Central Benzodiazepine Binding Sites Research Articles

Anxiolytic Potential of Chloroform Extract of Ziziphus mauritiana Lam. Leaves in Mice

Abstract: Introduction: With a tremendous spike in the cases of anxiety in recent years, scientists are looking for herbal drug alternatives for its treatment as all the conventional medication options available in the market have side effects and lead to dependence. Ethnopharmacological reports revealed the usefulness of Ziziphus mauritiana Lam. for anxiety and depression. Therefore, it was envisaged to explore this plant’s possible anxiolytic activity. Objectives: The purpose of the present study was to evaluate the anxiolytic activity of Z. mauritiana leaves based on its ethno pharmacological relevance. Materials and Methods: Anxiolytic activity of the chloroform leaf extract of this plant was determined with the use of animal models elevated plus maze, mirrored chamber and light/dark box test. The chloroform extract was given orally to mice at 200 and 400 mg/kg and number of entries and time spent in the animal models was observed. The extract was then subjected to PTLC and a pentacyclic triterpenoid (RJ11) was isolated which was then characterized using spectroscopic techniques. Molecular docking studies of isolated compound RJ11 was done on GABAA receptors. Results: The chloroform extract showed significant anxiolytic effect in mirrored chamber, elevated plus maze and light/ dark models at the dose of 400mg/kg. Molecular docking studies of isolated compound RJ11 on GABAA receptors revealed good binding affinity and interactions with central benzodiazepine binding site on GABAA that may be responsible for the anxiolytic effect of this plant. Conclusion: The chloroform extract of leaves of Z. mauritiana Lam. possess anxiolytic effect which may be attributed to binding affinity of pentacyclic triterpenoid present in this plant with GABAA receptors on its benzodiazepines receptor site. Keywords: Ziziphus mauritiana Lam., Chloroform extract, Anxiolytic, Triterpenoid, Molecular docking study.

Non-sedating benzodiazepines cause paralysis and tissue damage in the parasitic blood fluke Schistosoma mansoni.

Parasitic flatworm infections (e.g. tapeworms and fluke worms) are treated by a limited number of drugs. In most cases, control is reliant upon praziquantel (PZQ) monotherapy. However, PZQ is ineffective against sexually immature parasites, and there have also been several concerning reports on cestode and trematode infections with poor PZQ cure-rates, emphasizing the need for alternative therapies to treat these infections. We have revisited a series of benzodiazepines given the anti-schistosomal activity of meclonazepam (MCLZ). MCLZ was discovered in the 1970's but was not brought to market due to dose-limiting sedative side effects. However, in the decades since there have been advances in our understanding of the benzodiazepine GABAA receptor sub-types that drive sedation and the development of sub-type selective, non-sedating ligands. Additionally, the sequencing of flatworm genomes reveals that parasitic trematodes and cestodes have lost GABAAR-like ligand gated anion channels, indicating that MCLZ's anti-parasitic target is distinct from the human receptors that drive sedation. Therefore, we have screened a library of classical and non-sedating 1,4-benzodiazepines against Schistosoma mansoni and identified a series of imidazobenzodiazepines that immobilize worms in vitro. One of these hits, Xhe-II-048 also disrupted the parasite tegument, resulting in extensive vacuole formation beneath the apical membrane. The hit compound series identified has a dramatically lower (~1000×) affinity for the human central benzodiazepine binding site and is a promising starting point for the development of novel anti-schistosomal benzodiazepines with minimal host side-effects.

GABAergic modulation with classical benzodiazepines prevent stress-induced neuro-immune dysregulation and behavioral alterations

