The effects of morphine and Δ-9-tetrahydrocannabinol on motor activity in rats

Acute treatment of rats either by high doses of morphine or ⁹-tetrahydrocannabinol (THC) decreased locomotor activity. Naloxone reversed morphine-induced depression completely and reversed THC-induced depression only partially. On day 3 of treatment, tolerance developed to the locomotor inhibitory action of THC or morphine and partial cross-tolerance was observed to the depressant action of THC. Naloxone slightly depressed locomotor activity in THC-tolerant rats, but increased motor activity in morphine-tolerant rats.     

Cannabis constituents modulate ∆9-tetrahydrocannabinol-induced hyperphagia in rats

Rationale  

The hyperphagic effect of ∆⁹-tetrahydrocannabinol (∆⁹THC) in humans and rodents is well known. However, no studies have investigated the importance of ∆⁹THC composition and any influence other non-∆⁹THC cannabinoids present in Cannabis sativa may have. We therefore compared the effects of purified ∆⁹THC, synthetic ∆⁹THC (dronabinol), and ∆⁹THC botanical drug substance (∆⁹THC-BDS), a ∆⁹THC-rich standardized extract comparable in composition to recreationally used cannabis.     

Increased sensitivity to Δ9-THC-induced rewarding effects after seven-week exposure to electronic and tobacco cigarettes in mice

Cigarette (CIG) smoking often precedes the use of illegal drugs. Electronic-cigarettes (e-CIGs) have been promoted as a means of stopping smoking and reducing the harmful effects of CIGs on the population. However, although e-CIGs eliminate some of the morbidity associated with combustible tobacco, they are still nicotine-delivery devices. In order to study whether the nicotine delivered via e-CIG acts as “a gateway drug” to the use of cannabis, we analysed the behavioural and molecular effects of 7 weeks’ pre-exposure to air (AIR), e-CIGs or CIGs on addiction-related conditioned place preference (CPP) in mice using a sub-threshold (0.01 mg/kg) dose of delta-9-tetrahydrocannabinol (Δ⁹-THC), the principal psychoactive constituent of cannabis. After 8 and 66 days of withdrawal, this Δ⁹-THC dose was ineffective in inducing CPP in mice pre-exposed to pump-driven AIR, but very effective in mice pre-exposed to e-CIGs or CIGs. Exposure to e-CIGs or CIGs increases the expression of ΔFosB in the nucleus accumbens (NAc), which remains high during short-term e-CIG or CIG withdrawal and long-term CIG withdrawal and is not influenced by treatment with Δ⁹-THC. At the end of e-CIG or CIG exposure and during withdrawal, the mice also had a higher AMPA receptors GluA1/GluA2-3 ratio in the NAc. Chronic nicotine exposure increases sensitivity to rewarding effects of Δ⁹-THC in mice and produces long-lasting neurobiological changes regardless of the delivery method (CIG vs. e-CIG). The exposure to passive tobacco smoke or e-CIG vapours can similarly increase vulnerability to the effects of cannabis and possibly other drugs of abuse.     

Modulation of Δ9-tetrahydrocannabinol-induced hypothermia by fluoxetine in the rat

1. It has been suggested that the dose of Δ⁹-tetrahydrocannabinol (Δ⁹-THC) that induces hypothermia in the rat increases the release of brain 5-hydroxytryptamine (5-HT). In light of this, we investigated the hypothermia produced by Δ⁹-THC, and the effect the selective serotonin reuptake inhibitor fluoxetine has on this response.
2. A significant dose-dependent decrease in body temperature occurred after i.v. administration of 0.5 to 5 mg kg⁻¹ Δ⁹-THC; maximum decreases being 0.8±0.2°C to 2.9±0.3°C. This hypothermic response was attenuated by the cannabinoid CB₁ receptor antagonist SR 141716.
3. Fluoxetine (10 mg kg⁻¹ i.p.) alone caused a decrease in body temperature of 0.6±0.1°C (n=32, P<0.05) after 40 min. However, pretreatment with fluoxetine (10 mg kg⁻¹ i.p.) 40 min before Δ⁹-THC significantly reduced the Δ⁹-THC-induced hypothermia (n=7–8, P<0.05). Contrary to this antagonist-like effect, fluoxetine administered 40 min after Δ⁹-THC significantly potentiated the Δ⁹-THC-induced hypothermia, producing a maximum decrease of 3.2±0.3°C.
4. It is suggested that the effect of fluoxetine on the Δ⁹-THC-induced hypothermic response is dependent on the time of its administration relative to that of Δ⁹-THC. Pretreatment with fluoxetine increases extracellular 5-HT due to reuptake inhibition. Increased extracellular 5-HT can activate autoreceptors which may decrease serotonergic activity, thereby reducing the Δ⁹-THC-induced hypothermia. Conversely, when fluoxetine is adminstered after Δ⁹-THC, the reuptake block is thought to potentiate the already activated serotonegic system, hence potentiating the Δ⁹-THC-induced hypothermia.

