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PubMed2005

Spinal 5-HT1A receptors differentially influence nociceptive processing according to the nature of the noxious stimulus in rats: effect of WAY-100635 on the antinociceptive activities of paracetamol, venlafaxine and 5-HT.

The regulation of nociceptive processing by 5-HT at the spinal level is intricate since the neurotransmitter has been implicated in both pro and antinociception. The aim of our study was to investigate, according to the nature of the noxious stimulus, how the blockade of spinal 5-HT(1A) receptors could influence the antinociceptive actions of exogenous 5-HT as well as two analgesics involving endogenous 5-HT, paracetamol and venlafaxine. Rats were submitted either to the formalin test (tonic pain) or the paw pressure test (acute pain). WAY-100635 (40 microg/rat, i.t.), a selective 5-HT(1A) receptor antagonist, had no intrinsic action in either test. However, in the formalin test, it blocked the antinociceptive action of 5-HT (50 microg/rat, i.t.) and paracetamol (300 mg/kg, i.v.) in both phases of biting/licking behaviour and that of venlafaxine (2.5 mg/kg, s.c.) in the late phase only. In the paw pressure test, the combination of sub-effective doses of 5-HT (0.01 microg/rat, i.t.), paracetamol (50 mg/kg, i.v.) or venlafaxine (20 mg/kg, s.c.) with WAY-100635 led to a significant antinociceptive effect, which seems to depend on the reinforcement of the activity of inhibitory GABAergic interneurones. In conclusion, both direct stimulation of the spinal 5-HT(1A) receptors by 5-HT, and indirect stimulation using paracetamol or venlafaxine can differently influence pain transmission. We propose that the nature of the applied nociceptive stimulus would be responsible for the dual effect of the 5-HT(1A) receptors rather than the hyperalgesic state or the supraspinal integration of the pain message.

Acetaminophen↗
PubMed2005

Antihyperalgesic properties of the cannabinoid CT-3 in chronic neuropathic and inflammatory pain states in the rat.

CT-3 (ajulemic acid) is a synthetic analogue of a metabolite of Delta9-tetrahydrocannabinol that has reported analgesic efficacy in neuropathic pain states in man. Here we show that CT-3 binds to human cannabinoid receptors in vitro, with high affinity at hCB1 (Ki 6 nM) and hCB2 (Ki 56 nM) receptors. In a functional GTP-gamma-S assay CT-3 was an agonist at both hCB1 and hCB2 receptors (EC50 11 and 13.4 nM, respectively). In behavioural models of chronic neuropathic and inflammatory pain in the rat, oral administration of CT-3 (0.1-1 mg/kg) produced up to 60% reversal of mechanical hyperalgesia. In both models the antihyperalgesic activity was prevented by the CB1-antagonist SR141716A but not the CB2-antagonist SR144528. In the tetrad of tests for CNS activity, CT-3 (1-10 mg/kg, po) produced dose-related catalepsy, deficits in locomotor performance, hypothermia, and acute analgesia. Comparison of 50% maximal effects in the tetrad and chronic pain assays produced an approximate therapeutic index of 5-10. Pharmacokinetic analysis showed that CT-3 exhibits significant but limited brain penetration, with a brain/plasma ratio of 0.4 measured following oral administration, compared to ratios of 1.0-1.9 measured following subcutaneous administration of WIN55,212-2 or Delta9-THC. These data show that CT-3 is a cannabinoid receptor agonist and is efficacious in animal models of chronic pain by activation of the CB1 receptor. Whilst it shows significant cannabinoid-like CNS activity, it exhibits a superior therapeutic index compared to other cannabinoid compounds, which may reflect a relatively reduced CNS penetration.

Analgesics↗
PubMed2004

Unconditioned and conditioned anxiogenic effects of the cannabinoid receptor agonist CP 55,940 in the social interaction test.