ObjectivePsychosocial stress is associated with altered immunity, anxiety, and depression. Repeated social defeat (RSD), a model of social stress, triggers egress of inflammatory myeloid progenitor cells (MPCs; CD11b+/Ly6Chi) that traffic to the brain, promoting anxiety-like behavior. In parallel, RSD enhances neuroinflammatory signaling and long-lasting social avoidant behavior. Lorazepam and clonazepam are routinely prescribed anxiolytics that act by enhancing GABAergic activity in the brain. Besides binding to the central benzodiazepine binding site (CBBS) in the central nervous system (CNS), lorazepam binds to the translocator protein (TSPO) with high affinity causing immunomodulation. Clonazepam targets the CBBS and has low affinity for the TSPO. Here the aims were to determine if lorazepam and clonazepam would: (1) prevent stress-induced peripheral and central inflammatory responses, and (2) block anxiety and social avoidance behavior in mice subjected to RSD. MethodsC57/BL6 mice were divided into experimental groups, and treated with either lorazepam (0.10mg/kg), clonazepam (0.25mg/kg) or vehicle (0.9% NaCl). Behavioral data and tissues were collected the morning after the last cycle of RSD. ResultsLorazepam and clonazepam were effective in attenuating mRNA expression of CRH in the hypothalamus and corticosterone in plasma in mice subjected to RSD. Both drugs blocked stress-induced levels of IL-6 in plasma. Lorazepam and clonazepam had different effects on stress-induced enhancement of myelopoiesis and inhibited trafficking of monocytes and granulocytes in circulation. Furthermore, lorazepam, but not clonazepam, inhibited splenomegaly and the production of pro-inflammatory cytokines in the spleen following RSD. Additionally, lorazepam and clonazepam, blocked stress-induced accumulation of macrophages (CD11b+/CD45high) in the CNS. In a similar manner, both lorazepam and clonazepam prevented neuroinflammatory signaling and reversed anxiety-like and depressive-like behavior in mice exposed to RSD. ConclusionThese data support the notion that lorazepam and clonazepam, aside from exerting anxiolytic and antidepressant effects, may have therapeutic potential as neuroimmunomodulators during psychosocial stress. The reversal of RSD-induced behavioral outcomes may be due to the enhancement of GABAergic neurotransmission, or some other off-target effect. The peripheral actions of lorazepam, but not clonazepam, seem to be mediated by TSPO activation.

Cardiovascular responses to intravenous injection of a novel isoindolin-1-one derivate in conscious rats

An isoindolin-1-one derivate, JM-1232(−), was recently developed as a sedative and hypnotic agent with a strong affinity for the central benzodiazepine binding site of gamma-aminobutyric acid A (GABA A) receptors. The purpose of this study was to examine the effects of JM-1232(−) on the cardiovascular and sympathetic functions of conscious rats. We investigated the effect of JM-1232(−) on the mean arterial pressure (MAP), heart rate (HR), baroreflex activity, and plasma catecholamine levels in conscious rats. The intravenous (i.v.) administration of JM-1232(−) (0.1, 0.3, and 1.0 mg/kg/min) for 20 min decreased MAP and increased HR in intact rats. In sinoaortic denervated (SAD) rats, JM-1232(−) decreased MAP and HR. A decrease in MAP induced by JM-1232(−) was prevented by pre-treatment with hexamethonium and enhanced by SAD. An increase in HR induced by JM-1232(−) was prevented by pre-treatment with atropine, propranolol, or hexamethonium. A decrease in MAP and an increase in HR induced by JM-1232(−) were antagonized by co-administration of flumazenil. A high dose of JM-1232(−) decreased the plasma norepinephrine concentration, and a subdepressor dose of JM-1232(−) did not affect the baroreceptor reflex. These results show that the i.v. administration of JM-1232(−) decreased MAP mediated by benzodiazepine–GABA A receptors.

〚 3H〛Flunitrazepam binding to recombinant α 1β 2γ 2S GABA A receptors stably expressed in HEK 293 cells

The interaction of selected compounds with the binding of the benzodiazepine 〚 3H〛flunitrazepam to membranes isolated from human embryonic kidney (HEK) 293 cells, stably transfected with the α 1β 2γ 2S subtype of GABA A receptors, was studied. This subtype of GABA A receptors is the most common type of GABA A receptor found in the brain, and benzodiazepines are drugs known to enhance the effects of the inhibitory neurotransmitter gamma-amino butyric acid (GABA) by binding to the benzodiazepine binding sites which are part of the GABA A receptor complex. Scatchard analysis of binding data revealed the existence of a single type of binding site for 〚 3H〛flunitrazepam. GABA and thiopental enhanced, while the antagonist of central benzodiazepine binding sites – flumazenil, benzodiazepines such as clonazepam, flunitrazepam and diazepam, and the triazolopyridazine CI 218,872 – displaced with nanomolar potency the binding of 〚 3H〛flunitrazepam. A partial displacement was obtained with the antagonist of the peripheral benzodiazepine binding sites – PK 11195 – and with the neurosteroid dehydroepiandrosterone sulfate. The potency of drugs to enhance or inhibit 〚 3H〛flunitrazepam binding mainly corresponded to that observed for the modulation of the binding of 〚 3H〛flunitrazepam to the native type 1 benzodiazepine binding sites. This, as well as a high density of expressed binding sites, makes the cell line under study a very reliable and economical model for the testing of effects of different compounds at the GABA A receptor.

