-1788) reverses the increased anxiety and decreased seizure threshold seen when benzodiazepine treatment is withdrawn. In contrast are reports that flumaenil enhances BDZ-withdrawal responses. Indirect influences on the direction of flumazenil's effects on anxiety are the duration and dose of BDZ treatment, whether tolerance has developed to its anxiolytic effect and whether there is an anxiogenic response on drug withdrawal. However, we conclude that the crucial factor is the anxiety level of the animal: when this is high flumazenil becomes anxiolytic; when this is low flumazenil is anxiogenic. These bidirectional effects of flumazenil can be seen in drug-naive and BDZ-dependent animals. We propose a theory of benzodiazepine dependence that can account for anxiogenic responses on drug withdrawal and for flumazenil's bidirectional effects; central to this theory is the assumption that flumazenil normalises the benzodiazepine receptor, returning it to a baseline state. Thus it is whether an animal's score lies above or below this baseline that will determine the direction of flumazenil's effect. The clinical implications of this theory are discussed. We suggest that during the development of benzodiazepine dependence, two independent adaptive biochemical mechanisms are triggered: one underlying the development of tolerance to the anxiolytic responses, the other underlying the incidence of increased anxiety on drug withdrawal. It is only changes in the latter that are induced by the administration of flumazenil.

Characterisation of the phenomenon of "one-trial tolerance" to the anxiolytic effect of chlordiazepoxide in the elevated plus-maze.

In the elevated plus-maze test of anxiety the scores of control animals remain stable over repeated tests. However, a single prior exposure to the plus-maze renders an animal insensitive to the anxiolytic effects of chlordiazepoxide. This phenomenon of "one-trial tolerance" persisted even when the two trials were separated by as much as 2 weeks. It has previously been shown that the drug state of the animal on trial 1 is not important to the development of the phenomenon, but one-trial tolerance did not develop if a very high dose (75 mg/kg) of chlordiazepoxide was given on trial 1; it is suggested that this is due to the amnesic effects of the drug. The learning on trial 1 was not specific to a particular plus-maze and tolerance could be observed even when the maze on trial 1 was made from different material. The crucial experience on trial 1 was experience of an open arm of the maze. Whereas tolerance could be obtained as a result of a previous plus-maze experience, there was no evidence of an anxiogenic withdrawal response when rats were tested the following day undrugged. The phenomenon of one-trial tolerance is explained within our recently proposed two-factor theory of benzodiazepine dependence; it is suggested that one-trial tolerance provides a method for studying the mechanism underlying the development of tolerance to anxiolytic effects, independently from the mechanism underlying the development of withdrawal responses.

Evidence that the increased anxiety detected in the elevated plus-maze during chlordiazepoxide withdrawal is not due to enhanced noradrenergic activity.

Rats displayed a reduction in the percentage of time spent on the open arms of the elevated plus-maze 24-30 hours after withdrawal from chronic chlordiazepoxide treatment (10 mg/kg/day IP for 4 weeks). This indicated an anxiogenic response in this test. This anxiogenic response was not significantly reversed by DL-propranolol (5 and 10 mg/kg IP) or clonidine (0.02 and 0.04 mg/kg IP). These results provide no evidence to suggest that the anxiogenic effects of chlordiazepoxide withdrawal are mediated by an increase in noradrenergic activity. The possible involvement of multiple transmitter systems in benzodiazepine withdrawal symptomology is discussed.

Flumazenil but not nitrendipine reverses the increased anxiety during ethanol withdrawal in the rat.

After 7 day's gradual introduction of ethanol, rats were maintained for a further 4 weeks on a liquid diet containing 10% ethanol (mean daily dose 11.8 +/- 0.2 g/kg/day). Control-treated rats received liquid diet alone. Pairs of rats were tested in the social interaction test of anxiety 8 h after withdrawal. Withdrawal from ethanol significantly reduced the time spent in social interaction compared with controls, indicating an anxiogenic withdrawal response. Nitrendipine (50 mg/kg) had no effect on, whereas flumazenil (4 mg/kg) significantly reversed, this withdrawal response. This reversal appeared to be long-lasting as there was still no evidence of increased anxiety when rats were again withdrawn after 3 more days of ethanol diet.