The direct actions of cannabidiol and 2-arachidonoyl glycerol at GABAA receptors.

Cannabidiol (CBD) is a major non-intoxicating component of cannabis and possesses anti-epileptic, anxiolytic and anti-hyperalgesic properties. The mechanism of action of CBD in producing such effects remains unclear. Despite evidence that some endogenous and synthetic cannabinoids interact with GABAA receptors, no-one has yet investigated the effects of CBD. Here we used two-electrode voltage clamp electrophysiology to compare the actions of CBD with those of the major central endocannabinoid, 2-arachidonoyl glycerol (2-AG) on human recombinant GABAA receptors (synaptic α1-6ßγ2 and extrasynaptic α4ß2δ) expressed on Xenopus oocytes. CBD and 2-AG were positive allosteric modulators at α1-6ßγ2 receptors, with low micromolar potencies. The maximal level of enhancement seen with either CBD or 2-AG were on α2-containing GABAA receptor subtypes, with approximately a 4-fold enhancement of the GABA EC5 evoked current, more than twice the potentiation seen with other α-subunit receptor combinations. Further we observed ß-subunit selectivity, whereby modulatory activity was higher at ß2/ß3 over ß1 subunits. The ß1-subunit homologous mutant ß2(V436T) substantially diminished the efficacy of both drugs to a third of that obtained with wild-type ß2 subunit combinations, but without changing potency. The potency of CBD increased and efficacy preserved in binary α1/α2ß2 receptors indicating that their effects do not involve the classic benzodiazepine site. Exploration of extrasynaptic α4ß2δ receptors revealed that both compounds enhanced GABA EC5 evoked currents at concentrations ranging from 0.01-1µM. Taken together these results reveal a mode of action of CBD on specifically configured GABAA receptors that may be relevant to the anticonvulsant and anxiolytic effects of the compound.

Female neuregulin 1 heterozygous mice require repeated exposure to Δ9-tetrahydrocannabinol to alter sensorimotor gating function.

INTRODUCTION: The schizophrenia susceptibility gene neuregulin 1 (NRG1) confers vulnerability to the neurobehavioural eff ects of cannabinoids differently across sexes. Male but not female Nrg1 heterozygous (HET) mice display facilitation of prepulse inhibition (PPI) to acute Δ9-tetrahydrocannabinol (THC) exposure compared to WT controls. We aim to observe whether repeated administration of THC may overcome the acute insensitivity of female Nrg1 HET mice to THC exposure. METHODS: Female Nrg1 HET mice and WT controls were administered THC daily for 21 days, with PPI and anxiety-related behaviour in the light-dark test (LD) examined on the fi rst and last day of treatment and 21 days after cessation of dosing. RESULTS: Following repeated, but not acute THC exposure, female Nrg1 HET mice displayed THC-induced facilitation of PPI which was not observed in WT mice treated with THC. There were no residual eff ects of THC on PPI in either genotype when assessed 21 days following the final THC dose. An anxiogenic response to THC was evident following repeated, but not acute, administration in the LD test in both genotypes. DISCUSSION: These findings show that the acute insensitivity of female Nrg1 HET mice to THC-induced PPI facilitation may be overcome following repeated THC exposure.

The major plant-derived cannabinoid Δ(9)-tetrahydrocannabinol promotes hypertrophy and macrophage infiltration in adipose tissue.

Synthetic cannabinoid receptor agonists activate lipoprotein lipase and the formation of lipid droplets in cultured adipocytes. Here we extend this work by examining whether Δ(9)-tetrahydrocannabinol (THC), a major plant-derived cannabinoid, increases adipocyte size in vivo. Further, possibly as a consequence of hypertrophy, we hypothesize that THC exposure promotes macrophage infiltration into adipose tissue, an inflammatory state observed in obese individuals. Rats repeatedly exposed to THC in vivo had reduced body weight, fat pad weight, and ingested less food over the drug injection period. However, THC promoted adipocyte hypertrophy that was accompanied by a significant increase in cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) expression, an enzyme important in packaging triglycerides. We also showed that THC induced macrophage infiltration and increased expression of the inflammatory cytokine tumor necrosis factor alpha (TNF-α) in adipose tissue but did not induce apoptosis as measured by TUNEL staining. That THC increased adipocyte cell size in the absence of greater food intake, body weight and fat provides a unique model to explore mechanisms underlying changes in adipocyte size associated with a mild inflammatory state in fat tissue.

