György Lévay

mCPP-induced anxiety in the light-dark box in rats – a new method for screening anxiolytic activity

Abstract The activity of anxiolytic and other drugs in a light-dark test situation was studied in rats treated with the anxiogenic compound m-chlorophenyl-piperazine (mCPP). mCPP 0.5 mg/kg significantly diminished the exploratory activity of the animals in the light compartment of the apparatus. Drugs to be tested against mCPP-induced anxiety when studied alone (not in combination with mCPP) did not significantly alter the activity of rats in the light-dark apparatus, except yohimbine, which reduced the movement time values in the lit area. 1,4-Benzodiazepines [diazepam (0.1–4 mg/kg) and chlordiazepoxide (2–8 mg/kg)], 5-HT2A/2C antagonists [ritanserin (0.25–8 mg/kg) and deramciclane (0.5–8 g/kg)], the 5-HT3 antagonist MDL-72222 (3 mg/kg) and ethanol (2–4 mg/kg) significantly reduced the effect of mCPP. A dose-dependent increase in the exploratory activity of mCPP-treated animals was found in the 2,3-benzodiazepine girisopam (2.5–5 mg/kg)-treated groups. Tofisopam, another 2,3-benzodiazepine molecule, also showed activity against mCPP, although its effect was not statistically significant. The 5-HT1A partial agonist buspirone was also active in the dose range of 0.25–0.5 mg/kg, while the 5-HT1A full agonist 8-OH-DPAT was the only drug with presumed anxiolytic activity that clearly lacked any effect in this model. Imipramine, amitriptyline, morphine, naloxone, haloperidol, clozapine, amphetamine, yohimbine, carbamazepine and diphenylhydantoin were not effective. We conclude that mCPP-induced anxiety in the light-dark box is a potent and useful method for screening and detecting anxiolytic activity of a wide range of compounds with various modes of action.

Use of TTC staining for the evaluation of tissue injury in the early phases of reperfusion after focal cerebral ischemia in rats

BACKGROUND AND PURPOSE 2,3,5-Triphenyltetrazolium chloride (TTC) staining measures tissue viability used to evaluate infarct size. The goal of this study was to compare viability of neuronal tissue during the early phases of ischemia-reperfusion assessed either by perfusion of the brain with TTC solution transcardially, in vivo, or by staining brain slices, in vitro. METHODS The middle cerebral artery was occluded for 1 h in male SPRD rats and then reperfused for 0, 1, 4, 8, 16 and 24 h. Ischemic damage was evaluated by TTC staining, in vivo and in vitro, and by histology (Luxol Fast Blue and Fluoro-Jade staining, electron microscopy). RESULTS Core volume of tissue injury measured in vivo was large at 0 h and steadily decreased by 50% (p<0.001) up to 16 h, but substantially increased from 16 to 24 h of reperfusion. In contrast, a significant core volume appeared at 4 h only, in vitro, and gradually increased up to 24 h. Core volume was larger in vivo than in vitro at all times except at 16 h when the opposite was observed. Evans blue administered intracardially stained TTC-negative areas at 1 and 24 h. Histology covered the evolution of serious tissue injury but also demonstrated some morphologically preserved neurons in the infracted area at 24 h. CONCLUSIONS Formation of formazan from TTC can depend on both the staining method and the metabolic burden of the brain tissue causing uncertainties in the volume of ischemia-induced brain injury measured by TTC staining.

Chronic mild stress generates clear depressive but ambiguous anxiety-like behaviour in rats

A 3-week chronic mild stress (CMS) protocol decreased sucrose preference of rats and increased immobility in the forced swim test. It also induced social avoidance and increased grooming, but acted as if reducing anxiety in the plus-maze. Sucrose preference and social avoidance, but not other measures of the behaviour, showed significant correlation. We conclude that CMS-induced depression-like behaviour is associated with social avoidance, a seemingly anxiety-related measure, but not with other anxiety-like traits in rats.

Selective 5-HT1A and 5-HT7 antagonists decrease epileptic activity in the WAG/Rij rat model of absence epilepsy.

Recent studies have provided evidence that activation of 5-HT1A receptors increases epileptic activity in the WAG/Rij rat model of absence epilepsy, and additional data have suggested the involvement of 5-HT7 receptors as well. Therefore, we have tested the effects of the selective 5-HT1A receptor antagonist WAY-100635 and the selective 5-HT7 receptor antagonist SB-258719 on spontaneous epileptic activity. In general, both compounds reduced epileptic activity compared to vehicle. Significant decreases were found in the number of paroxysms and the cumulative and average duration of spike-wave discharges (SWDs), although the time courses of these effects induced by the two compounds were clearly different. These results provide evidence that activation of 5-HT1A and 5-HT7 receptors plays a significant role in regulating SWD activity in this animal model of absence epilepsy.