     

High-carbohydrate high-fat diet–induced metabolic syndrome and cardiovascular remodeling in rats

The prevalence of metabolic syndrome including central obesity, insulin resistance, impaired glucose tolerance, hypertension, and dyslipidemia is increasing. Development of adequate therapy for metabolic syndrome requires an animal model that mimics the human disease state. Therefore, we have characterized the metabolic, cardiovascular, hepatic, renal, and pancreatic changes in male Wistar rats (8-9 weeks old) fed on a high-carbohydrate, high-fat diet including condensed milk (39.5%), beef tallow (20%), and fructose (17.5%) together with 25% fructose in drinking water; control rats were fed a cornstarch diet. During 16 weeks on this diet, rats showed progressive increases in body weight, energy intake, abdominal fat deposition, and abdominal circumference along with impaired glucose tolerance, dyslipidemia, hyperinsulinemia, and increased plasma leptin and malondialdehyde concentrations. Cardiovascular signs included increased systolic blood pressure and endothelial dysfunction together with inflammation, fibrosis, hypertrophy, increased stiffness, and delayed repolarization in the left ventricle of the heart. The liver showed increased wet weight, fat deposition, inflammation, and fibrosis with increased plasma activity of liver enzymes. The kidneys showed inflammation and fibrosis, whereas the pancreas showed increased islet size. In comparison with other models of diabetes and obesity, this diet-induced model more closely mimics the changes observed in human metabolic syndrome.     

Bilobetin ameliorates insulin resistance by PKA-mediated phosphorylation of PPARα in rats fed a high-fat diet

BACKGROUND AND PURPOSE

The amelioration of insulin resistance by bilobetin is closely related to its hypolipidaemic effect. The aim of the present study was to determine the insulin-sensitizing mechanism of bilobetin by elucidating its effect on lipid metabolism.

EXPERIMENTAL APPROACH

Rats fed a high-fat diet were treated with bilobetin for either 4 or 14 days before applying a hyperinsulinaemic–euglycaemic clamp. Triglyceride and fatty acids labelled with radioactive isotopes were used to track the transportation and the fate of lipids in tissues. The activity of lipid metabolism-related enzymes and β-oxidation rate were measured. Western blot was used to investigate the phosphorylation, translocation and expression of PPARα in several tissues and cultured cells. The location of amino acid residues subjected to phosphorylation in PPARα was also studied.

KEY RESULTS

Bilobetin ameliorated insulin resistance, increased the hepatic uptake and oxidation of lipids, reduced very-low-density lipoprotein triglyceride secretion and blood triglyceride levels, enhanced the expression and activity of enzymes involved in β-oxidation and attenuated the accumulation of triglycerides and their metabolites in tissues. Bilobetin also increased the phosphorylation, nuclear translocation and activity of PPARα accompanied by elevated cAMP level and PKA activity. Threonine-129–alanine and/or serine-163–alanine mutations on the PPARα genes and PKA inhibitors prevented the effects of bilobetin on PPARα. However, cells overexpressing PKA appeared to stimulate the phosphorylation, nuclear translocation and activity of PPARα.

CONCLUSIONS AND IMPLICATIONS

Bilobetin treatment ameliorates hyperlipidaemia, lipotoxicity and insulin resistance in rats by stimulating PPARα-mediated lipid catabolism. PKA activation is crucial for this process.     