In spite of the addictive properties of cannabinoids, under certain circumstances, they can evoke strong anxiogenic and aversive responses in humans and in animal tests of anxiety. Effects of different doses of CP 55,940 (10, 20, and 40 microg/kg) were tested in the low-light, familiar (LF) apparatus test condition of the social interaction test. The 40-microg/kg dose of CP 55,940 significantly decreased the time spent in social interaction, indicating an anxiogenic effect. This dose also had an independent effect of reducing locomotor activity. In rats tested undrugged 24 h after testing with 40 microg/kg, there was a significant anxiogenic effect, indicating conditioned anxiety. The group of rats injected with 40 microg/kg immediately after the social interaction test showed an unexpected significant anxiolytic effect when tested undrugged 24 h later. In an additional experiment, rats were tested in the high-light, familiar (HF) apparatus test condition after 10 or 40 microg/kg, and only those that were tested after 40 microg/kg showed an anxiogenic effect on the test day and a conditioned anxiogenic effect when tested undrugged 24 h later. Once again, those injected with 40 microg/kg after the social interaction test displayed an anxiolytic effect when tested undrugged 24 h later. We provide the first evidence for unconditioned and conditioned anxiogenic-like responses to a cannabinoid agonist in the social interaction test.

Animals↗
PubMed2004

The effect of venlafaxine on behaviour, body weight and striatal monoamine levels on sleep-deprived female rats.

Partial sleep deprivation is clinically associated with fatigue, depressive symptoms and reduced memory. Previously, it has been demonstrated that venlafaxine, an atypical antidepressant, increases the levels of noradrenaline and serotonin in rat hippocampus. The aim of this study was to evaluate the effects of venlafaxine on depression, anxiety, locomotor activity and memory in a model of REM sleep (REMs) deprivation in rats. We have also studied the influence of venlafaxine on monoamine levels in the striatum. Six groups of animals (N=20 each) were treated with saline or venlafaxine (1 or 10 mg/kg) during 10 days, submitted or not to REMs deprivation and studied with the forced swimming test of Porsolt (STP), plus-maze, passive avoidance and open-field tests right after sleep deprivation. Animals were also studied for passive avoidance 24 h later (rebound period). Brain samples for monoamine measurements were collected either immediately after REMs deprivation or after 24 h. Both REMs deprivation and venlafaxine showed an antidepressant effect. An anxiolytic effect was also observed after REMs deprivation. Previous treatment with venlafaxine blocked the antidepressant and anxiolytic effects of REMs deprivation. REMs deprivation alone and treatment with venlafaxine 10 mg/kg increased locomotor activity, and this effect was inhibited by venlafaxine in REMs deprived rats. Both venlafaxine treatment and REMs deprivation induced weight loss. Venlafaxine treatment, but not REMs deprivation, induced an increase in striatal dopamine (DA) levels. The combination of REMs deprivation and venlafaxine treatment was associated with an increase in serotonin turnover 24 h after rebound sleep. In this study, venlafaxine treatment hindered most behavioral effects of REMs deprivation and was associated with an interference on dopamine and serotonin systems in the striatum.

Animals↗
PubMed2006

Multi-target strategies for the improved treatment of depressive states: Conceptual foundations and neuronal substrates, drug discovery and therapeutic application.

Major depression is a debilitating and recurrent disorder with a substantial lifetime risk and a high social cost. Depressed patients generally display co-morbid symptoms, and depression frequently accompanies other serious disorders. Currently available drugs display limited efficacy and a pronounced delay to onset of action, and all provoke distressing side effects. Cloning of the human genome has fuelled expectations that symptomatic treatment may soon become more rapid and effective, and that depressive states may ultimately be "prevented" or "cured". In pursuing these objectives, in particular for genome-derived, non-monoaminergic targets, "specificity" of drug actions is often emphasized. That is, priority is afforded to agents that interact exclusively with a single site hypothesized as critically involved in the pathogenesis and/or control of depression. Certain highly selective drugs may prove effective, and they remain indispensable in the experimental (and clinical) evaluation of the significance of novel mechanisms. However, by analogy to other multifactorial disorders, "multi-target" agents may be better adapted to the improved treatment of depressive states. Support for this contention is garnered from a broad palette of observations, ranging from mechanisms of action of adjunctive drug combinations and electroconvulsive therapy to "network theory" analysis of the etiology and management of depressive states. The review also outlines opportunities to be exploited, and challenges to be addressed, in the discovery and characterization of drugs recognizing multiple targets. Finally, a diversity of multi-target strategies is proposed for the more efficacious and rapid control of core and co-morbid symptoms of depression, together with improved tolerance relative to currently available agents.

Adrenergic Uptake Inhibitors↗
PubMed2005

Trazodone addition for insomnia in venlafaxine-treated, depressed inpatients: a semi-naturalistic study.

