Potent Anxiolytic-like Effects Research Articles

Anxiolytic-like property of risperidone and olanzapine as examined in multiple measures of fear in rats

Atypical antipsychotics are also used in the treatment of anxiety-related disorders. Clinical and preclinical evidence regarding their intrinsic anxiolytic efficacy has been mixed. In this study, we examined the potential anxiolytic-like effects of risperidone and olanzapine, and compared them with haloperidol, chlordiazepoxide (a prototype of sedative–anxiolytic drug) or citalopram (a selective serotonin reuptake inhibitor). We used a composite of two-way avoidance conditioning and acoustic startle reflex model and examined the effects of drug treatments during the acquisition phase (Experiment 1) or extinction phase (Experiments 2 and 3) on multiple measures of conditioned and unconditioned fear/anxiety-like responses. In Experiment 4, we further compared risperidone, olanzapine, haloperidol, citalopram and chlordiazepoxide in a standard elevated plus maze test. Results revealed three distinct anxiolytic-like profiles associated with risperidone, olanzapine and chlordiazepoxide. Risperidone, especially at 1.0 mg/kg, significantly decreased the number of avoidance responses, 22 kHz ultrasonic vocalization, avoidance conditioning-induced hyperthermia and startle reactivity, but did not affect defecations or time spent on the open arms. Olanzapine (2.0 mg/kg, sc) significantly decreased the number of avoidance responses, 22 kHz vocalization and amount of defecations, but it did not inhibit startle reactivity and time spent on the open arms. Chlordiazepoxide (10 mg/kg, ip) significantly decreased the number of 22 kHz vocalization, avoidance conditioning-induced hyperthermia and amount of defecations, and increased time spent on the open arms, but did not decrease avoidance responses or startle reactivity. Haloperidol and citalopram did not display any anxiolytic-like property in these tests. The results highlight the importance of using multiple measures of fear-related responses to delineate behavioral profiles of psychotherapeutic drugs.

Assessing anxiolytic-like effects of selective serotonin reuptake inhibitors and serotonin-noradrenaline reuptake inhibitors using the elevated plus maze in mice

The anxiolytic-like effects of selective serotonin reuptake inhibitors (SSRIs; paroxetine, fluvoxamine) and serotonin-noradrenaline reuptake inhibitors (SNRIs; milnacipran, venlafaxine) were compared with those of benzodiazepines (diazepam, chlordiazepoxide) and tricyclic antidepressants (imipramine, amitriptyline) using the elevated plus maze in mice. Paroxetine and fluvoxamine had no significant effects on the time spent in open arms and the number of open arm entries, even at a dose of 20 mg/kg p.o. On the other hand, milnacipran and venlafaxine showed a dose-dependent increase in the time spent in open arms and the number of open-arm entries. Significant effects were observed at doses of 10 and 20 mg/kg p.o. for both drugs. Diazepam and chlordiazepoxide showed potent anxiolytic-like effects, whereas imipramine and amitriptyline caused no anxiolytic-like effects. Like diazepam and chlordiazepoxide, milnacipran and venlafaxine increased the distance moved in open arms at the same dose levels showing anxiolytic-like effects. From these results, it may be concluded that SNRIs caused anxiolyic-like effects similar to benzodiazepines.

Synthesis, in Vivo Evaluation, and Molecular Modeling Studies of New Pyrazolo[5,1-c][1,2,4]benzotriazine 5-Oxide Derivatives. Identification of a Bifunctional Hydrogen Bond Area Related to the Inverse Agonism

A new series of pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide 8-alkyloxy-/aryloxy-/arylalkyloxy and 8-aryl-/arylalkylderivatives variously substituted at the 3-position were synthesized and binding studies at the benzodiazepine site on GABA(A) receptor were carried out. The pharmacological profile was identified for compounds 10, 11, 16(+), 16(-), and 17 by considering six potential benzodiazepine actions: motor coordination, anticonvulsant action, spontaneous motility and explorative activity, potential anxiolytic-like effects, mouse learning and memory modulation, and finally, ethanol-potentiating action. Compound 17 stands out as the compound that improves mouse memory processes selectively, safely, and in a statistically significant manner. From a ligand-based pharmacophoric model, we identified a hydrogen bond interaction area HBp-3 near the lipophilic area. This new pharmacophoric model allowed us to identify four structural compound typologies and thus to rationalize the affinity data of all compounds.