Life cycle assessment of the production and use of polypropylene tree shelters.

A detailed Life Cycle Assessment (LCA) has been conducted for the manufacture, use and disposal of polypropylene tree shelters, which are used to protect young seedlings in the first few years of growth. The LCA was conducted using Simapro software, the Ecoinvent database and ReCiPe assessment methodology. Detailed information on materials, manufacturing, packaging and distribution of shelters was obtained from Tubex Ltd. in South Wales, UK. Various scenarios based on different forest establishment methods, with or without tree shelters were derived and analysed using data from published literature and independent sources. The scenarios included commercial forestry in northern temperate conditions, amenity forest establishment in temperate conditions, and forest establishment in semi-arid conditions. For commercial forestry, a reduction in required seedling production and planting as well as additional time-averaged wood production led to significant benefits with tree shelters, both compared to unprotected and fenced cases. For the amenity forest scenarios, tree shelter use had a net environmental impact, while for semi-arid forestry, the benefits of reduction in water use outweighed shelter production impacts. The current practice of in-situ degradation was compared to collection and disposal and it was found that in-situ degradation was slightly preferable in terms of overall environmental impact. Use of biopolymer-based shelters would improve the environmental performance slightly.

Wafer-level self-aligned nano tubular structures and templates for device applications.

A method to fabricate self-aligned nano-scale tubular structures is introduced and investigated. These tubular structures, can be fabricated on wafer-level using common CMOS technologies, are robust and cannot be removed through standard etching or resist strip techniques. This method has shown the potential to be used in different nano device applications since the size of nano-scale tubular structures is adjustable. In addition, these structures can be fabricated as nano-scale templates in advanced device applications.

Composition and Use of Cannabis Extracts for Childhood Epilepsy in the Australian Community.

Recent surveys suggest that many parents are using illicit cannabis extracts in the hope of managing seizures in their children with epilepsy. In the current Australian study we conducted semi-structured interviews with families of children with diverse forms of epilepsy to explore their attitudes towards and experiences with using cannabis extracts. This included current or previous users of cannabis extracts to treat their child's seizures (n = 41 families), and families who had never used (n = 24 families). For those using cannabis, extracts were analysed for cannabinoid content, with specific comparison of samples rated by families as "effective" versus those rated "ineffective". Results showed that children given cannabis extracts tended to have more severe epilepsy historically and had trialled more anticonvulsants than those who had never received cannabis extracts. There was high variability in the cannabinoid content and profile of cannabis extracts rated as "effective", with no clear differences between extracts perceived as "effective" and "ineffective". Contrary to family's expectations, most samples contained low concentrations of cannabidiol, while Δ9-tetrahydrocannabinol was present in nearly every sample. These findings highlight profound variation in the illicit cannabis extracts being currently used in Australia and warrant further investigations into the therapeutic value of cannabinoids in epilepsy.

Author Correction: Composition and Use of Cannabis Extracts for Childhood Epilepsy in the Australian Community.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Heterozygous neuregulin 1 mice display greater baseline and Delta(9)-tetrahydrocannabinol-induced c-Fos expression.

Cannabis use may increase the risk of developing schizophrenia by precipitating the disorder in genetically vulnerable individuals. Neuregulin 1 (NRG1) is a schizophrenia susceptibility gene and mutant mice heterozygous for the transmembrane domain of this gene (Nrg1 HET mice) exhibit a schizophrenia-related phenotype. We have recently shown that Nrg1 HET mice are more sensitive to the behavioral effects of the main psychoactive constituent of cannabis, Delta(9)-tetrahydrocannabinol (THC). In the present study, we examined the effects of THC (10 mg/kg i.p.) on neuronal activity in Nrg1 HET mice and wild type-like (WT) mice using c-Fos immunohistochemistry. In the lateral septum, THC selectively increased c-Fos expression in Nrg1 HET mice with no corresponding effect being observed in WT mice. In addition, THC promoted a greater increase in c-Fos expression in Nrg1 HET mice than WT mice in the central nucleus of the amygdala, the bed nucleus of the stria terminalis and the paraventricular nucleus of the hypothalamus. Consistent with Nrg1 HET mice exhibiting a schizophrenia-related phenotype, these mice expressed greater drug-free levels of c-Fos in two regions thought to be involved in schizophrenia, the shell of the nucleus accumbens and the lateral septum. Interestingly, the effects of genotype on c-Fos expression, drug-free or following THC exposure, were only observed when animals experienced behavioral testing prior to perfusion. This suggests an interaction with stress was necessary for the promotion of these effects. These data provide neurobiological correlates for the enhanced behavioral sensitivity of Nrg1 HET mice to THC and reinforce the existence of cannabinoid-neuregulin 1 interactions in the CNS. This research may enhance our understanding of how genetic factors increase individual vulnerability to schizophrenia and cannabis-induced psychosis.