A mouse model of anxiety molecularly characterized by altered protein networks in the brain proteome

Recently, several attempts have been made to describe changes related to certain anxiety states in the proteome of experimental animal models. However, these studies are restricted by limitations regarding the number and correct identification of separated proteins. Moreover, the application of a systems biology approach to discover the molecular mechanisms of anxiety requires genetically homogenous inbred animal models. Therefore, we developed a novel mouse model of anxiety using a combination of crossbreeding (inbred for 35 generations) and behavioral selection. We found significant changes in 82 proteins in the total brain proteome compared to the control proteome. Thirty-four of these proteins had been previously identified in other anxiety, depression or repeated psychosocial stress studies. The identified proteins are associated with different cellular functions, including synaptic transmission, metabolism, proteolysis, protein biosynthesis and folding, cytoskeletal proteins, brain development and neurogenesis, oxidative stress, signal transduction. Our proteomics data suggest that alterations in serotonin receptor-associated proteins, in the carbohydrate metabolism, in the cellular redox system and in synaptic docking are all involved in anxiety.

5-HT2C receptors inhibit and 5-HT1A receptors activate the generation of spike–wave discharges in a genetic rat model of absence epilepsy

The present study was conducted to investigate the role of 5-HT(2C) and 5-HT(1A) receptors in the generation of spike-wave discharges (SWD) in the genetic absence epilepsy model Wistar Albino Glaxo rats from Rijswijk, Netherlands (WAG/Rij rats). We have determined the effects of the 5-HT(2C) receptor preferring agonist m-chlorophenyl-piperazine (m-CPP), the selective 5-HT(2C) receptor antagonist SB-242084, the selective 5-HT(1A) receptor antagonist WAY-100635, two selective serotonin re-uptake inhibitors (SSRI, fluoxetine and citalopram) and their combinations in this model. The 5-HT(2C) agonist m-CPP caused marked, dose-dependent decreases in the cumulative duration and number of SWD administered either intraperitoneally (0.9 and 2.5 mg/kg) or intracerebroventricularly (0.05 and 0.1 mg/kg). Treatment with SB-242084 (0.2 mg/kg, ip) alone failed to cause any significant change in SWD compared to vehicle. Pretreatment with SB-242084 (0.2 mg/kg, ip) eliminated the effects of m-CPP on SWD. Fluoxetine (5.0 mg/kg, ip) alone caused moderate increase in SWD. After pretreatment with SB-242084, the effect of fluoxetine was significantly enhanced. The combination of SB-242084 and citalopram (2.5 mg/kg, ip) caused a similar effect, namely an increase in SWD. In contrast, pretreatment with WAY-100635 significantly attenuated the effect of fluoxetine. In conclusion, these results indicate that the increase in endogenous 5-HT produces a dual effect on SWD; the inhibition of epileptiform activity is mediated by 5-HT(2C), the activation by 5-HT(1A) receptors.

Comparison of anticonvulsive and acute neuroprotective activity of three 2,3-benzodiazepine compounds, GYKI 52466, GYKI 53405, and GYKI 53655

GYKI 52466 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine], a non-competitive AMPA [alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate] and kainate receptor antagonist and its two analogues, GYKI 53405 [1-(4-aminophenyl)-3-acetyl-4-methyl-3,4-dihydro-7,8-methylenedioxy-5H-2,3-benzodiazepine] and GYKI 53655 [1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-3,4-dihydro-7,8-methylenedioxy-5H-2,3-benzodiazepine] were investigated in two seizure models and in MgCl2 induced global cerebral ischaemia, as an acute neuroprotective model. The ED(50) values of GYKI 52466 for suppression of the tonic and clonic phases of sound-induced seizures were 3.6 and 4.3 mg/kg, respectively. The corresponding data for GYKI 53405 were 1.1 and 3.1 mg/kg, while ED(50) values of GYKI 53655 were 1.3 and 2.0 mg/kg, respectively. The inhibition of seizure evoked by maximal electroshock was also found to be remarkable: the ED(50) values of GYKI 52466 and its two analogues were 6.9, 2.6, and 2.2 mg/kg, respectively. All compounds prolonged the survival times in MgCl2 induced global cerebral ischaemia test in a dose-dependent fashion, with PD(50) (dose of 50% prolongation) values of 24.1, 8.3, and 8.2 mg/kg intraperitoneal, respectively. In audiogenic seizure model the duration of anticonvulsant action of 10 mg/kg GYKI 52466 and 5 mg/kg GYKI 53405, GYKI 53655 were examined, too. The effect of GYKI 52466 decreased to 50% after 2 h, while the analogues showed more than 80% seizure suppression 3 h after treatment. After 6 h the effect of GYKI 53655 decreased to zero, while the effect of GYKI 52466, remained on the 50% level.