Anxiolytic-like Effects of Saponin and Polysaccharide Fractions Extracted from White and Red Ginsengs in the Elevated Plus-Maze Model

Ginseng has been widely used for the management of anxiety and emotional instability, but there is little experimental evidence supporting these clinical applications. The anxiolytic-like effect of ginseng saponin and polysaccharide fractions of white (WG) and red ginsengs (RG) was investigated using the elevated plus-maze test. The saponin (SF) and polysaccharide (PF) fractions were orally administered to male ICR mice for 3 days and behavioral test for the anxiolytic activity were performed. SF significantly increased the time-spent open arms and number into the in the open arm entries. However, PF weakly increased the time-spent in the open arms, but did not increase number into the open arm entries. The WG showed more potent anxiolytic-like effect than that of RG.. The anxiolytic-like activities were antagonized by flumazenil, but not by esmolol. These findings suggest the saponin fractions of WG and RG promote the anxiolytic-like activity by antagonizing GABA/benzodiazepine receptors in mice.

Behavioral effects of modafinil in marmoset monkeys

Modafinil is increasingly used in sleep disturbances in general and in neurodegenerative diseases and is recently being used in healthy people for attention control. However, the application of modafinil is possibly not only restricted to alertness enhancing effects. More insight in this compound may lead to new applications. Not all behavioral aspects have been studied sufficiently; therefore, more detailed investigations on modafinil's positive and aversive behavioral effects are addressed in this paper. Determination of effects of modafinil in marmoset monkeys with observational methods and with behavioral tests measuring locomotor activity, hand-eye coordination, response to a threat situation and startle response. Two hours after oral administration of modafinil in doses of 50, 100, 150, and 225 mg/kg, animals were observed and tested in the behavioral test systems. Locomotor activity was increased after 100 mg/kg modafinil in the Bungalow test and after 100, 150, and 225 mg/kg, as found in the movement parameters of the human threat test. Moreover, modafinil showed anxiolytic-like effects in the human threat test. No other side effects were observed, nor were the hand-eye coordination and startle response affected. Besides psychostimulation, modafinil has no aversive effects in the doses used in the domains measured. The potential anxiolytic-like effects of modafinil may create new possibilities for the therapeutic use of modafinil.

Peptide S is a novel potent inhibitor of voluntary and fast-induced food intake in rats

Peptide S (NPS or PEPS) and its cognate receptor have been recently identified both in the central nervous system and in the periphery. NPS/PEPS promotes arousal and has potent anxiolytic-like effects when it is injected centrally in mice. In the present experiment, we tested by different approaches its central effects on feeding behaviour in Long–Evans rats. PEPS at doses of 1 and 10 μg injected in the lateral brain ventricle strongly inhibited by more than 50% chow intake in overnight fasted rats with effects of longer duration with the highest dose ( P < 0.0001). A similar decrease was observed for the spontaneous intake of a high-energy palatable diet (−48%; P < 0.0001). This anorexigenic effect was comparable to that induced by corticotropin-releasing hormone in fasted rats at equimolar doses. However, peptide S did not modify food intake stimulated by neuropeptide Y (NPY) at equimolar doses. It also did not affect the fasting concentrations of important modulators of food intake like leptin, ghrelin, and insulin in circulation. This study therefore showed that peptide S is a new potent anorexigenic agent when centrally injected. Its inhibitory action appears to be independent of the NPY, ghrelin, and leptin pathways. Development of peptide S agonists could constitute a new approach for the treatment of obesity.