Effects of newly-developed benzodiazepine ligands on noise-induced mitochondrial damage in the rat

In this study we measured the ability of three newly-synthesized N-arylalkylindol-3-ylglyoxylylamide derivatives, which have recently been characterized as partial agonists at central benzodiazepine binding sites, to prevent the rat cardiac mitochondrial alterations resulting from acute loud noise exposure. In particular, we evaluated the effects of these new compounds on the ultrastructural damage induced by noise stress on the right atrium and ventricle after 6 and 12 hr of loud noise exposure. In parallel experiments, we measured the affinity of these compounds for peripheral benzodiazepine binding sites. Following a single injection of the test products, we observed a cardioprotective effect which was more marked after 6 hr compared with 12 hr of noise exposure. Confirming our recent data showing that full agonists at benzodiazepine receptors produce cardioprotection, we demonstrate in this study that partial agonists, like indolylglyoxylylamides, can also produce a cardioprotective effect. Based on their greater affinity in binding studies, the protective activity seems to be related more to their action at central than at peripheral benzodiazepine receptors.

Chromosomes 4 and 13 in beta-carboline-induced seizures in mice: benzodiazepine binding.

Methyl beta-carboline-3-carboxylate (beta-CCM) is a ligand for the benzodiazepine (BZD) binding site of the GABA-A receptors with convulsive properties. We provided evidence for the involvement of a fragment of mouse chromosomes 4 and 13 in beta-CCM-induced seizures in a previous paper. Here, we analyzed, through [3H]-flumazenil binding, whether central BZD binding sites could be involved in the physiological processes underlying these differences of genetic sensitivities. In the JE/Le strain, where the effects of the chromosome 4 fragment can be analyzed, we found associations between [3H]-flumazenil binding and the convulsive action of beta-CCM. On the contrary, this no longer holds true in C3XtEso strain, where the effects of the chromosome 13 fragment were observed.

Pharmacological profile of apigenin, a flavonoid isolated from Matricaria chamomilla

Dried flowers of Matricaria chamomilla L. are largely used to provide sedative as well as spasmolytic effects. In the present study, we examined in particular the pharmacological property of a fraction isolated from a methanolic extract of M. chamomilla, which was identified by HPLC–MS–MS analysis as apigenin. By radioreceptor binding assays, we demonstrated the ability of the flavone to displace a specific radioligand, [ 3H]Ro 15-1788, from the central benzodiazepine binding site. Electrophysiological studies performed on cultured cerebellar granule cells showed that apigenin reduced GABA (gamma-aminobutyric acid)-activated Cl − currents in a dose-dependent fashion. The effect was blocked by co-application of Ro 15-1788, a specific benzodiazepine receptor antagonist. Accordingly, apigenin reduced the latency in the onset of picrotoxin-induced convulsions. Moreover, apigenin injected i.p. in rats reduced locomotor activity, but did not demonstrate anxiolytic, myorelaxant, or anticonvulsant activities. The present results seem to suggest that the inhibitory activity of apigenin on locomotor behaviour in rats cannot be ascribed to an interaction with GABA A–benzodiazepine receptor but to other neurotransmission systems, since it is not blocked by Ro 15-1788.

Brain benzodiazepine binding in aged rats

Membrane [3H]flunitrazepam binding to central and peripheral benzodiazepine binding sites was studied in four brain areas (cerebellum, cortex, striatum and midbrain) of young (age 2–4 months) and aged (>24 months) rats. A generalized reduction in the density of central binding sites (Bmax) was observed in all brain areas examined in aged rats. This reduction is irrelevant of the brain area and, according to literature, may correspond to cell loss and\\or differential expression of mRNAs coding for the subunits of the GABA\\benzodiazepine receptor complex during ageing. In the case of the peripheral binding sites, there was a decrease of Bmax in all brain areas with the exception of the cerebellum. However, the percent reduction of peripheral binding sites varied significantly among the different brain areas. These data suggest a differential effect of ageing on brain benzodiazepine binding which may reflect the special role for each brain area during ageing.