Chronic ?-tetrahydrocannabinol during adolescence increases sensitivity to subsequent cannabinoid effects in delayed nonmatch-to-position in rats

Early-onset marijuana use has been associated with short- and long-term deficits in cognitive processing. In human users, self-selection bias prevents determination of the extent to which these effects result only from drug use. This study examined the long-term effects of Δ⁹-tetrahydrocannabinol (Δ⁹-THC), the major psychoactive constituent of marijuana, in a delayed nonmatch-to-position task (DNMP). Male Long-Evans rats were injected daily with 10 mg/kg Δ⁹-THC during or after adolescence [postnatal days (PN) 21-50 or PN50-79, respectively] or with vehicle. On PN91, training in DNMP was initiated. Successful acquisition and pharmacological challenge began on approximately PN300. Decreases in accuracy were observed at lower doses of Δ⁹-THC in Δ⁹-THC-treated rats (versus vehicle-treated rats). Administration of chronic Δ⁹-THC at a younger age tended to enhance this effect. While anandamide did not decrease accuracy in any group, rats treated with Δ⁹-THC during adolescence initiated fewer trials at the 30 mg/kg dose of anandamide than did rats in the other two groups. To the extent tested, these differences were pharmacologically selective for cannabinoids, as scopolamine (positive control) decreased accuracy at the same dose in all groups and amphetamine (negative control) did not affect accuracy in any of the groups at doses that did not impair overall responding. These results suggest that repeated administration of a modest dose of Δ⁹-THC during adolescence (PN21-50) or shortly thereafter (PN50-79) produces a long-term increase in latent sensitivity to cannabinoid-induced impairment of performance in a complex operant task.     

Pharmacological specificity of Δ9-tetrahydrocannabinol discrimination in rats

While many previous studies have shown that a variety of cannabinoids substitute and cross-substitute for ⁹-tetrahydrocannabinol (THC) in drug discrimination procedures, few have systematically examined potential THC-like effects of non-cannabinoid compounds. The purpose of the present study was to delineate further the pharmacological specificity of THC discrimination. Rats were trained to discriminate THC (3.0 mg/kg) from vehicle. Following determination of a dose-effect curve with THC, substitution tests with selected compounds from a variety of pharmacological classes, includingl-phenylisopropyl adenosine, dizocilpine, dextromethorphan, clozapine, buspirone, MDL 72222, muscimol, midazolam and chlordiazepoxide, were performed. Whereas THC produced full dose-dependent substitution, substitution tests with non-cannabinoid drugs resulted in less than chance (50%) levels of responding on the THC-appropriate lever, with the exception of (+)-MDMA (2.5 mg/kg, 50%) and diazepam (3.0 mg/kg, 67%). These results are consistent with those of previous studies and suggest that the discriminative stimulus effects of THC exhibit pharmacological specificity.     

Role of exercise training on insulin resistance and TNF-α in high-fat diet rats

This study investigated the effect of exercise training on insulin resistance and serum and adipose TNF-α in high-fat diet-induced insulin-resistant rats. Thirty male Wistar rats were randomly divided into two groups: normal control group (NC; n = 8) that accepted normal chow and high-fat diet group (HF; n = 22) that fed on high-fat diet to induce insulin resistance model. The HF group was randomly assigned to two subgroups after 18 weeks: sedentary group (SE; n = 10) and exercise training group (ET; n = 12) that performed swimming exercise training for 6 weeks, while both groups continued high-fat diet. Changes of body weight, lipid profile, and fasting plasma glucose and insulin were measured. The insulin sensitivity index (ISI) was calculated. Serum concentration of TNF-α was detected by ELISA. The expression of TNF-α mRNA and protein in adipose tissue was examined by using real-time fluorescence quantitative polymerase chain reaction (PCR) and Western blot, respectively. After 18 weeks, compared with the NC group, body weight, blood lipid, glucose, and insulin in the HF group were significantly elevated, while the ISI decreased obviously, which suggested that insulin resistance appeared in the HF group. After exercise training for 6 weeks, compared with the SE group, both ISI and serum TNF-α concentration in the ET group were decreased significantly; however, the expression levels of TNF-α mRNA and protein in adipose tissue increased by 27.5% and 20.5%, respectively. In conclusion, exercise training ameliorates insulin resistance. The reduction of the level of serum TNF-α and the increased expression of TNF-α in adipose tissue by exercise training may be involved in this mechanism.     