Tandem mass spectrometric data–FAAH inhibitory activity relationships of some carbamic acid O‐aryl esters

We have recently described a class of systemically active inhibitors of the intracellular activity of fatty acid amide hydrolase (FAAH) and traced extensive structure-activity relationships. These compounds, characterized by an N-alkyl carbamic acid O-aryl ester structure, exert potent anxiolytic-like effects in animal models. In the present study, possible relationships between mass spectrometric parameters (related to the propensity of the C(O)--O bond to be cleaved) and FAAH-inhibitory potency were tested. With this aim, a set of our products was analyzed by electrospray ionization mass spectrometry and the protonated molecules were decomposed by low-energy collisions. The experiments were performed by ion trap mass spectrometry, which led to a step-by-step energy deposition, thus favouring the lowest critical energy decomposition channels. For all compounds, breakdown curves relative to [MH](+) ions and to the fragment implying C(O)--O bond cleavage were obtained. The crossing point between these curves was related to the energetics of decomposition and the values found for the investigated compounds were linearly correlated (r(2) = 0.797) with their FAAH-inhibitory activity. This indicates that the energetics of the C(O)--O bond cleavage may be relevant in explaining FAAH inhibition.

Melatonin MT1/2 receptor stimulation reduces cortical overflow of cholecystokinin-like material in a model of anticipation of social defeat in the rat

The involvement of cholecystokinin (CCK) in the potential anxiolytic-like effects of melatonin and of the antitumor MT1/2 receptor agonist, S23478, was assessed by measuring the cortical outflow of CCK-like material (CCKLM) in a rat model of anticipation of social defeat. After repeated social defeats by a male Tryon Maze Dull (TMD) rat, Sprague–Dawley (SD) rats were implanted for microdialysis in the frontal cortex and placed in the same environment as for the defeated sessions, but no confrontation with the TMD rat was allowed. Anticipation of social defeat induced anxiety-like behaviors (immobility, ultrasonic vocalization, defensive postures) associated with a significant increase (~+90%) in cortical CCKLM outflow in SD rats. Acute pretreatment with melatonin (5 or 40 mg/kg i.p.) or S23478, at 5 mg/kg i.p., had no or only minor effects on anxiety-like behaviors and did not affect CCKLM overflow. In contrast, at 40 mg/kg i.p., S23478 significantly reduced the duration of immobility and vocalization as well as the cortical CCKLM overflow (–30%) in defeated SD rats, and both effects were prevented by the MT1/2 receptor antagonist S22153 (40 mg/kg i.p.). These data indicated that MT1/2 receptor stimulation can exert anxiolytic-like effects associated with inhibition of cortical CCKergic neurotransmission in rats anticipating social defeat.

The NK 1 receptor antagonist NKP608 lacks anxiolytic-like activity in Swiss-Webster mice exposed to the elevated plus-maze

The selective non-peptide NK 1 receptor antagonist NKP608 has been shown to exert potent anxiolytic-like effects in the rat and gerbil social interaction tests. In vitro binding of NKP608 in cortical, striatal and rest-of-brain tissue samples from mice, rats and gerbils indicated comparable pIC 50 values for rats and mice (in all three tissues) and only slightly higher values for gerbils. It would therefore be expected that doses previously found to produce anxiolytic-like effects in rats and gerbils would also be active in mice. The present study evaluated NKP608 in one of the most widely-used animal models of anxiety, the mouse elevated plus-maze. Two consecutive experiments were conducted in which the effects of NKP608 (0.0003–10.0 mg/kg, p.o.) were compared to those produced by the prototypical benzodiazepine anxiolytic, chlordiazepoxide (CDP, 15 mg/kg, p.o.). Ethological scoring methods were used to provide comprehensive behavioural profiles for each compound. In both experiments, acute CDP treatment resulted in significant anxioselective effects, i.e., reductions in measures of open arm avoidance without any alteration in general activity levels (closed arm entries and rearing). Although the results of Experiment 1 (0.001–10.0 mg/kg NKP608) suggested a weak anxiolytic-like action of NKP608 at 0.001 mg/kg (significant increase in percent open arm entries), Experiment 2 failed both to replicate this effect or to find any behavioural activity at lower (0.0003 mg/kg) or higher (0.03 mg/kg) doses. Present findings suggest that the anxiolytic efficacy of this NK 1 receptor antagonist may be test-specific and thus limited to particular subtypes of anxiety. These new data are also discussed in relation to the general difficulty of relating the behavioural profiles of NK 1 receptor antagonists to their potency at NK 1 receptors.