L-364,718 AND L-365,260, two CCK antagonists, have no affinity for central benzodiazepine binding sites in chickens

It has recently been demonstrated that L-365,260, a CCK-B antagonist in mammals, causes an increase in food intake in chickens. In contrast, L-364,718, a CCK-A antagonist in mammals, shows this effect only at very high dose levels. It has been shown that L-365,260 has very low affinity for chicken CCK receptors. Thus, the mechanism of action of L-365,260 remains unknown. As L-365,260 is a benzodiazepine derivative, one may hypothesize that it would be acting on benzodiazepine binding sites. The aims of this work were to establish the existence of benzodiazepine binding sites in the chicken brain, and to check the possibility that L-365, 260 was acting on these receptors, determining the affinity of L-364,718 and L-365, 260 for them. We have found specific binding for tritiated flunitrazepam (a benzodiazepine agonist) ([ 3H]-flunitrazepam) in chicken brain membranes. A single binding site was detected with a K d of 3.58 ± 0.97 nM and a B max of 451.6 ± 23.3 mol/mg protein. L-365,260 and L-364,718 exhibited very low affinity for these binding sites (K i = 1.17 × 10 −6 ± 0.16 × 10 −6 M and K i > 10 −5 M, respectively). Thus, these results demonstrate that the increase in food intake caused by L-365,260 in the chicken is not due to a direct action on benzodiazepine receptors. Other possible explanations for its effect are discussed.

MK-801 alters the GABA A receptor complex and potentiates flurazepam's antiseizure efficacy

MK-801 is an uncompetitive allosteric antagonist that interferes with glutamate-gated calcium ion conductance through the NMDA receptor-associated ionophore. In an outbred strain of mouse, MK-801 elicits episodes of explosive “popping” behaviors that may serve as a preclinical screening paradigm for novel antipsychotic medications. This investigation examined the effects of MK-801, at doses associated with the elicitation of popping, on the GABA A receptor complex in cerebral cortex, and flurazepam's ability to antagonize electrically precipitated seizures. Twenty four hours after MK-801 administration, there was an increased density of the radiolabeled antagonist-preferring conformation of the central benzodiazepine binding site and a potentiation of flurazepam's antiseizure efficacy. The data show that interference with NMDA receptor-mediated calcium ion conductance is associated with a relatively selective change in the GABA A receptor complex in cerebral cortex, and has functional behavioral consequences. Moreover, the data provide additional evidence for a delicate balance between GABAergic and glutamatergic transmission. Disturbance of this balance can have behavioral consequences for the animal.

Temporal lobe central benzodiazepine binding in unilateral mesial temporal lobe epilepsy

PET-demonstrated decreases in [11C]flumazenil binding occur in anterior mesial temporal structures on the side of epileptogenesis in unilateral mesial temporal lobe epilepsy. We performed quantitative autoradiography on anterior mesial and lateral temporal specimens from 11 subjects with unilateral mesial temporal lobe epilepsy and six neurologically normal controls to identify the predominant in vitro correlates of the decreased [11C]flumazenil binding. In anterior mesial temporal regions exhibiting the greatest neuronal cell loss, decreases in agonist and antagonist binding to type 1 and 2 (central) benzodiazepine binding sites were highly correlated with neuronal cell counts. Cell loss and decreased binding were particularly prominent in the lateral portion of hippocampal region CA1, adjacent to CA2. Lateral temporal central benzodiazepine binding was diffusely increased, achieving statistical significance in cortical laminae V and VI. These findings suggest that the predominant source of PET-demonstrated decreases in [11C]flumazenil binding in mesial temporal epilepsy is hippocampal sclerosis, rather than down-regulation of central benzodiazepine binding sites on surviving hippocampal neurons.

Positron emission tomography measures of benzodiazepine binding in Alzheimer's disease.

