Discovery of Natural Ah Receptor Antagonists from Salvia miltiorrhiza Bunge and Synthesis of Analogs for Tumor Immunotherapy.

IDO/TDO/Kyn/AhR signaling plays a crucial role in regulating innate and adaptive immunity, and targeting Ah receptor (AhR) inhibition can potentially redirect immune cells toward an antitumoral phenotype. Therefore, AhR is an attractive drug target for novel small molecule cancer immunotherapies. In this study, natural products tanshinolic A-D (1-4), the first adducts composed of ortho-naphthoquinone-type tanshinone and phenolic acid featuring a unique 1,4-benzodioxan hemiacetal structure, were isolated and characterized from the roots of Salvia miltiorrhiza Bunge. Luciferase reporter gene assay revealed that these adducts exhibited significant AhR inhibitory activity. A linear strategy was developed to construct a cis-3,4-disubstituted 1,4-benzodioxan hemiacetal structure. Encouragingly, in both in vitro and in vivo experiments, (±)-13e demonstrated the ability to inhibit tumor cell proliferation, promote INF-γ secretion in CD8+ T cells, and inhibit PD-1/PD-L1 signal transduction, which could exert tumor inhibition properties by inhibiting AhR activity, positioning it as a promising candidate for tumor immunotherapy.

alpha 2-adrenoceptor-mediated hyperpolarization of locus coeruleus neurons: intracellular studies in vivo.

Intracellular recordings in vivo from noradrenergic neurons in the rat locus coeruleus showed that membrane potential was hyperpolarized by the administration of clonidine (an alpha 2-adrenoceptor agonist) or after a burst of spikes evoked by intracellular pulses; both types of hyperpolarization were associated with a decrease in membrane input resistance, and both could be blocked by the alpha 2-adrenoceptor antagonist piperoxane. These results suggest that a hyperpolarization of membrane potential mediated by an alpha 2-adrenoceptor underlies both clonidine- and activation-induced inhibition of locus coeruleus cell firing.

Fluparoxan: A Comprehensive Review of its Discovery, Adrenergic and CNS Activity and Treatment of Cognitive Dysfunction in Central Neurodegenerative Diseases.

BACKGROUND: The design, medicinal chemistry, pharmacokinetics and development of the highly selective α2-adrenoceptor antagonist fluparoxan are reviewed. METHOD: The drug's activity and selectivity in vitro, its efficacy in animals and its excellent oral pharmacokinetics and central α2-adrenoceptor antagonist activity in man, are evaluated as well as its ability to increase extracellular levels of noradrenaline, dopamine and acetylcholine in vivo. CONCLUSION: Furthermore, its potential for the treatment of central neurodegenerative diseases is highlighted, in particular its improvement of cognitive dysfunction in schizophrenia and in models of Alzheimer's disease.

Effect of piperoxane on serum prolactin: possible role of epinephrine-mediated synapses in the inhibition of prolactin secretion.

Intravenous administration of 2,5 mg/kg piperoxane produced a rapid and significant increase in serum PRL concentrations in four non-human primates. This PRL increase was maximal 15 min after piperoxane infusion and significant, when compared with baseline levels, in the +15, +30, +45, +60, and +90-min samples. The iv administration of 5 mg/kg piperoxane also produced a rapid and significant increase, whereas saline 0.5 mg/kg or 1.0 mg/kg, did not change serum PRL levels. The iv administration of 10 microgram/kg clonidine, but not saline, produced a rapid and significant reduction in serum PRL levels. PRL levels were significantly reduced +15, +30, and +60 min after the clonidine infusion. Pretreatment with a bolus of 10 microgram/kg clonidine at -15 min caused a significant attenuation of the piperoxane-induced elevation in serum PRL in two monkeys. These data support the hypothesis that alpha-adrenergic receptors are involved in the inhibition of PRL secretion. These data are compatible with noradrenergic or adrenergic mechanisms which remain to be defined.

Suppression of vasopressin secretion by clonidine: effect of alpha-adrenoceptor antagonists.