Cannabis reward: biased towards the fairer sex?

In contrast to drugs such as alcohol, amphetamine and cocaine, cannabis use in humans has proven difficult to model in laboratory animals. Recent breakthrough discoveries of intravenous THC self-administration in rhesus monkeys and self-administration of the synthetic cannabinoid agonist WIN 55,212-2 in rats have allowed new studies of the genetic, neural and environmental determinants of cannabis use. In the present issue of BJP, Fattore and colleagues further demonstrate genetic (strain) differences in WIN 55,212-2 self-administration in rats, with Long Evans (LE) and Lister Hooded (LH), but not Sprague-Dawley, rats self-administering this drug. They then show that female LE and LH rats self-administer more WIN 55,212-2 than male rats. Ovariectomy abolished this sex difference, suggesting a permissive role for oestrogen in cannabis reward. This accompanying Commentary reviews recent progress in animal models of cannabis use and highlights the role of genetic, developmental and endocrine factors in driving cannabis use and dependence.

The multidrug transporter ABCG2 (BCRP) is inhibited by plant-derived cannabinoids.

BACKGROUND AND PURPOSE: Cannabinoids are used therapeutically for the palliation of the adverse side effects associated with cancer chemotherapy. However, cannabinoids also inhibit both the activity and expression of the multidrug transporter, P-glycoprotein in vitro. Here we address the interaction of cannabinol (CBN), cannabidiol (CBD) and delta 9-tetrahydrocannabinol (THC) with the related multidrug transporter, ABCG2. EXPERIMENTAL APPROACH: Cannabinoid inhibition of Abcg2/ABCG2 was assessed using flow cytometric analysis of substrate accumulation and ATPase activity assays. The cytotoxicity and chemosensitization by cannabinoids was determined with cell viability assays. Expression of cannabinoid and vanilloid receptors was assessed using reverse transcriptase polymerase chain reaction, and cannabinoid modulation of ABCG2 expression was examined using immunoblotting. KEY RESULTS: CBN, CBD and THC increased the intracellular accumulation of the Abcg2/ABCG2 substrate, mitoxantrone, in an over-expressing cell line. The THC metabolite, (-)-11-nor-9-carboxy-delta 9-THC was much less potent. The plant cannabinoids inhibited both basal and substrate stimulated ATPase activity of human ABCG2. Cannabinoid cytotoxicity occurred in the absence of known cannabinoid cell surface receptors, and only at concentrations higher than those required for Abcg2/ABCG2 inhibition. Sub-toxic concentrations of the cannabinoids resensitized the overexpressing cell line to the cytotoxic effect of Abcg2/ABCG2 substrates, mitoxantrone and topotecan. This occurred in the absence of any effect on ABCG2 expression. CONCLUSIONS AND IMPLICATIONS: Cannabinoids are novel Abcg2/ABCG2 inhibitors, reversing the Abcg2-mediated multidrug-resistant phenotype in vitro. This finding may have implications for the co-administration of cannabinoids with pharmaceuticals that are ABCG2 substrates.

Heterozygous neuregulin 1 mice are more sensitive to the behavioural effects of Delta9-tetrahydrocannabinol.