To evaluate the integrity of neurons and neuropil in metabolically affected association cortices of patients with Alzheimer's disease by measuring central benzodiazepine binding sites with the use of positron emission tomography. In patients with Alzheimer's disease, we determined the cerebral distribution of flumazenil tagged with carbon 11 ([11C]flumazenil), a ligand that binds to the gamma-aminobutyric acid A (GABAA) receptor complex, and the distribution of fludeoxyglucose tagged with fluorine 18 fludeoxyglucose F 18), a measure of local cerebral glucose metabolism. Tracer kinetic analysis was applied to quantify data in regions of interest. These data were compared with those of normal control subjects. Patients with probable Alzheimer's disease ([11C]flumazenil, n = 13; fludeoxyglucose F 18, n = 11) and normal elderly control subjects ([11C]flumazenil, n = 6; fludeoxyglucose F 18, n = 10). Significant decreases of the [11C]flumazenil transport rate were found in hypometabolic parietal and temporal association cortices, but [11C]flumazenil binding was not significantly decreased. When measured in living patients, association cortical benzodiazepine binding sites are relatively preserved, suggesting structurally intact cortical neuropil underlying the glucose hypometabolism identified in Alzheimer's disease.

Environmental stress-induced functional modification of the central benzodiazepine binding site.

The central benzodiazepine binding site mediates the therapeutically relevant pharmacologic actions of benzodiazepine agonists, including reducing anxiety, sedation, muscle relaxation, and antagonism of seizure production. Benzodiazepines potentiate the ability of gamma-aminobutyric acid (GABA) to promote membrane chloride ion conductance by binding reversibly to a distinct site on the GABAA receptor complex. Although the role of this central benzodiazepine binding site in mediating the pharmacologic actions of benzodiazepine agonists has been shown conclusively, its participation in an animal's response to stress is less certain. Data are reviewed consistent with an environmental stress-induced modification of the structure and function of the central benzodiazepine binding site. Modifications show brain anatomic regional selectivity. The mechanisms of these modifications include rapid enzyme-mediated phosphorylations of the GABAA receptor complex and the selective transcription of individual polypeptide subunits. The potential relevance of the data on environmental stress-induced functional modifications of the central benzodiazepine binding site to the development of newer medications and improved understanding of the pathophysiology of stress-related neuropsychiatric disorders is discussed.

Prevention by eliprodil (SL 82.0715) of traumatic brain damage in the rat. Existence of a large (18h) therapeutic window

The neuroprotective potential of eliprodil (SL 82.0715), an N-methyl- d-aspartate (NMDA) receptor antagonist acting at the polyamine modulatory site, in brain trauma was examined in a rat model of lateral fluid-percussion brain injury. The volume of the lesion was assessed histologically by measuring, at 7-days post-injury, the area of brain damage at 10 coronal planes. Eliprodil (10 mg/kg i.p.) when given 15 min, 6 h and 24 h after fluid percussion (1.6 atm) and then b.i.d. for the following 6 days, reduced by 60% the volume of cortical damage. A similar neuroprotection was obtained when the first administration of eliprodil was delayed by up to 12 h after the brain insult. Moreover, when the treatment with this compound was started at 18 h post-injury, cortical damage was still significantly reduced by 33%. Autoradiographic studies showed that eliprodil treatment (10 mg/kg, i.p.), initiated 15 min after the trauma, also caused a marked reduction of the loss of the neuronal markerω 1−2 (central benzodiazepine) binding sites of the increase in the glial/macrophage marker peripheral type benzodiazepine binding sites in the cerebral cortex. In contrast, dizocilpine (a blocker of the cationic channel coupled to the NMDA receptor) when administered 6 h and 24 h after fluid percussion and then b.i.d. for the following 6 days induced a non significant reduction of the volume of the lesion at the highest tolerated dose (0.6 mg/kg i.p.). These results demonstrate the neuroprotective activity of eliprodil in experimental brain trauma using neuropathology as an endpoint and indicate that three is a very large time window for therapeutic intervention, consistent with the delayed nature of the neuronal loss, in this condition.

Evidence for more than two central benzodiazepine receptors in rat spinal cord

Competitive binding assays were performed in rat spinal cord membranes at 0° C against [ 3H]Ro15-1788. The displacement curves of Ro15-1788 and alpidem produced pseudo-Hill slopes of 1.0 and 0.5, respectively. In addition, 5–10% of the specifically bound [ 3H]Ro15-1788 could not be displaced by > 100 μM of alpidem. These two pieces of evidence strongly suggest the presence of at least three central benzodiazepine binding sites in the spinal cord.