Studies were performed in anesthetized dogs to determine if the diuretic effect of clonidine results from inhibition of vasopressin secretion. Intravenous clonidine (30 microgram/kg) decreased plasma vasopressin concentration (as measured by RIA) from 10.9 +/- 1.5 to 5.0 +/- 1.1 ng/ml (P less than 0.01) in association with a transient increase in arterial blood pressure and a decrease in heart rate. Intravenous administration of two alpha-adrenoceptor antagonists, piperoxane and phentolamine, virtually abolished the pressor effect of clonidine but did not prevent the suppression of plasma vasopressin concentration. Clonidine decreased plasma vasopressin concentration from 11.9 +/- 3.1 to 3.3 +/- 1.0 pg/ml in the phentolamine-treated dogs (P less than 0.01) and from 18.1 +/- 4.5 to 12.4 +/- 3.6 pg/ml in the piperoxane-treated dogs (P less than 0.05). These results provide direct evidence that the diuretic effect of clonidine results from inhibition of the secretion of vasopressin. This inhibition does not appear to be a consequence of the pressor effect of the drug but may result from a direct action in the central nervous system.

Comparison of the discriminative stimulus properties of clonidine and amphetamine in rats.

Rats were trained to discriminate d-amphetamine (1.0 mg/kg) or clonidine (0.06 mg/kg) from saline in a standard, two-lever procedure with food reinforcement (n = 6). The similarity between the discriminable properties of amphetamine and clonidine was both partial and asymmetrical. Cross tests with amylobarbitone and chlordiazepoxide in rats trained with clonidine suggested that a major component of the clonidine stimulus may be general sedation. Pretreatment with the alpha 2-adrenoreceptor antagonist, piperoxane partially antagonized the discriminative stimulus produced by clonidine. The alpha 1 antagonist, phenoxybenzamine failed to alter the clonidine stimulus. Although amphetamine and clonidine may share some elements in common, this may not represent a noradrenergic component in the amphetamine discriminative stimulus since noradrenergic mediation of the clonidine stimulus was not established.

Interaction between central alpha 1- and alpha 2-adrenoceptors on sympathetic tone in rats.

The interaction between piperoxan and alpha 2-agonists on sympathetic tone was studied in rats. The sympatho-inhibitory effect of alpha 2-agonists (clonidine, guanfacine, B-HT 933) was assessed by recording heart rate in normotensive bilaterally-vagotomized rats. Clonidine (3 micrograms/kg, i.c.v.) and B-HT 933 (100 micrograms/kg, i.c.v.) induced a bradycardia which was fully reversed by piperoxan (30 micrograms/kg, i.c.v.). However, in rats treated with guanfacine, piperoxan induced a partial recovery of the bradycardic effect. The injection of a small dose of the specific alpha 1-adrenoceptor blocking drug, AR-C 239 (10 micrograms/kg, i.c.v.) which, by itself did not modify heart rate, completely inhibited the reversal effect of piperoxan in rats treated with clonidine, B-HT 933 or guanfacine. In rat brainstem membranes, B-HT 933 was found to bind to both alpha 1- and alpha 2-adrenoceptors and was as potent as clonidine in competing for alpha 1-sites bound by [3H]prazosin. On the other hand, in bilaterally vagotomized rats, piperoxan (30 micrograms/kg, i.c.v.) induced an increase in blood pressure and heart rate which was inhibited by previous administration of AR-C 239 (10 micrograms/kg, i.c.v.). These data suggest that, by inhibiting central alpha 2-adrenoceptors, piperoxan unmasks central alpha 1-adrenoceptor stimulation by endogenous catecholamines leading to an increase in the sympathetic tone, but a full recovery in heart rate could be observed only with the mixed alpha 1- and alpha 2-adrenoceptor agonists, clonidine and B-HT 933. In addition, these data further indicate that alpha 1-adrenoceptors are implicated in a tonic control of the sympathetic nerve activity in normotensive rats.

Antagonists of adenosine and alpha-2-adrenoceptors reverse the anticonvulsant effects of tizanidine in DBA/2 mice.

The anticonvulsant activity of 5-chloro-4-(2-imidazolin-2yl-amino)-2,1,3-benzothiazole, tizanidine, was studied following intraperitoneal (i.p.) administration in DBA/2 mice (which show sound-induced seizures). Protection against sound-induced seizures was observed after tizanidine, (0.5-4 mg/kg i.p.). The ED50 values for suppression of the tonic, clonic and wild running phases of sound-induced seizures were 0.54, 0.76 and 1.43 mg/kg, respectively. This protective action was significantly reduced by pretreatment with aminophylline (25 mg/kg i.p.), yohimbine (1 mg/kg i.p.) or piperoxan (20 mg/kg i.p.). Methysergide, a serotonin antagonists, did not significantly reduce the anticonvulsant effects of tizanidine. The present experiments suggest an involvement of purinergic and adrenergic mechanisms in the anticonvulsant action of tizanidine.