Effect of two noncompetitive AMPA receptor antagonists GYKI 52466 and GYKI 53405 on vigilance, behavior and spike-wave discharges in a genetic rat model of absence epilepsy

The present study was conducted to investigate the effects of two noncompetitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists, GYKI 52466 and GYKI 53405 (the racemate of talampanel) on the generation of spike-wave discharges (SWD) parallel with the vigilance and behavioral changes in the genetic absence epilepsy model of WAG/Rij rats. Intraperitoneal (i.p.) administration of GYKI 52466 (1-[4-aminophenyl]-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine; 3, 10 and 30 mg/kg, i.p.), the prototypic compound of the 2,3-benzodiazepine family, caused a fast dose-dependent increase in the number and cumulative duration of SWD. These changes were accompanied by dose-dependent increase in duration of light slow wave sleep (SWS1) and passive awake, vigilance states associated with the presence of SWD. In addition a short, transient behavioral activation occurred that was followed by strong ataxia and immobility, decrease of active wakefulness and increase in deep slow wave sleep. GYKI 53405 (7-acetyl-5-(4-aminophenyl)-8-methyl-8,9-dihydro-7H-1,3-dioxolo[4,5-b][2,3]benzodiazepine, the racemate of talampanel, 16 mg/kg, i.p.) failed to affect any measure of SWD and vigilance. When used as a pretreatment, GYKI 52466 (10 mg/kg) slightly attenuated SWD-promoting effects of the 5-HT1A receptor agonist 8-OH-DPAT, it decreased cumulative duration and average time of paroxysms. In conclusion, AMPA receptors play moderate role in regulation of epileptic activity, and some of these effects are connected to their effects on vigilance in this model.

Pharmacological evaluation of the stress-induced social avoidance model of anxiety

We have shown earlier that mild electric shocks induce a lasting social avoidance in male rats. Here we investigated whether shock-induced social avoidance can be developed into a laboratory model of stress-induced anxiety. The putative new model would assess sub-chronic, stress-induced anxiety (as opposed to tests based on natural fear) in a heterologous context (as opposed to classical fear conditioning). A single exposure to mild electric shocks induced a robust social avoidance that lasted more than 5 days. Low doses of chlordiazepoxide (0.5, 1 mg/kg), diazepam (0.5, 1, 5 mg/kg), buspirone (0.3, 1 mg/kg), and fluoxetine (1, 3, 5 mg/kg) abolished this effect, whereas the anxiogenic compound m-chlorophenylpiperazine (0.5-3 mg/kg) induced social avoidance in unshocked rats. These effects were produced at doses that did not affect locomotion in the open field. Haloperidol (0.05, 0.1, 1, 5 mg/kg) influenced social avoidance at sedative doses only. The sensitivity of the model to anxiolytic agents was compromised at high (sedating) doses. Taken conjointly, these data show that shock-induced social avoidance can be used to assess the anxiolytic potential of compounds. In addition to predictive validity, the model appears to show construct and face validity as well: stress is among the etiological factors of, whereas social avoidance simulates the social deficits seen in, a variety of anxiety disorders. The model may be used to study the effects of anxiolytics on sub-chronic states of stress-induced anxiety.

Reduction of cerebral infarct size by non-competitive AMPA antagonists in rats subjected to permanent and transient focal ischemia

Antagonists of 2-amino-3(3-hydroxy-5-methyl-4-isoxazolyl) propionic acid (AMPA) receptors can considerably reduce brain damage after cerebral ischemia, but effectiveness of selective AMPA antagonists has been questioned recently. Therefore, we evaluated the antiischemic efficacy of [+/-]-7-acetyl-5-[4-aminophenyl]-7,8-dihydro-8-cyano-8-methyl-9H-1,3-dioxolo-[4,5-h]-2,3-benzodiazepine (EGIS-8332) and GYKI 53405, two selective, non-competitive AMPA antagonists in two rat models of focal cerebral ischemia. Permanent focal ischemia was produced by electrocoagulation of the middle cerebral artery (MCA). EGIS-8332 and GYKI 53405 were administered 30 min after MCA occlusion at doses of 1, 3 or 10 mg/kg i.p. In transient focal ischemia, MCA was occluded for 1 h and reperfused for 24 h using the intraluminal filament technique and the compounds were given at 3x10 mg/kg i.p. 60, 120 and 180 min following occlusion. In permanent focal ischemia, EGIS-8332 decreased the volume of cerebral infarction both at 10 mg/kg i.p. (36.4%, p<0.01) and at 3 mg/kg i.p. (26.4%, p<0.05) in a dose-dependent manner. GYKI 53405 produced a similar antiischemic effect at 10 mg/kg i.p. (36.4%, p<0.01), but it was ineffective at 3 mg/kg i.p. (6.5%, p=0.57). In transient focal ischemia, EGIS-8332 reduced the volume of necrotic brain tissue (38.7%, p<0.01) and GYKI 53405 was similarly effective (32.6%, p<0.05). Both compounds afforded neuroprotection in the cortical and subcortical regions of the MCA territory. Selective, non-competitive AMPA antagonists administered after the ischemic insult can produce effective neuroprotective action in experimental models of focal cerebral ischemia; therefore, these compounds may be useful as therapeutic agents for the treatment of stroke and neurodegenerative disorders.