In this paper, we present the results of a prospective semi-naturalistic study of the addition of trazodone for insomnia to a 4 week, 300mg/day venlafaxine treatment in 50 depressed inpatients. The Montgomery and Asberg depression rating scale was used as a rating instrument. The study is designated as semi-naturalistic due to the fact that, although the venlafaxine treatment regimen was strictly defined, the timing of the trazodone introduction and the dosage were determined by the clinicians. The indication was based on the persistency of insomnia despite the use of authorized sedative co-medication (zopiclone as a hypnotic, clorazepate as an anxiolytic). Among the 42 patients who completed the study, 27 did not receive trazodone (G1) while 15 did (G2). Although the two groups were not clinically different at study entry, G2 patients showed less improvement than G1 patients during venlafaxine treatment alone, both in terms of insomnia (MADRS item 4) and inner tension (MADRS item 3). After trazodone introduction, insomnia improved and the median (interquartile range) of this item in G1 and G2 patients showed no statistically significant difference on Day 28 (G1: 0 (0-1); G2: 0 (0-2)). However, inner tension did not improve and the median (interquartile range) was higher on Day 28 in G2 patients (G1: 1 (0-2); G2: 2 (1-4); P < 0.05). Thus, trazodone is probably used for patients who develop not only insomnia, but also inner tension/anxiety during venlafaxine treatment. However, it alleviates only the first symptom, not the second.

Adult↗
PubMed2004

Cannabinoid agonist, CP 55,940, facilitates intake of palatable foods when injected into the hindbrain.

Cannabinoids have been shown to influence food intake, and until recently, the neural pathways mediating these effects have remained obscure. It has been previously shown that intracerebroventricular injection of delta-9-tetrahydrocannabinol (Delta(9)-THC) causes increased consumption of palatable foods in rats, and we postulated the involvement of the hindbrain in this cannabinoid-induced food intake. Cannulated rats (both female and male groups) trained to consume sweetened condensed milk received either lateral or fourth ventricle injections of CP 55,940 and were presented with sweetened condensed milk 15 min after injection. Rats were injected over a range of doses between 100 pg and 10 microg per rat. Milk intake was recorded for a total of 3 h. Lateral ventricle injection of CP 55,940 increased milk intake at doses in the microgram range. However, CP 55,940 was effective in increasing food intake at nanogram doses when injected into the fourth ventricle. Finally, male rats appeared to be more sensitive to CP 55,940 than female rats inasmuch as milk consumption was increased at the 1 ng dose in male rats, whereas only the 10 ng dose was effective in females. These results indicate that CP 55,940 may act in the hindbrain to influence feeding behavior in rats.

Animals↗
PubMed2005

Differential distribution of leaf chemistry in eucalypt seedlings due to variation in whole-plant nutrient availability.

We investigated the effects of whole-plant nutrient-availability on the degree of distribution of some plant primary and secondary (nitrogen, fibre, flavonols, gallotannins and cineole) chemicals across young, mature and old leaves of seedlings of Eucalyptus nitens. Four treatments that ranged from low to high nutrient-application rates resulted in mean whole-plant foliar concentrations of 0.63%, 0.85%, 1.11% and 1.82% nitrogen dry matter (N%DM) for treatments A, B, C and D, respectively. Within-plant distribution (across the leaf age profile of young, mature and old leaves within a eucalypt seedling) of N%DM ranged from zero in treatment A to a wide range of distribution in treatment D (low N%DM concentrations in old leaves to high N%DM concentrations in young leaves). Similarly, the distribution of fibre ranged from zero in treatment A to a wide range of distribution in treatment D, but with high concentrations in old leaves and low concentrations in young leaves. In contrast, flavonols (weakly) and gallotannins had a wide range of distribution in treatment A (low concentrations in old leaves to high concentrations in young leaves) but were little or not distributed in the other treatments. Again in contrast, cineole was strongly distributed between old and young leaves (low concentrations in old leaves to high concentrations in young leaves) across all treatments while concentrations in mature leaves reflected one or other leaf age depending on treatment. Protein precipitable phenols in treatments A, B and C were high in young, and low in old leaves; whereas in treatment D they were low and similar between leaves of different ages.

Cyclohexanols↗
PubMed2006

Phylogenetic fragrance patterns in Nicotiana sections Alatae and Suaveolentes.