Inhibitory effects of clonidine and tizanidine on release of substance P from slices of rat spinal cord and antagonism by alpha-adrenergic receptor antagonists.

Effects of clonidine and tizanidine, which have antinociceptive and alpha 2-agonistic actions, were studied on the release of substance P from slices of spinal cord from the rat. Veratridine-evoked depolarization induced a 2-3-fold increase in the release of substance P from the slices of spinal cord. Exposure of the cord tissue to 10 microM clonidine and tizanidine significantly reduced the release of substance P. The inhibitory effects of clonidine and tizanidine were attenuated by pre-exposure of the tissue to 10 microM piperoxane, which has alpha 2-antagonistic activity and the inhibitory effect of clonidine was attenuated by 10 microM yohimbine. Moreover, the inhibitory effects of clonidine and tizanidine were also blocked by a small dose of prazosin, an antagonist for alpha 1- and alpha 2B-receptors. None of the antagonists had any effect on release of substance P, when given alone. These results suggest that alpha 2B-adrenoceptors are involved in the inhibitory effects of clonidine and tizanidine on the release of substance P.

Pharmacological antagonism of facilitatory but not inhibitory effects of serotonin and norepinephrine on excitability of spinal motoneurons.

Serotonin (5HT) and norepinephrine (NE) produced long-lasting facilitation of glutamate-evoked activity of spinal motoneurons when applied iontophoretically with small ejection currents into the ventral horn. The facilitation was usually preceded by a brief period of inhibition at the onset of current application to the monoamine-containing barrels. This inhibition did not outlast the current application. Ejection of hydrogen ions produced only inhibition of glutamate-evoked activity with no subsequent facilitation at current offset. The 5HT antagonists, methysergide and metergoline, blocked the facilitation, but not the inhibition of motoneuron excitability caused by 5HT. Similarly, the alpha-adrenergic antagonists, piperoxane and phentolamine, blocked the facilitatory, but not the inhibitory, effects of NE on excitability of motoneurons. Since the inhibitory effects of 5HT and NE could not be blocked with the antagonists used, and since ejection of hydrogen ions also produced inhibition, non-specific causes for the inhibitory effects of 5HT and NE could not be rejected. However, the facilitatory effects of 5HT and NE on excitability of motoneurons were readily blocked by antagonists and were, therefore, attributed to actions on separate 5HT and NE receptors in the ventral horn.

Effect of methoxy substitution on the adrenergic activity of three structurally related alpha 2-adrenoreceptor antagonists.

We have recently reported the synthesis and alpha 2-antagonist activity of the methoxy derivative 2 [2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline] and described the enhanced potency of this compound over the parent 1,4-benzodioxan, idazoxan, in reversing the inhibition caused by alpha 2-adrenoreceptor agonists of the electrically induced twitch in the rat or mouse vas deferens. It was of interest to us to discover whether a similar substitution in the structurally related alpha 2-adrenoreceptor antagonists piperoxan, prosympal, and fenmetazole would similarly enhance potency. We subsequently discovered that this was not so and potency was decreased markedly. In particular, that of the methoxy derivative of piperoxan was ca. 220 times less than the parent structure.

In vivo voltammetric monitoring of noradrenaline release and catecholamine metabolism in the hypothalamic paraventricular nucleus.