RATIONALE: Cannabis use may precipitate schizophrenia especially if the individual has a genetic vulnerability to this mental disorder. Human and animal research indicates that neuregulin 1 (Nrg1) is a susceptibility gene for schizophrenia. OBJECTIVES: The aim of this study was to investigate whether dysfunction in the Nrg1 gene modulates the behavioural effects of Delta(9)-tetrahydrocannabinol (THC), the major psychotropic component of cannabis. MATERIALS AND METHODS: Heterozygous Nrg1 transmembrane-domain knockout mice (Nrg1 HET) were treated with acute THC (0, 5 or 10 mg/kg i.p.) 30 min before being tested using open field (OF), hole board (HB), light-dark (LD), elevated plus maze (EPM), social interaction (SI) and prepulse inhibition (PPI) tests. RESULTS: Nrg1 HET mice showed differences in baseline behaviour with regard to locomotor activity, exploration and anxiety. More importantly, they were more sensitive to the locomotor suppressant actions of THC compared to wild type-like (WT) mice. In addition, Nrg1 HET mice expressed a greater THC-induced enhancement in % PPI than WT mice. The effects of THC on anxiety-related behaviour were task-dependent, with Nrg1 HET mice being more susceptible than WT mice to the anxiogenic effects of THC in LD, but not in the EPM, SI and OF tests. CONCLUSIONS: Nrg1 HET mice were more sensitive to the acute effects of THC in an array of different behaviours including those that model symptoms of schizophrenia. It appears that variation in the schizophrenia-related neuregulin 1 gene alters the sensitivity to the behavioural effects of cannabinoids.

The effects of cannabinoids on P-glycoprotein transport and expression in multidrug resistant cells.

Cannabis is the most widely used illicit drug in the world. Cannabinoids are used therapeutically by some patients as they have analgesic, anti-emetic and appetite stimulant properties which palliate adverse symptoms. Use of these agents in an oncology setting raises the question of whether they act to modulate the effectiveness of concurrently administered anti-cancer drugs. The transporter, P-glycoprotein (P-gp) confers multiple drug resistance (MDR) by effluxing a diverse array of anti-cancer agents. This study was undertaken to examine the effect of cannabinoids on P-gp. Unlike the known P-gp inhibitor, PSC833, short 1h exposure to three plant-derived cannabinoids, cannabinol (CBN), cannabidiol (CBD) and Delta(9)-tetrahydrocannabinol (THC) and the synthetic cannabinoid receptor agonist, WIN55, 212-2 (WIN) did not inhibit the efflux of the P-gp substrate Rhodamine 123 (Rh123) in either a drug-selected human T lymphoblastoid leukaemia cell line (CEM/VLB(100)) or in a mouse fibroblast MDR1 transfected cell line (77.1). However, in CEM/VLB(100) cells, prolonged 72 h exposure to the cannabinoids, THC and CBD, decreased P-gp expression to a similar extent as the flavonoid, curcumin (turmeric). This correlated with an increase in intracellular accumulation of Rh123 and enhanced sensitivity of the cells to the cytotoxic actions of the P-gp substrate, vinblastine. Taken together, these results provide preliminary evidence that cannabinoids do not exacerbate P-gp mediated MDR. Further, plant-derived cannabinoids are moderately effective in reversing MDR in CEM/VLB(100) cells by decreasing P-gp expression.

Neural correlates of interactions between cannabidiol and Δ(9) -tetrahydrocannabinol in mice: implications for medical cannabis.

BACKGROUND AND PURPOSE: It has been proposed that medicinal strains of cannabis and therapeutic preparations would be safer with a more balanced concentration ratio of Δ(9) -tetrahydrocannabinol (THC) to cannabidiol (CBD), as CBD reduces the adverse psychotropic effects of THC. However, our understanding of CBD and THC interactions is limited and the brain circuitry mediating interactions between CBD and THC are unknown. The aim of this study was to investigate whether CBD modulated the functional effects and c-Fos expression induced by THC, using a 1:1 dose ratio that approximates therapeutic strains of cannabis and nabiximols. EXPERIMENTAL APPROACH: Male C57BL/6 mice were treated with vehicle, CBD, THC or a combination of CBD and THC (10 mg·kg(-1) i.p. for both cannabinoids) to examine effects on locomotor activity, anxiety-related behaviour, body temperature and brain c-Fos expression (a marker of neuronal activation). KEY RESULTS: CBD potentiated THC-induced locomotor suppression but reduced the hypothermic and anxiogenic effects of THC. CBD alone had no effect on these measures. THC increased brain activation as measured by c-Fos expression in 11 of the 35 brain regions studied. CBD co-administration suppressed THC-induced c-Fos expression in six of these brain regions. This effect was most pronounced in the medial preoptic nucleus and lateral periaqueductal gray. Treatment with CBD alone diminished c-Fos expression only in the central nucleus of the amygdala compared with vehicle. CONCLUSIONS AND IMPLICATIONS: These data confirm that CBD modulated the pharmacological actions of THC and provide new information regarding brain regions involved in the interaction between CBD and THC.