Transfer of the discriminative stimulus effects of Δ9-THC and nicotine from one operant response to another in rats

Objective  

Transfer of the discriminative stimulus effects of two drugs from one operant (original-response) to a topographically different response (transfer-response) that was spared drug discrimination training was investigated.     

Stimulus effects of Δ9-THC and its interaction with naltrexone and catecholamine blockers in rats

Rats trained in a T-shaped maze to discriminate the effects of i.p. injections of ⁹-tetrahydrocannabinol (⁹-THC, 4mg/kg) and the effects of the vehicle were tested for antagonism and generalization to the ⁹-THC stimulus by naltrexone (4 mg/kg), haloperidol (0.32 mg/kg), propranolol (20 mg/kg), and phenoxybenzamine (10 mg/kg). None of these drugs blocked the ⁹-THC stimulus, nor were they found to generalize to ⁹-THC.     

Antagonism of discriminative stimulus effects of Δ9-THC and (R)-methanandamide in rats

Rationale In previous drug discrimination studies we observed surmountable antagonism by Δ⁹-tetrahydrocannabinol (THC) in the presence of constant doses of SR-141716 [N-(piperidin-1-yl)-5-(4-chloro-phenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (0.3 and 1 mg/kg), but there was only marginal evidence for surmountable antagonism with combinations of SR-141716 and (R)-methanandamide, a chiral analog of the endocannabioid anandamide. Objective Here we examine antagonism where the cannabinoid CB1 receptor agonist [Δ⁹-THC and (R)-methanandamide] dose is held constant (i.e., the training dose) and the antagonist {i.e., SR-141716 and AM-251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; 2 ml/kg]} dose varied. We also tested the cannabinoid CB2 receptor antagonist SR-144528 {N-[(1S)-endo-1,3,3-trimethylbicyclo(2.2.1)heptan-2-yl]5-(4-chloro-3-methyl-phenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide}. Methods Different groups of rats were trained to discriminate between vehicle and three different doses of Δ⁹-THC (1.8, 3, and 5.6 mg/kg, presumably reflecting different efficacy demands) as well as 10 mg/kg (R)-methanandamide. Dose-generalization tests involved different doses of the cannabinoid CB1 receptor agonists. Antagonist tests varied the dose of the antagonist (range: 0.1 and 3 mg/kg for SR-141716 and AM-251, and 1 to 10 mg/kg for SR-144528). Results SR-141716 and AM-251 doses dependently blocked the agonist-induced discriminative stimulus effects. SR-141716 tended to be slightly more potent than AM-251. The effective dose 50 (ED₅₀) of SR-141716 was higher in the 5.6 mg/kg Δ⁹-THC-trained group relative to the two other Δ⁹-THC-trained groups. The cannabinoid CB2 receptor antagonist SR-144528 combined with the training dose of 1.8 mg/kg Δ⁹-THC, as well as when combined with the training dose of 10 mg/kg (R)-methanandamide, did not markedly change drug-appropriate (agonist) responses. Conclusion Data support that the discriminative stimulus effects of (R)-methanandamide and its overlap with the Δ⁹-THC cue are, indeed, CB1 receptor mediated events as revealed in antagonism tests with the selective central CB1 receptor antagonists SR-141716 and AM-251. The activation of cannabinoid CB2 receptors appears to be insignificant for these discriminations.     

Naloxone-induced or postwithdrawal abstinence signs in Δ9-Tetrahydrocannabinol-Tolerant rats

Acute stressing procedures cause a shortlasting increase in the levels of guanosie 3,5-monophosphate (cGMP) in mouse brain without significantly influencing the concentrations of adenosine 3,5-monophosphate (cAMP). Animals were pretreated with various centrally acting drugs before being stressed in order to study the involvement of specific neurotransmitters in the stress-induced rise of cGMP levels. Centrally depressant drugs affecting different synaptic mechanisms, such as chlorpromazine, reserpine, haloperidol, diazepam, and pentobarbital, inhibited the cGMP increase elicited by stress. Pretreatment with atropine, diphenhydramine, antazoline, cyproheptadine, phentolamine, bunitrolol, and indomethacin had no significant effect. Clonidine and both the (-)-and (+)-isomers of propranolol inhibited the stress-induced cGMP increase in a doserelated manner. Our results suggest that norepinephrine, serotonin, acetylcholine, or prostaglandins are not involved in the elevation of cGMP levels elicited by acute stress. Participation of other neurotransmitter(s), such as dopamine or GABA, cannot be excluded.     