The paraventricular hypothalamic nucleus receives a dense noradrenergic innervation. Electrochemically treated carbon fibre electrodes were implanted in the paraventricular nucleus of anaesthetized rats and their locations were histologically controlled after each experiment. Differential normal pulse voltammograms showed an oxidation peak at +50 mV. This peak was mainly due to 3,4-dihydroxyphenylacetic acid synthesized by noradrenergic terminals since: it appeared at the same oxidation potential as 3,4-dihydroxyphenylacetic acid in vitro; it was rapidly suppressed after inhibition of tyrosine hydroxylase by alpha-methyl-p-tyrosine or monoamine oxidase by pargyline; blockade of dopamine-beta-hydroxylase by FLA 63 induced a marked increase in this signal, whereas this drug was without effect in dopaminergic terminals fields (striatum, zona incerta); stimulation of alpha 2 noradrenergic receptors by clonidine (50 micrograms/kg) decreased the peak height and this effect was reversed by piperoxane (30 mg/kg). This oxidation peak corresponded to a 3,4-dihydroxyphenylacetic acid concentration of 2 microM. On the other hand, when recorded from rats which were treated with pargyline 3 h before recording, a small peak appeared at +100 mV. This signal was attributed to the oxidation of extracellular noradrenaline on the basis of the following arguments: it appeared at the same potential as noradrenaline in vitro; desipramine (25 mg/kg) induced a 4-fold increase in this peak height; piperoxan (2 mg/kg) enhanced this signal and reversed the decrease induced by clonidine (50 micrograms/kg); electrical stimulations (bipolar electrode, square pulses, 0.3 ms, 200 microA, 15 Hz for 40 s) in the rostral part of the A1 group were followed by an immediate, short-lasting 4-fold increase in the signal.

The antinociceptive action of some beta-adrenoceptor agonists in mice.

The antinociceptive actions of several beta-adrenoceptor agonist drugs have been studied in mice by use of a modified abdominal constriction test. All the drugs studied had high antinociceptive activity, with ID50 values in the nmol kg-1 range. (-)-Isoprenaline and (+/-)-isoxsuprine were the most potent, being about ten times more active than salbutamol, the least potent drug studied. All these drugs produced their action very rapidly and appear to act within the peritoneum. (-)-Isoprenaline had about six times the potency of the (+)-isomer. (+/-)-Propranolol caused rightward shifts, usually parallel, of the dose-response curves for (-)-isoprenaline. (+)-Propranolol was more than ten times less potent than the racemic drug. Practolol also caused parallel, rightward shifts of the dose-response curves for (-)-isoprenaline, and was about twice as potent as (+/-)-propranolol, whether given by subcutaneous or intraperitoneal injection. Atenolol and ICI 118551 had intermediate potencies. Propranolol, practolol and ICI 118551 were all considerably less potent in antagonizing the antinociceptive actions of fenoterol and RO363, than (-)-isoprenaline. None of these antagonist drugs showed more than a slight ability to discriminate between the beta 1- and beta 2-selective agonist drugs. No evidence was found for the involvement of opioid, dopamine, or alpha-adrenoceptors in the antinociceptive action of the beta-adrenoceptor agonist drugs. Evidence for and against the involvement of beta-adrenoceptors is discussed, and it is concluded that if these receptors do mediate the antinociceptive action they appear to be atypical.

The effects of piperoxan on uptake of noradrenaline and overflow of transmitter in the isolated blood perfused spleen of the cat.

1 The competitive alpha-adrenoceptor blocking agent, piperoxan, in concentrations up to 2 x 10(-4) M, produced large dose-dependent increases in transmitter overflow from the isolated blood perfused spleen of the cat following nerve stimulation at 10 hertz. 2 At concentrations greater than 2 x 10(-4) M, piperoxan produced a rise in perfusion pressure, a contraction of the splenic capsule, and a marked dose-dependent decrease in transmitter overflow. 3 Phenoxybenzamine (10(-4) M) and desmethylimipramine (3 x 10(-5) M) produced further increases in transmitter overflow when added after piperoxan. 4 Piperoxan (5.8 to 6.6 x 10(-6) M) had no effect on the recovery of 3H in the venous blood following the close arterial infusion or injection of (3H)-(--)-noradrenaline, indicating that the drug does not inhibit uptake of the amine. 5 Piperoxan produced dose-dependent inhibition of responses of the splenic vasculature to close arterial injection of 1 microgram of (--)-noradrenaline but was much less effective at inhibiting responses to nerve stimulation. At 2 x 10(-6) M piperoxan produced a considerable reduction of the response to injected noradrenaline but potentiated the response to nerve stimulation. 6 In isolated strips of cat splenic capsule, piperoxan produced a shift to the right of the dose-response curve to noradrenaline with no change of the maximum response. There was no evidence of a postsynaptic sensitizing effect of the type observed in the rat vas deferens.

The effects of labetalol (AH 5158) on adrenergic transmission in the cat spleen.