Design, synthesis, and structure-activity relationships of alkylcarbamic acid aryl esters, a new class of fatty acid amide hydrolase inhibitors.

Fatty acid amide hydrolase (FAAH), an intracellular serine hydrolase enzyme, participates in the deactivation of fatty acid ethanolamides such as the endogenous cannabinoid anandamide, the intestinal satiety factor oleoylethanolamide, and the peripheral analgesic and anti-inflammatory factor palmitoylethanolamide. In the present study, we report on the design, synthesis, and structure-activity relationships (SAR) of a novel class of potent, selective, and systemically active inhibitors of FAAH activity, which we have recently shown to exert potent anxiolytic-like effects in rats. These compounds are characterized by a carbamic template substituted with alkyl or aryl groups at their O- and N-termini. Most compounds inhibit FAAH, but not several other serine hydrolases, with potencies that depend on the size and shape of the substituents. Initial SAR investigations suggested that the requirements for optimal potency are a lipophilic N-alkyl substituent (such as n-butyl or cyclohexyl) and a bent O-aryl substituent. Furthermore, the carbamic group is essential for activity. A 3D-QSAR analysis on the alkylcarbamic acid aryl esters showed that the size and shape of the O-aryl moiety are correlated with FAAH inhibitory potency. A CoMSIA model was constructed, indicating that whereas the steric occupation of an area corresponding to the meta position of an O-phenyl ring improves potency, a region of low steric tolerance on the enzyme active site exists corresponding to the para position of the same ring. The bent shape of the O-aryl moieties that best fit the enzyme surface closely resembles the folded conformations observed in the complexes of unsaturated fatty acids with different proteins. URB524 (N-cyclohexylcarbamic acid biphenyl-3-yl ester, 9g) is the most potent compound of the series (IC(50) = 63 nM) and was therefore selected for further optimization.

The Gerbil Elevated Plus-Maze I: Behavioral Characterization and Pharmacological Validation

Several neurokinin NK1 receptor antagonists currently being developed for anxiety and depression have reduced affinity for the rat and mouse NK1 receptor compared with human. Consequently, it has proven difficult to test these agents in traditional rat and mouse models of anxiety and depression. This issue has been overcome, in part, by using non-traditional lab species such as the guinea pig and gerbil, which have NK1 receptors closer in homology to human NK1 receptors. However, there are very few reports describing the behavior of gerbils in traditional models of anxiety. The aim of the present study was to determine if the elevated plus-maze, a commonly used anxiety model, could be adapted for the gerbil. Using a specially-designed elevated plus-maze, gerbils exhibited an ‘anxious’ behavioral profile similar to that observed in rats and mice, i.e., reduced entries into, and time spent exploring, an open, aversive arm. The anxiolytic drugs diazepam (0.03–3 mg/kg i.p.), chlordiazepoxide (0.3–10 mg/kg i.p.), and buspirone (0.3–30 mg/kg s.c.) increased open arm exploration and produced anxiolytic-like effects on risk-assessment behaviors (reduced stretch-attend postures and increased head dips). Of particular interest, the antidepressant drugs imipramine (1–30 mg/kg p.o.), fluoxetine (1–30 mg/kg, p.o.) and paroxetine (0.3–10 mg/kg p.o.) each produced some acute anxiolytic-like activity, without affecting locomotor activity. The antipsychotic, haloperidol, and the psychostimulant, amphetamine, did not produce any anxiolytic-like effects (1–10 mg/kg s.c). The anxiogenic β-carboline, FG-7142, reduced time spent in the open arm and head dips, and increased stretch-attend postures (1–30 mg/kg, i.p.). These studies have demonstrated that gerbils exhibit an anxiety–like profile on an elevated plus-maze, and that the gerbil elevated plus-maze may have predictive validity for anxiolytics, and antidepressants with potential anxiolytic-like effects.