Utilization of family characteristics in nutritional classification of preschool children.

Methodologies useful in classifying preschool children into nutritional categories are presented and evaluated with respect to classification accuracy. The methodologies are illustrated with data from a study conducted by the National Nutrition Council of the Philippines on the relationships of family characteristics and preschool children's nutritional levels. We have demonstrated that reasonably good classification ability can be achieved with rather simple statistical methodologies.

A comparison of delta 9-THC and anandamide induced c-fos expression in the rat forebrain.

Rats were injected with the cannabinoid receptor agonists delta 9-THC (5 mg/kg) or anandamide (20 mg/kg) and assessed for changes in body temperature and locomotor activity. Their brains were then examined for the expression of the immediate early gene c-fos. Similar reductions in body temperature and locomotor activity were seen with delta 9-THC and anandamide although there was evidence, in line with previous reports, to suggest a shorter duration of action of anandamide. delta 9-THC and anandamide caused equally high levels of c-fos expression in the paraventricular nucleus of the hypothalamus and the lateral septum. Both drugs also increased c-fos expression in the central nucleus of the amygdala although the effect was greater with delta 9-THC. Only delta 9-THC caused significant increases in c-fos expression in the nucleus accumbens and caudate-putamen. These differences may be linked to differential activation of cannabinoid receptor subtypes or to differences in efficacy in activating second messenger systems linked to cannabinoid receptors. These findings complement evidence of qualitative differences in the actions of anandamide and delta 9-THC emerging from tests of drug discrimination, cross-tolerance, conditioned place preference and anxiety.

The distribution of cannabinoid-induced Fos expression in rat brain: differences between the Lewis and Wistar strain.

Previous studies have suggested that cannabis-like drugs produce mainly aversive and anxiogenic effects in Wistar strain rats, but rewarding effects in Lewis strain rats. In the present study we compared Fos expression, body temperature effects and behavioral effects elicited by the cannabinoid CB(1) receptor agonist CP 55,940 in Lewis and Wistar rats. Both a moderate (50 microg/kg) and a high (250 microg/kg) dose level were used. The 250 microg/kg dose caused locomotor suppression, hypothermia and catalepsy in both strains, but with a significantly greater effect in Wistar rats. The 50 microg/kg dose provoked moderate hypothermia and locomotor suppression but in Wistar rats only. CP 55,940 caused significant Fos immunoreactivity in 24 out of 33 brain regions examined. The most dense expression was seen in the paraventricular nucleus of the hypothalamus, the islands of Calleja, the lateral septum (ventral), the central nucleus of the amygdala, the bed nucleus of the stria terminalis (lateral division) and the ventrolateral periaqueductal gray. Despite having a similar distribution of CP 55,940-induced Fos expression, Lewis rats showed less overall Fos expression than Wistars in nearly every brain region counted. This held equally true for anxiety-related brain structures (e.g. central nucleus of the amygdala, periaqueductal gray and the paraventricular nucleus of the hypothalamus) and reward-related sites (nucleus accumbens and pedunculopontine tegmental nucleus). In a further experiment, Wistar rats and Lewis rats did not differ in the amount of Fos immunoreactivity produced by cocaine (15 mg/kg). These results indicate that Lewis rats are less sensitive to the behavioral, physiological and neural effects of cannabinoids. The exact mechanism underlying this subsensitivity requires further investigation.

Effects of pre-exposure and co-administration of the cannabinoid receptor agonist CP 55,940 on behavioral sensitization to cocaine.

Rats given cocaine (15 mg/kg, i.p.) every second day over a 2-week period displayed a progressively greater locomotor response to the drug over days indicating behavioral sensitization. When the cannabinoid receptor agonist CP 55,940 ((-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hyd roxypropyl)cyclohexanol) (10, 25 or 50 microg/kg) was administered under a similar regime, no such sensitization was observed. Rather, the two highest doses of CP 55,940 (25 and 50 microg/kg) caused locomotor suppression that lasted throughout administration. When rats pre-exposed 10 times to CP 55,940 were challenged with cocaine (15 mg/kg), no exaggerated locomotor response to cocaine was evident relative to non pre-exposed rats. When these rats were subsequently re-tested with CP 55,940, the cannabinoid continued to produce a dose-dependent suppression of locomotor activity. Finally, when CP 55,940 (50 microg/kg) was co-administered with cocaine, it significantly reduced the locomotor hyperactivity produced by the drug but did not block the development of behavioral sensitization. These results show that CP 55,940 does not sensitize locomotor activity with repeated administration in the same way as cocaine, and that pre-exposure or concurrent exposure to CP 55,940 does not enhance sensitivity to the subsequent behavioral effects of cocaine.