We analyzed floral volatiles from eight tobacco species (Nicotiana; Solanaceae) including newly discovered Brazilian taxa (Nicotiana mutabilis and "Rastroensis") in section Alatae. Eighty-four compounds were found, including mono- and sesquiterpenoids, nitrogenous compounds, benzenoid and aliphatic alcohols, aldehydes and esters. Floral scent from recent accessions of Nicotiana alata, Nicotiana bonariensis and Nicotiana langsdorffii differed from previously published data, suggesting intraspecific variation in scent composition at the level of biosynthetic class. Newly discovered taxa in Alatae, like their relatives, emit large amounts of 1,8-cineole and smaller amounts of monoterpenes on a nocturnal rhythm, constituting a chemical synapomorphy for this lineage. Fragrance data from three species of Nicotiana sect. Suaveolentes, the sister group of Alatae, (two Australian species: N. cavicola, N. ingulba; one African species: N. africana), were compared to previously reported data from their close relative, N. suaveolens. Like N. suaveolens, N. cavicola and N. ingulba emit fragrances dominated by benzenoids and phenylpropanoids, whereas the flowers of N. africana lacked a distinct floral scent and instead emitted only small amounts of an aliphatic methyl ester from foliage. Interestingly, this ester also is emitted from foliage of N. longiflora and N. plumbaginifolia (both in section Alatae s.l.), which share a common ancestor with N. africana. This result, combined with the synapomorphic pattern of 1,8 cineole emission in Alatae s.s., suggests that phylogenetic signal explains a major component of fragrance composition among tobacco species in sections Alatae and Suaveolentes. At the intraspecific level, interpopulational scent variation is widespread in sect. Alatae, and may reflect edaphic specialization, introgression, local pollinator shifts, genetic drift or artificial selection in cultivation. Further studies with genetically and geographically well-defined populations are needed to distinguish between these possibilities.

Cyclohexanols↗
PubMed2004

Mirtazapine and venlafaxine in the management of collateral psychopathology during alcohol detoxification.

Symptoms of anxiety and depression are common in a large proportion of alcohol-abusing/dependent individuals during alcohol detoxification. The aim of this study was to examine the impact of a combined psychotherapeutic-psychopharmacological (either with mirtazapine or venlafaxine) treatment of these symptoms during the early withdrawal phase of alcohol compared to a group treated only with psychotherapy. A total of 60 alcohol-dependent/abusing subjects randomly assigned to three groups (psychotherapy, psychotherapy plus mirtazapine, psychotherapy plus venlafaxine) were studied. Assessment of psychopathology and global functioning throughout a 4-5-week detoxification period was done by the Hamilton Anxiety Rating Scale (HARS), the Hamilton Depression Rating Scale (HDRS), and the Global Assessment Scale (GAS). At baseline, high scores of anxiety and depression were recorded (HARS: controls: 33.1+/-7.8, mirtazapine: 33.2+/-12.6, venlafaxine: 36.6+/-5.4; HDRS: controls: 39.5+/-7.4, mirtazapine: 37.9+/-7.8, venlafaxine: 41.9+/-4.5). A marked improvement (p<0.000) was evidenced in all groups by the end of the detoxification period. However, patients on mirtazapine improved significantly more compared to the other two groups (HARS: controls: 9.6+/-7.6, mirtazapine: 4.3+/-4.4*, venlafaxine: 7.2+/-4.1, *p=0.011; HDRS: controls: 8.6+/-7.9, mirtazapine: 3.8+/-3.2*, venlafaxine: 8.2+/-3.5, *p=0.017; GAS: controls: 79.5+/-9.4, mirtazapine: 87.5+/-5.5**, venlafaxine: 83.0+/-8.0, **p=0.006). It is concluded that addition of mirtazapine, but not venlafaxine, to a standard psychotherapy-oriented alcohol detoxification treatment may facilitate the detoxification process by minimizing psychological discomfort. Consequently, it may prove to be a facilitator for the long-term abstinence from alcohol.

Adult↗
PubMed2004

The effect of chronic antidepressant treatment on serum brain-derived neurotrophic factor levels in depressed patients: a preliminary study.