Role of CRF1 and CRF2 receptors in fear and anxiety

Fear and anxiety are common emotions that can be triggered by stress. This paper reviews the work examining the role played by specific corticotropin-releasing factor (CRF) receptors in mediating the expression of these emotions. Several lines of evidence taken from CRF1 transgenic knockout mice, CRF1 antisense oligonucleotide studies, and CRF1 receptor antagonist work suggest that the anxiety inducing effects of CRF are mediated by the CRF1 receptor. Of these three methodological approaches, the work using transgenic CRF1 knockout mice appears to be the most consistent. In contrast, the work using specific CRF1 antagonists has produced somewhat varied results that may be explained, in part, by the testing method. When animals are stressed prior to behavioral testing, CRF1 receptor antagonists appear to have anxiolytic-like effects. In addition, chronic dosing with CRF1 antagonists may have more potent anxiolytic-like effects, especially in animal models of spontaneous anxiety, than acute dosing procedures. Spontaneous anxiety is defined as behavior that is elicited entirely by the testing situation without current or prior aversive or explicitly induced stress. CRF1 antisense oligonucleotide work is difficult to interpret because of potential toxicological side effects produced by the antisense oligonucleotide and, in some cases, the absence of verifiable reductions in CRF1 receptor densities after treatment. Similar methods—CRF2 knockouts, CRF2 antisense oligonucleotides, and CRF2 antagonists—were used to evaluate the function of CRF2 receptors in emotionality. In comparison to the large number of CRF1 receptor studies, fewer CRF2 receptor investigations have been conducted and these studies have yielded mixed results. However, recent work demonstrating a robust reduction in CRF2 receptors using a CRF2 antisense oligonucleotide with minimal toxicity, and dose response studies using a peptide CRF2 antagonist suggest that CRF2 receptors play a role in stress-induced and spontaneous anxiety. Furthermore, inhibiting the actions of both CRF1 and CRF2 receptors produces a greater reduction in stress-induced behavior than inhibition of either receptor alone. Thus, current data suggest that CRF1 and CRF2 receptors are involved in the mediation of fear and anxiety behavior.

Pharmacological Characterization of 6-Bromo-3′-nitroflavone, a Synthetic Flavonoid with High Affinity for the Benzodiazepine Receptors

6-Bromo-3′-nitroflavone is a synthetic flavone derivative that selectively recognizes benzodiazepine receptors and has potent anxiolytic-like effects. Here, we describe in detail its pharmacological characterization. When IP injected in mice, 6-bromo-3′-nitroflavone (0.01–0.3 mg/kg) had an anxiolytic-like effect in the elevated plus-maze test. This effect was blocked by the specific benzodiazepine receptor antagonist, flumazenil. In addition, it exhibited anxiolytic-like actions when given orally (1 mg/kg). 6-Bromo-3′-nitroflavone did not exhibit myorelaxant effects (up to 30 mg/kg, IP). Unlike diazepam, this flavonoid produced no anterograde amnesia in a one-trial inhibitory avoidance learning. On the other hand, 6-bromo-3′-nitroflavone possessed mild anticonvulsant activity (0.1 mg/kg, IP) and provoked sedative-depressant actions only at doses 100–1000 times higher than those producing anxiolytic-like effects. 6-Bromo-3′-nitroflavone (0.1–1 mM) produced a lower potentiation of γ-amino-butyric acid (GABA)-stimulated 36Cl − influx (126–138%) in comparison to diazepam (0.1 mM: 166%) in cerebral cortical membrane vesicles. Taken together, these findings suggest that 6-bromo-3′-nitroflavone has anxiolytic-like action possibly behaving as a partial agonist of the benzodiazepine receptors.