1. The competitive alpha- and beta-adrenoceptor blocking agent labetalol, in concentrations up to 10(-4) M, produced dose-dependent increases in transmitter overflow from the isolated blood perfused spleen of the cat following nerve stimulation at 10 and 30 Hz. 2. At concentrations above 10(-4) M labetol produced a pronounced decrease in transmitter overflow. 3. Labetalol (1.5 X 10(-4) M) increased the recovery of 3H label in the venous blood following the close-arterial infusion of [3H]-(-)-noradrenaline indicating that the drug inhibits uptake of the amine. 4. Both labetalol (3.8 X 10(-5) M) and piperoxan (7.4 X 10(-6) M) produced parallel shifts to the right of the dose-response curves to noradrenaline and oxymetazoline in isolated strips of cat splenic capsule. In this preparation both drugs acted as competitive postsynaptic alpha-adrenoceptor blocking agents. 5. Labetalol (3.3 X 10(-5) M) increased the transmitter overflow following stimulation of the splenic nerves with 200 impulses at 10 Hz. The overflow could be further increased by subsequent addition of piperoxan (7.2 X 10(-6 M). Piperoxan (5.7 X 10(-6) M) alone produced a marked increase in transmitter overflow which could be further increased by subsequent addition of desmethylimipramine (DMI; 3.2 X 10(-5) M). Cocaine (1.5 X 10(-5) M) or DMI (5.4 X 10(-5 M) produced a small increase in transmitter overflow which was not further increased by addition of labetalol (2.8 X 10(-5) M). 6. Labetalol produced a biphasic effect on the responses of the isolated blood perfused spleen of the cat to nerve stimulation. With low doses (up to 10(-4) M) vascular responses were potentiated and with high doses (greater than 10(-4) M) inhibited. The potentiation was related to uptake blockade and the inhibition to decreased transmitter overflow and postsynaptic alpha-adrenoceptor blockade. 7. Labetalol appears to act as a postsynaptic alpha-adrenoceptor antagonist in the isolated blood perfused spleen of the cat with little effect on presynaptic alpha-adrenoceptors. The moderate elevation of transmitter overflow by the drug is related to the inhibitory effect of the drug on neuronal uptake rather than on presynaptic alpha-adrenoceptors.

The pharmacology of fluparoxan: a selective alpha 2-adrenoceptor antagonist.

1. This paper describes the pharmacology of the novel alpha 2-adrenoceptor antagonist fluparoxan (GR 50360) which is currently being studied clinically as a potential anti-depressant. Idazoxan and yohimbine were included in many studies for comparison. 2. In the rat isolated, field-stimulated vas deferens and the guinea-pig isolated, field-stimulated ileum preparations, fluparoxan was a reversible competitive antagonist of the inhibitory responses to the alpha 2-adrenoceptor agonist UK-14304 with pKB values of 7.87 and 7.89 respectively. In the rat isolated anococcygeus muscle, fluparoxan was a much weaker competitive antagonist of the contractile response to the alpha 1-adrenoceptor agonist phenylephrine with a pKB of 4.45 giving an alpha 2: alpha 1-adrenoceptor selectivity ratio of greater than 2500. 3. In the conscious mouse, fluparoxan (0.2-3.0 mg kg-1) was effective by the oral route and of similar potency to idazoxan in preventing clonidine-induced hypothermia and antinociception. In the rat, UK-14304-induced hypothermia (ED50 = 1.4 mg kg-1, p.o. or 0.5 mg kg-1, i.v.) and rotarod impairment (ED50 = 1.1 mg kg-1 p.o. or 1.3 mg kg-1, i.v.) were antagonized by fluparoxan. Fluparoxan, 0.67-6 mg kg-1, p.o., also prevented UK-14304-induced sedation and bradycardia in the dog. 4. In specificity studies fluparoxan had low or no affinity for a wide range of neurotransmitter receptor sites at concentrations up to at least 1 x 10(-5) M. It displayed weak affinity for 5-HT1A (pIC50 = 5.9) and 5-HT1B (pKi = 5.5) binding sites in rat brain. 5. We conclude that fluparoxan is a highly selective and potent alpha 2-adrenoceptor antagonist. The density of rat brain [3H]-dihydroalprenolol binding sites was reduced by 26% when fluparoxan was administered chronically for 6 days at a dose of 12 mg kg- 1 orally twice daily. The down-regulation of beta-adrenoceptors by fluparoxan is consistent with its antidepressant potential.

Effects of clonidine, piperoxane and locus coeruleus lesion on the serotonergic and dopaminergic systems in raphe and caudate nucleus.