Recent studies suggested a role of brain-derived neurotrophic factor (BDNF) in depression. While BDNF levels are lower in depressed patients, antidepressant treatment increases serum BDNF levels of depressed patients. Our study aims to test the effect of chronic venlafaxine treatment on serum BDNF levels in patients with a major depressive disorder. Ten patients diagnosed as major depressive disorder according to DSM-IV are included in the study. Two of the patients had their first episode and were drug-naive, the other eight patients were drug-free for at least 4 weeks. The severity of depression was assessed with Hamilton Depression Rating Scale (HDRS). The control group consisted of ten age- and sex-matched subjects without any psychiatric disorder. Blood samples were collected at the baseline and after 12 weeks of antidepressant treatment (during remission). At the baseline the mean serum BDNF level was 17.9+/-9.1 ng/ml and the mean HDRS score was 23.2+/-4.6. Serum BDNF levels of the study group were significantly lower than in the control group (31.6+/-8.6 ng/ml). At the end of the study, the mean serum BDNF level was 34.6+/-7.1 ng/ml whereas the mean HDRS score was 8.2+/-3.9. From the baseline to the remission after 12 weeks of treatment, the increase in serum BDNF level and the decrease in HDRS score were statistically significant, respectively. When we compared the serum BDNF level of depressed patients at remission to that of the controls, there was no statistically significant difference. This study shows that venlafaxine treatment of depression improves serum BDNF level which may be considered as a nonspecific peripheral marker of depression.

Adolescent↗
PubMed2005

Low-dose venlafaxine in three adolescents and young adults with autistic disorder improves self-injurious behavior and attention deficit/hyperactivity disorders (ADHD)-like symptoms.

In our clinical practice, we have had good experiences with venlafaxine in the treatment of self-injurious behavior (SIB) and attention deficit/hyperactivity disorders (ADHD)-like symptoms in patients with pervasive developmental disorders (PDD), and we report here three cases of possible therapeutic response: (A) a 17-year-old boy with autism and severe behavioral symptoms, including aggression toward self or property, SIB and hyperactivity, who appeared to respond to low-dose venlafaxine (18.75 mg/day); (B) a 23-year-old woman with autism hyperactivity who appeared to respond to low-dose venlafaxine (18.75 mg/day); (C) a 17-year-old girl with autism hyperactivity who appeared to respond to low-dose venlafaxine (18.75 mg/day). Follow-ups occurred respectively 18, 36 and 6 months after treatment initiation, making it possible to observe the stability of the clinical improvement in these cases.

Adolescent↗
PubMed2006

A comparison of the efficacy and tolerability of reboxetine and sertraline versus venlafaxine in major depressive disorder: a randomized, open-labeled clinical trial.

The aim of the study was to compare the efficacy and tolerability of the combination of reboxetine and sertraline to venlafaxine XR (extended release) in major depressive disorder (MDD). The study consisted of 40 patients with MDD, aged 18-65 years. Patients were evaluated six times during a 10-week period. Treatment was started as venlafaxine XR 75 mg/day once a day (od) or reboxetine 4 mg/day twice a day (bid)+sertraline 50 mg/day od. In the second week, venlafaxine XR was increased to 150 mg/day od and reboxetine 8 mg/day bid while sertraline was kept at the same dose. The Hamilton Depression Rating Scale (HDRS), Montgomery and Asberg Depression Rating Scale, Clinical Global Impressions-Severity of Illness and Clinical Global Impressions-Global Improvement Scale were applied on each visit. Beginning from the second visit, both groups showed significant declines in each scale. There were no significant differences between treatment response rates. Remission rates defined as HDRS<or=10 were significantly higher in the venlafaxine XR group at visit 4 only. However, when remission was accepted as HDRS<or=7, no significant difference was observed. Side effect frequency was similar between the treatment groups. We may suggest that the reboxetine+sertraline combination is not superior to venlafaxine treatment.

Adolescent↗
PubMed2006

Mycosporine-like amino acids in the zooxanthella-ciliate symbiosis Maristentor dinoferus.