Effects of the cannabinoid receptor agonist CP 55,940 and the cannabinoid receptor antagonist SR 141716 on intracranial self-stimulation in Lewis rats.

Lewis rats were trained to self-stimulate the medial forebrain bundle (MFB) using a rate-frequency paradigm. They were then tested for the effects of the cannabinoid receptor agonist CP 55,940, the selective cannabinoid receptor antagonist SR 141716 and the dopamine D1 receptor antagonist SCH 23390. CP 55,940 (0, 10, 25 and 50 microg/kg i.p.) had no effect on MFB self-stimulation behaviour as assessed by the M50, the stimulation frequency at which half-maximal response rates were obtained. With SR 141716, only a very high dose (20 mg/kg i.p.) caused a significant inhibition of the rewarding efficacy of the stimulation. This was seen as an increase in the M50. All other doses of SR 141716 (0, 1, 3, 10 mg/kg i.p.) were ineffective in modulating the M50. By comparison, a relatively low dose (0.06 mg/kg i.p.) of SCH 23390 caused a large increase in M50. These results indicate a relatively modest influence, if any at all, of exogenous or endogenous cannabinoids on reward-relevant neurotransmission.

Endoscopic therapy of adenomas of the papilla of Vater. A retrospective analysis with long-term follow-up.

AIMS: To compare the efficacy and the complication rate between endoscopic snare resection of adenomas of Vater's papilla and endoscopic palliation. METHODS: In a retrospective, non randomized manner, we compared long-term results of our endoscopic strategies in 36 patients with histologically confirmed adenoma of Vater's papilla submitted either to local endoscopic snare resection (n=18) or to simple endoscopic palliation (n= 18), respectively. RESULTS: Between 1985 and 1998 results were reviewed. Median age was 76.5 (range 42-89) years in the palliation, and 64.0 (23-89) years in the endoscopic snare resection group. Median duration of follow-up was 33 (6-135) and 75.0 (27-123) months, respectively. The incidence of adenocarcinoma of Vater's papilla was 1 per 52.8 patient-years after endoscopic snare resection and 1 per 15.5 patient-years in the group treated with endoscopic palliation. Compared to the results of endoscopic palliation (prosthesis, sphincterotomy), we found a significant reduction of carcinoma-related death (p=0.0045, McNemar) and adenoma carcinoma-sequence (p=0.007, McNemar) after snare resection. CONCLUSIONS: This retrospective study suggests that complete endoscopic snare resection of adenomas of Vater's papilla will lead to a lower rate of adenoma-carcinoma sequence, to a lower carcinoma-related death rate and probably improves patient survival. These results should be proven prospectively.

Low-dose midazolam attenuates predatory odor avoidance in rats.

Previous studies have shown that predatory odors are a potent anxiogenic stimulus for rodents, yet the ability of benzodiazepines to block odor-induced anxiety remains uncertain. The present study reevaluated this issue using a novel apparatus that, in contrast to previous studies, allowed rats to hide from the odor in a small wooden "hide box" placed within a larger arena. The odor stimulus used was a fabric cat collar that had been worn by a domestic cat for a period of 3 weeks. The experiment was divided into three phases on successive days: 1) habituation, where all rats were placed in the apparatus without cat odor present; 2) conditioning, where rats were presented with the cat odor in the apparatus; and 3) test, where rats previously exposed to the odor were tested for a conditioned avoidance response in the absence of the odor. Results showed that rats exposed to the cat collar displayed a robust avoidance response, spending about 70% of a 20-min session in the hide box compared to 25% in control rats. This avoidance response was completely reversed in rats given a low dose (0.375 mg/kg) of midazolam. During the test phase, rats exposed to the cat odor on the previous day showed elevated levels of hiding when returned to the test apparatus without the cat odor present. This conditioned avoidance was significantly attenuated in rats who had received midazolam (0.375 mg/kg) during cat odor exposure but not in rats given the same dose during the test. These results show that low-dose midazolam is an effective anxiolytic agent in rats during exposure to predatory odor.