The anxiolytic-like effects of the neurosteroid allopregnanolone: interactions with GABA A receptors

The neurosteroid 3α-hydroxy-5α-pregnan-20-one (allopregnanolone) was administered systemically to rats which were tested in the Geller-Seifter conflict paradigm, an established animal model of anxiety. Allopregnanolone was found to produce significant anxiolytic-like effects at a dose of 8 mg/kg. When three ligands that function at different sites on the γ-aminobutyric acid/benzodiazepine receptor-chloride ionophore complex (GABA A receptors) were examined in conjunction with allopregnanolone, the anti-conflict effects of allopregnanolone were effectively reversed only by the benzodiazepine receptor inverse agonist RO15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4 H-imidazo [1,5-α]-[1,4]benzodiazepine-3-carboxylate). Since this inverse agonist has been reported to inhibit the GABA A-activated chloride flux in neuronal membranes, it is likely that the stimulation of the chloride channel in GABA A receptors is an important component of the effects of allopregnanolone. In contrast, the benzodiazepine receptor antagonist flumazenil (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4 H-imidazo [1,5-α]-[1,4]benzodiazepine-3-carboxylate) did not block the anxiolytic-like actions of allopregnanolone, indicating that allopregnanolone does not bind at the benzodiazepine site directly. Isopropylbicyclophosphate, which binds at the picrotoxinin site on the GABA A receptors and blocks the behavioral actions of ethanol, also dose-dependently reversed the anti-conflict effect of this neurosteroid. The results suggest that allopregnanolone may be working either at a site specific for the benzodiazepine receptor inverse agonist RO15-4513 or at the picrotoxinin site to produce its potent anxiolytic-like behavioral effects. © 1997 Elsevier Science B.V.

Interaction of Orally Administered 5-{3-[((25)-1,4-Benzodioxan-2-ylmethyl)amino]propoxy}-1,3-benzodioxole (MKC-242) with 5-HT1A Receptors in Rat Brain

The present study was carried out to clarify whether orally administered 5-{3-[((25)-1,4-benzodioxan-2-ylmethyl)amino]propoxy}-l,3-benzodioxole (MKC-242), a serotonin1A (5-HTiA)-receptor agonist having potent anxiolytic-like and antidepressant-like effects in animal models, binds to 5-HTiA receptors in rat brain. Quantitative autoradiography showed that orally administered MKC-242 (0.1-0.5 mg/kg) caused a significant decrease in [3H]8-hydroxy-2-(di-h-propylamino)tetralin (8-OH-DPAT) binding in the hippocampus and dorsal raphe nucleus sections. The decrease in the binding by MKC-242 was observed up to 4 hr after administration, and the effective doses were similar to those in its anxiolytic-like effect in the animal models. Repeated treatment of MKC-242 (0.5 mg/kg/day, p.o.) or buspirone (30 mg/kg/day, p.o.) for 2 weeks did not affect [3H]8-OH-DPAT binding in both sections. These results suggest that orally administered MKC-242 at the low doses that do not show 5-HTiA-receptor-mediated in vivo responses such as the hypothermic effect, adrenocortical effect and the decrease in 5-HT turnover passes the blood-brain barrier and subsequently binds to 5-HT1A receptors in rat brain. In addition, they indicate that repeated stimulation of the receptors by the agonists does not affect the number of the binding sites.

Preclinical pharmacology of FG5893: a potential anxiolytic drug with high affinity for both 5-HT 1A and 5-HT 2A receptors

The effects of FG5893 were evaluated by several different methods; rats were used as experimental animals. Receptor binding studies revealed that FG5893 (2-(4-(4,4-bis(4-fluorophenyl)butyl)-1-piperazinyl)-3-pyridinecarboxylic acid methyl ester) binds with high affinity to both 5-HT 1A ( K i = 0.7 nM) and 5-HT 2A receptors ( K i = 4.0 nM) but has only low affinity for the 5-HT 2C receptor ( K i = 170 nM). FG5893 dose dependently reduced body temperature, and this effect was inhibited by pretreatment with (±)-pindolol. FG5893 (0.1 mg/kg) significantly inhibited head twitch behaviour induced by DOI (1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane) and FG5893 was also a potent inhibitor of ultrasound vocalization in rat pups (0.3 mg/kg) and of a passive avoidance response (0.1 mg/kg) in mature animals. FG5893 inhibited the cage-leaving response and induced part of the 5-HT behavioural syndrome, but only at very high doses (5 and 10 mg/kg, respectively). At increased doses (1 mg/kg), FG5893 also elicited corticosterone release and reduced the immobility time in the forced-swim test (1 mg/kg). Together, these data indicate that the mixed 5-HT 1A receptor agonist/5-HT 2A receptor antagonist FG5983 is a potent stimulator of presynaptic 5-HT 1A receptors but is less active at the postsynaptic site. FG5893 had potent anxiolytic-like effects both on separation-induced ultrasound vocalization in rat pups and on a passive avoidance response. At increased doses, FG5893 possessed an antidepressant-like property.