To assess the influences of central noradrenergic neurons on both serotonergic and dopaminergic systems, the neurochemical effects of clonidine, piperoxane, and 6-hydroxydopamine were examined. Using quantitative fluorescence histochemistry and high performance liquid chromatography, we have demonstrated that clonidine, much like apomorphine, preferentially augmented intracellular serotonin (5-HT) fluorescence in the dorsal raphe without affecting 5-HT cells in the median raphe nucleus. Clonidine also produced a significant decrease of extraperikaryal catecholamine (CA) fluorescence in the same region. Piperoxane, at a dose having no significant effect alone, antagonized the effects of clonidine on 5-HT and CA. 6-Hydroxydopamine lesions of the locus coeruleus produced a similar increase of 5-HT fluorescence in the dorsal raphe and decrease of CA fluorescence in both the dorsal and median raphe. Biochemically, clonidine decreased while piperoxane increased a measure of 5-HT turnover in the corresponding terminal region of the dorsal raphe, the striatum. Similarly, dopamine turnover was also decreased by clonidine and increased by piperoxane in the striatum. These effects may be mediated by noradrenergic projections from the locus coeruleus to both the dorsal raphe and the substantia nigra. These results support the hypothesis that the effects of clonidine on serotonergic and dopaminergic neurons are indirectly mediated through noradrenergic receptor stimulation.

Central noradrenergic neurons: a locus for the functional interplay between alpha-2 adrenoceptors and opiate receptors.

The locus coeruleus (LC) contains the largest clusters of noradrenergic neurons in the brain. Single-cell recordings in rats show that LC neurons can be inhibited by clonidine acting via alpha-2 adrenoceptors; morphine and opiate peptides are also inhibitory but act via separate opiate receptors. During states of opiate withdrawal LC neurons become hyperactive; under these conditions clonidine can normalize activity via alpha-2 adrenoceptors even when opiate receptors are blocked. These single-cell studies provide a possible mechanism for the clinical efficacy of clonidine in relieving the signs and symptoms of opiate withdrawal in human addicts.

Anxiolytics antagonize yohimbine's discriminative stimulus properties.

Male Sprague-Dawley rats were trained to discriminate 3.2 mg/kg yohimbine HCl from saline in a two-lever operant procedure. Generalization tests indicated that piperoxane, another alpha 2-adrenergic blocker with anxiogenic properties in humans, produces yohimbine-like discriminati-effects.. In contrast to yohimbine and piperoxane, many other agents were discriminated as vehicle, including corynanthine, raubasine, phentolamine, prazosin, WB-4101, mianserin, tolazoline, and mezilamine. Diazepam caused a dose-related antagonism of yohimbine's stimulus properties. A partial antagonism of yohimbine cueing was also obtained with meprobamate, phenobarbital, chlordiazepoxide, and clonazepam. These results suggest that yohimbine discrimination in rats may be a useful model for detecting agents with anxiolytic activity.

Possible cholinergic-dopaminergic link in memory facilitation induced by oxotremorine in mice.

The immediate post-trial injection of the centrally active muscarinic agonist oxotremorine (0.025, 0.050 and 0.100 mg/kg, IP) can facilitate the retention of a passive-avoidance response in mice, as indicated by performance on a retention test 24 h later. Injections given 10 min after training also significantly facilitated retention, but given 120 min after training did not affect retention. These findings suggest an action of oxotremorine on memory mechanisms. The enhanced retention was neither the result of a punishing effect of oxotremorine nor of a nonspecific proactive pharmacological action of the drug. The memory facilitation produced by oxotremorine (0.050 mg/kg, IP) was not antagonized by pretreatment with phentolamine (10 mg/kg, 30 min, IP), phenoxybenzamine (10 mg/kg, 120 min, IP) or piperoxane (20 mg/kg, 30 min, IP). The alpha-noradrenergic blocking agents had no effect by themselves. On the other hand, the immediate post-trial injection of oxotremorine (0.050 mg/kg, IP) did not enhance retention when mice were pretreated with haloperidol (0.5 mg/kg, 120 min, IP). Haloperidol injected either before training or before the retention test did not alter performance during the retention test. This suggests that haloperidol impairs neither acquisition of the avoidance response nor its retrieval. Thus, it is probable that haloperidol pretreatment impaired oxotremorine-induced memory facilitation. We suggest a possible participation of brain catecholamines in memory facilitation induced by oxotremorine in mice.