Occupancy of the central benzodiazepine binding site during stress alters the stress-induced reduction of flurazepam's antiseizure efficacy : John Mastropaolo, Deborah Olin Norris, David A. O'Connor, Stephen I. Deutsch Department of Veterans Affairs Medical Center, Washington, DC 20422

Occupancy of the central benzodiazepine binding site during stress alters the stress-induced reduction of flurazepam's antiseizure efficacy : John Mastropaolo, Deborah Olin Norris, David A. O'Connor, Stephen I. Deutsch Department of Veterans Affairs Medical Center, Washington, DC 20422

Central Benzodiazepine<i>-</i>Binding Sites in Human Cerebral Structures Associated with Memory Processes

This work describes the distribution of the central benzodiazepine-binding sites (BZD-BS) in several structures recently described as involved in memory processes. The study was performed on 4 postmortem human brains by in vitro quantitative autoradiography using <sup>3</sup>H-flunitrazepam as a ligand. Very high densities of BZD-BS (1,500–2,000 fmol/mg protein) were observed in the anterior and posterior cingulate cortex, in the indusium griseum and in the accessory basal nucleus of the amygdala, and moderate concentrations (500–700 fmol/mg) in the magnocellular region of the mediodorsal thalamic nucleus. In the classical cerebral memory circuits, high densities of BZD-BS (1,000 to 1,500) were also found, except in the septum where the density of BZD-BS (400 to 600) was not significantly different from that in control structures not involved in memory. No evidence for lateralization of the sites was observed.

Benzodiazepines inhibit neutrophil chemotaxis and superoxide production in a stimulus dependent manner; PK-11195 antagonizes these effects

Diazepam, which binds both central (neuronal) and peripheral (non-neuronal) benzodiazepine binding sites, and Ro5-4864, a ligand selective for benzodiazepine peripheral binding sites (PBS), both inhibited the FMLP induced chemotaxis in human neutrophils at concentrations as low as 10−8 M. A selective peripheral benzodiazepine antagonists, PK-11195 (10−5 M), partially reversed the benzodiazepine inhibition of chemotaxis. Diazepam also inhibited the superoxide production induced by FMLP, NaF, and A23187, but not that induced by PMA whose stimulant action was insensitive even to 10−4 M diazepam. The FMLP-induced superoxide production was most sensitive to diazepam inhibition (ID50 = 2.25 × 10−6 M diazepam); the effect of NaF was slightly less sensitive (ID50 = 1.34 × 10−5 M diazepam); and the effect of A23187 was least sensitive as it was suppressed only at 10−4 M diazepam concentrations. Like diazepam, Ro5-4864 inhibited the FMLP-induced superoxide production, and PK-11195 (10−5 M) significantly antagonized both diazepam and Ro5-4864 inhibition.Binding studies showed the presence of a saturable benzodiazepine ‘peripheral’ type binding site (PBS) on human neutrophils with a Kdof 1.2 ± 0.06 × 10−8M (± SEM), and a Bmaxof 1028 ± 86.2 fmol/106Cells (± SEM) for [3H]Ro5-4864; the binding was displaceable by PK-11195, Ro5-4864 and diazepam but not by clonazepam.

The Effect of Medetomidine on GABA and Benzodiazepine Receptors in Vivo: Lack of Anxiolytic but some Evidence of Possible Stress‐Protective Activity

Medetomidine, a new selective alpha 2-adrenoceptor agonist, potentiated bicuculline seizures in mice. In vivo pretreatment with medetomidine in mouse cerebral cortex reduced dose-dependently (2.5-100 micrograms/kg) GABA-potentiated 3H-flunitrazepam binding. The affinity of 3H-muscimol was also reduced by medetomidine. This effect of medetomidine on GABA-potentiated benzodiazepine binding was reversed by pretreatment with atipamezole (1 mg/kg), a specific alpha 2-antagonist. In an elevated plus-maze model of anxiety medetomidine (0.5-10 micrograms/kg) was inactive both in rats and mice and did not antagonize the behavioural effects of an anxiogenic beta-carboline, DMCM. However, at lower doses medetomidine (10 but not 50 micrograms/kg) antagonized the swimming stress caused increase of central benzodiazepine binding sites (labeled with 3H-Ro 15-1788) in mouse cerebral cortex. The increase of peripheral benzodiazepine binding sites on brain and heart cryostat cut slices caused by stress was also antagonized by pretreatment with medetomidine. The behavioural and biochemical data obtained in this study are evidence that medetomidine does not have anxiolytic effect but may have, in lower doses, stress-protective activity.