Coral reef organisms living in mutualistic symbioses with phototrophic dinoflagellates are widespread in shallow UV-transparent waters. Maristentor dinoferus is a recently discovered species of marine benthic ciliate that hosts symbiotic dinoflagellates of the genus Symbiodinium. In this study, we tested this ciliate for the occurrence of mycosporine-like amino acids, a family of secondary metabolites that minimize damage from exposure to solar UV radiation by direct screening. Using high-performance liquid chromatography and liquid chromatography coupled to mass spectrometry, five mycosporine-like amino acids (shinorine, palythenic acid, palythine, mycosporine-2-glycine, and porphyra-334) were identified in aqueous methanolic extracts of the symbiosis. This is the first report of mycosporine-like amino acids in a marine ciliate.

Amino Acids↗
PubMed2004

Predictors of improved mood over time in clinical trials for major depression.

The objective of this exploratory analysis was to use a repeated measures modeling approach to identify potential predictors of improved mood over time in patients with major depression. Fifty-one subjects with major depressive disorder (MDD) were enrolled in a 1-week single blind placebo lead-in, followed by an 8-week, double-blind placebo-controlled treatment with either fluoxetine or venlafaxine. Hierarchical linear regression models were used to identify baseline and placebo lead-in predictors of change in repeated measures of the Hamilton Depression Rating Scale (HDRS) during treatment. Non-specific predictors of improved mood included decreased prefrontal activity during placebo lead-in as measured by quantitative electroencephalographic cordance, lower pretreatment depressed mood on the HDRS, shorter duration of current episode, increased verbalization of suicidal thoughts, no family history of mood disorders, less severe middle insomnia, higher guilt, lower somatic anxiety, and younger age. Moderators of improved mood included somatization, paranoid ideation, and self-reported depressed mood. We also found that change in prefrontal cordance after randomization mediated the effects of middle insomnia, suicidal thoughts, and family history of mood disorders. We recommend the use of repeated measures modeling, and the exploration of relationships among biological and psychological factors, for future analyses of clinical trial data.

Adult↗
PubMed2004

Efficacy of open-label venlafaxine in subjects with major depressive disorder: associations with neuroendocrine response to serotonergic and noradrenergic probes.

An open-label pilot study explored the relationship between severity of depressive symptoms and venlafaxine dose required for clinical efficacy in outpatients with major depressive disorder (MDD). The utility of the neuroendocrine response to serotonergic (ipsapirone) and noradrenergic (clonidine) probes as predictors of venlafaxine dosage required for effective treatment was also explored. Nineteen medically healthy medication-free outpatients over 18 years of age who met criteria for MDD were studied. Participants received either a 20-mg dose of ipsapirone orally, a 0.002-mg/kg intravenous dose of clonidine, or placebo. Following a 1-week single-blind placebo lead-in, all subjects were treated with immediate release venlafaxine. Low-dose responders were defined as those subjects experiencing a >50% decrease in depression score on 37.5 mg, b.i.d., and high-dose responders were defined as those subjects experiencing similar improvement on venlafaxine doses of 75 mg, b.i.d., or higher. Subjects responding to low-dose treatment had a lower mean baseline Hamilton depression score than subjects requiring high-dose treatment. Neuroendocrine and temperature responses to clonidine or ipsapirone challenges were not significantly different in the high- vs. low-dose responders. Evaluation of models of "serotonergic-responsive" and norepinephrine-responsive" depression requires larger numbers of patients.

Administration, Oral↗
PubMed2005

Effects of paroxetine and venlafaxine on immune parameters in patients with obsessive compulsive disorder.

BACKGROUND: Obsessive-compulsive disorder (OCD) has been associated with an altered activity of the immune system. This study was carried out to investigate whether treatment with paroxetine and venlafaxine modifies the immune function in OCD and whether this modification is related to treatment outcome. METHODS: Ex vivo production of TNF-alpha, IL-4, IL-6, IL-10, and IFN-gamma in whole blood cultures, and NK-cell activity and peripheral blood NK-cell-, monocytes-, T-cell-, and B-cell- percentages were measured in 42 outpatients with OCD who participated in a 12-week, double-blind SRI trial. RESULTS: Paroxetine and venlafaxine treatment did not affect immune parameters and clinical response was not directly related to altered activity of the immune system. Responders, defined as having a >or=25% decrease on the Y-BOCS, differed from non-responders with regard to the change of TNF-alpha values. CONCLUSIONS: The present results suggest that paroxetine and venlafaxine do not affect immune parameters and that clinical response is not related to altered activity of the immune system in OCD. Nonetheless, our data yielded interesting preliminary results that warrant further discussion and investigation.

Adult↗