Interactions of lesopitron (E-4424) with central 5-HT 1A receptors: in vitro and in vivo studies in the rat

Previous studies have shown that the 5-HT 1A receptor ligand, lesopitron (E-4424, 2-[4-[4-(4-chloro-1-pyrazolyl)butyl]-1-piperazinyl]pyrimidine), exerts potent anxiolytic-like effects in rodents and monkeys (Costall et al., 1992, J. Pharmacol. Exp. Ther. 262, 90). In an attempt to determine whether these effects are really mediated through the interaction of lesopitron with central 5-HT 1A receptors, we investigated the agonistic and/or antagonistic nature of this interaction under in vitro and in vivo conditions in the rat. In vitro binding and autoradiographic studies with [ 3H]8-hydroxy-2-(di- n-propylamino)tetralin ([ 3H]8-OH-DPAT) and [ 3H]lesopitron as radioligands confirmed that lesopitron binds to 5-HT 1A receptors in the rat brain with a relatively high affinity (p K i = 7.35). As expected of a full agonist at postsynaptic 5-HT 1A receptors, lesopitron (IC 50 = 125 nM) inhibited forskolin-stimulated adenylate cyclase activity in rat hippocampal membranes to the same extent as 5-HT, and this effect was preventable by potent 5-HT 1A receptor antagonists such as (−)-tertatolol, (−)-propranolol and N-tert-butyl-3,4-(2-methoxyphenyl)piperazin-1-yl-2-phenyl-propanamide ((+)-WAY 100135). As previously shown for agonists acting at the somato-dendritic 5-HT 1A autoreceptors in the dorsal raphe nucleus, lesopitron inhibited the firing of serotoninergic neurons both in vitro (in brainstem slices, IC 50 = 120 nM) and in vivo (in chloral hydrate-anaesthetized rats, ID 50 = 35 μg/kg i.v.), and this effect was preventable by (−)-tertatolol. Interestingly, the inhibition of the discharge due to lesopitron lasted for only a few minutes both in vitro and in vivo whereas the anxiolytic-like properties of this drug lasted for hours after a single injection in mice (Costall et al., 1992). In addition, the doses required for the stimulation of pre- and postsynaptic 5-HT 1A receptors were markedly higher than those producing significant anxiolytic-like effects in rodents (Costal et al., 1992). It is therefore unlikely that the anxiolytic-like properties of lesopitron involve its stimulatory action at central 5-HT 1A receptors.

The benzodiazepine antagonist flumazenil blocks the effects of CCK receptor agonists and antagonists in the elevated plus-maze.

Peripheral administration of the unsulphated cholecystokinin octapeptide (CCK-8us) led to an anxiogenic-like action in the elevated plus-maze model of anxiety in rats. Devazepide and L-365,260 showed potent anxiolytic-like effects at similar doses. The fact that devazepide is 1000 times more potent as a CCK-A receptor antagonist than L-365,260, whereas the two compounds are nearly equipotent at the CCK-B receptor subtype, suggests that CCK-B rather than CCK-A receptors are involved in these effects. Similar results were obtained in mice using the two-compartment test. In the elevated plus-maze, the benzodiazepine antagonist, flumazenil, which was inactive when given alone, significantly antagonized the anxiogenic-like activity of CCK-8us and the anxiolytic-like effects of devazepide and L-365,260. These results suggest a complex interaction between benzodiazepine and CCK receptor mechanisms in the regulation of anxiety states.