Effects of Δ9-tetrahydrocannabinol in individuals with a familial vulnerability to alcoholism

Background and aims

A family history (FH) of alcoholism accounts for approximately 50 % of the risk of developing alcohol problems. Several lines of preclinical evidence suggest that brain cannabinoid receptor (CB₁R) function may mediate the effects of alcohol and risk for developing alcoholism including the observations that reduced CB₁R function decreases alcohol-related behaviors and enhanced CB₁R function increases them. In this first human study, we probed CB₁R function in individuals vulnerable to alcoholism with the exogenous cannabinoid Δ⁹-tetrahydrocannabinol (Δ⁹-THC).

Design, setting, and participants

Healthy volunteers (n?=?30) participated in a three test day study during which they received 0.018 and 0.036 mg/kg of Δ⁹-THC, or placebo intravenously in a randomized, counterbalanced order under double-blind conditions.

Measurements

Primary outcome measures were subjective “high,” perceptual alterations, and memory impairment. Secondary outcome measures consisted of stimulatory and depressant subjective effects, attention, spatial memory, executive function, Δ⁹-THC and 11-hydroxy-THC blood levels, and other subjective effects. FH was calculated using the Family Pattern Density method and was used as a continuous variable.

Findings

Greater FH was correlated with greater “high” and perceptual alterations induced by Δ⁹-THC. This enhanced sensitivity with increasing FH was specific to Δ⁹-THC’s rewarding effects and persisted even when FH was calculated using an alternate method.

Conclusions

Enhanced sensitivity to the rewarding effects of Δ⁹-THC in high-FH volunteers suggests that alterations in CB₁R function might contribute to alcohol misuse vulnerability.     

A follow-up study: acute behavioural effects of Δ9-THC in female heterozygous Neuregulin 1 transmembrane domain mutant mice

Rationale  

Heavy cannabis use is linked with an increased risk for schizophrenia. We showed previously that male heterozygous neuregulin 1 transmembrane domain (Nrg1 HET) mice are more sensitive to some effects of the psychotropic cannabis constituent Δ⁹-tetrahydrocannabinol (THC). We report data from a follow-up study in female Nrg1 HET mice, investigating THC effects on behaviours with some relevance to schizophrenia.     

Maternal exposure to Δ9-tetrahydrocannabinol impairs female offspring glucose homeostasis and endocrine pancreatic development in the rat

Recent reports indicate that 7% of pregnant mothers in North America use cannabis. This is concerning given that in utero exposure to Δ9-tetrahydrocannabinol (Δ9-THC), the main psychoactive component in cannabis, causes fetal growth restriction and may alter replication and survival of pancreatic β-cells in the offspring. Accordingly, we hypothesized that maternal exposure to Δ9-THC during pregnancy would impair postnatal glucometabolic health of offspring. To test this hypothesis, pregnant Wistar rats were treated with daily intraperitoneal injections of either 3 mg/kg Δ9-THC or vehicle from gestational day 6 to birth. Offspring were subsequently challenged with glucose and insulin at 5 months of age to assess glucose tolerance and peripheral muscle insulin sensitivity. Female offspring exposed to Δ9-THC in utero were glucose intolerant, associated with blunted insulin response in muscle and increased serum insulin concentration 15 min after glucose challenge. Additionally, pancreata from male and female offspring were harvested at postnatal day 21 and 5 months of age for assessment of endocrine pancreas morphometry by immunostaining. This analysis revealed that gestational exposure to Δ9-THC reduced the density of islets in female, but not male, offspring at postnatal day 21 and 5 months, culminating in reduced β-cell mass at 5 months. These results demonstrate that fetal exposure to Δ9-THC causes female-specific impairments in glucose homeostasis, raising concern regarding the metabolic health of offspring, particularly females, exposed to cannabis in utero.     

Perinatal Δ9-tetrahydrocannabinol exposure in rats modifies the responsiveness of midbrain dopaminergic neurons in adulthood to a variety of challenges with dopaminergic drugs

The present study has been designed to explore further the existence of a persistent, but ‘silent’ alteration in the adult functionality of midbrain dopaminergic neurons following perinatal cannabinoid exposure. To this end, we evaluated the responsiveness of these neurons, measured at the neurochemical or behavioral levels, to pharmacological challenges with a variety of dopaminergic drugs administered to adult male and female rats that had been exposed to Δ⁹-tetrahydrocannabinol (THC) or vehicle during the perinatal period. Results were as follows: In the first experiment, we tested the magnitude of motor inhibition caused by administration of dopaminergic receptor antagonists. The most interesting observation was that the administration of SCH 23390, a D₁ antagonist, produced a more marked motor inhibition, reflected by a greater decrease in the ambulation measured in an open-field test, in adult animals of both sexes when they had been exposed perinatally to THC. This did not occur with the motor inhibition caused by sulpiride, a D₂ antagonist. In the second experiment, we evaluated the sensitivity of midbrain dopaminergic neurons to amphetamine (AMPH), which causes, through different mechanisms, a decrease in dopamine (DA) metabolism. The most interesting observation was that adult females, when exposed perinatally to THC, exhibited a trend to lesser response to AMPH, in terms of decreasing DA metabolism, than oil-exposed females. This was observed in dopaminergic terminals reaching the limbic forebrain area, but not in those terminals reaching the striatum, and was a specific effect for THC-exposed adult females because it was not observed in THC-exposed adult males. In the third experiment, we evaluated the in vivo synthesis of DA in midbrain dopaminergic neurons by analyzing the magnitude of l-3,4-dihydroxyphenylalanine (l-DOPA) accumulation caused by the blockade of l-DOPA decarboxylase with NSD 1015. The most worthy finding was that, as occurred in the above experiment, adult females, when exposed perinatally to THC, tended to exhibit a higher ability to synthesize DA in vivo in the limbic forebrain but not in the striatum, as reflected by the increased l-DOPA accumulation observed after NSD 1015 administration. As in the above experiment, this was not seen in males. In summary, our results are consistent with the possible existence of subtle and sexually dimorphic changes in the sensitivity of midbrain dopaminergic neurons in adulthood caused by the exposure to THC during perinatal development. These silent changes could be revealed after the administration of drugs which specifically act on key processes of dopaminergic neurotransmission, such as the synthesis, reuptake and catabolism of DA and its binding to receptors.     

Discriminative effects of combinations of Δ9-Tetrahydrocannabinol and pentobarbital in pigeons

The purpose of the present study is to examine potentially additive effects of pentobarbital and ⁹-Tetrahydrocannabinol ⁹-THC using a drug discrimination paradigm. Three groups of pigeons were trained to discriminate between the effects induced by i.m. administrations of either (a) 0.25 mg/kg ⁹-THC and vehicle, (b) 4 mg/kg pentobarbital and saline, and (c) ⁹-THC and pentobarbital. Test probes under extinction conditions produced orderly dose generalization gradients among the drug-vs nondrug-trained animals. ED₅₀ for pentobarbital was 1.60 mg/kg and ED₅₀ for ⁹-THC was 0.10 mg/kg. Tests in birds discriminating between pentobarbital and ⁹-THC suggested a sharpening of the drug cue effects; tests with the vehicles resulted in approximately a random key selection (33%–66%) while tests with combinations of the two training drugs suggested that ⁹-THC was the more salient cue in this group. Tests with combinations of various doses of pentobarbital and ⁹-THC in the drug-vs nondrug-trained birds did not increase responding on the respective drug-training associated key. Thus the cue effects of pentobarbital and ⁹-THC were not summational under these experimental conditions. In conclusion, rather low doses of pentobarbital and ⁹-THC are effective as discriminative cues in pigeons and the cues thus induced are different. Combinations of the two drugs are not necessarily summational, and the pentobarbital vs the ⁹-THC discriminations augmented the discriminable effects of the two drugs. In addition, the analeptic drug, bemegride, antagonizes the pentobarbital (4 mg/kg) stimulus in the group trained to discriminate between this barbiturate and saline, which agrees with earlier drug antagonism data obtained among mammals (gerbils and rats), required to discriminate between barbiturates and the nondrug condition.