The 12 Rs Framework as a Comprehensive, Unifying Construct for Principles Guiding Animal Research Ethics.

Animal research ethics and animal welfare in science have become progressively tightly regulated, and ethical integrity and scientific quality, as well as social responsiveness and responsibility have become key requirements for research to be approved, funded, published, and accepted. The multitude of factors to contemplate has in some instances not only become complex, requiring a team approach, but often perceived as confusing and overwhelming. To facilitate a process of simplistic yet comprehensive conceptualization, we developed the 12 Rs Framework to act as a mind map to guide scientists, oversight structures, and other stakeholders through the myriad of ethical considerations. It unfolds into three domains of twelve encompassing ethical principles, values, and other considerations, including the animal welfare, social values, and scientific integrity domains, whilst also recognizing the diversity of local context, legal requirements, values, and cultures around the globe. In the end, it can be seen as a unifying ethical framework to foster and promote animal research ethics.

The neuropsychiatric manifestations of COVID-19: Interactions with psychiatric illness and pharmacological treatment.

The recent outbreak of the corona virus disease (COVID-19) has had major global impact. The relationship between severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection and psychiatric diseases is of great concern, with an evident link between corona virus infections and various central and peripheral nervous system manifestations. Unmitigated neuro-inflammation has been noted to underlie not only the severe respiratory complications of the disease but is also present in a range of neuro-psychiatric illnesses. Several neurological and psychiatric disorders are characterized by immune-inflammatory states, while treatments for these disorders have distinct anti-inflammatory properties and effects. With inflammation being a common contributing factor in SARS-CoV-2, as well as psychiatric disorders, treatment of either condition may affect disease progression of the other or alter response to pharmacological treatment. In this review, we elucidate how viral infections could affect pre-existing psychiatric conditions and how pharmacological treatments of these conditions may affect overall progress and outcome in the treatment of SARS-CoV-2. We address whether any treatment-induced benefits and potential adverse effects may ultimately affect the overall treatment approach, considering the underlying dysregulated neuro-inflammatory processes and potential drug interactions. Finally, we suggest adjunctive treatment options for SARS-CoV-2-associated neuro-psychiatric symptoms.

Pre-pubertal, low-intensity exercise does not require concomitant venlafaxine to induce robust, late-life antidepressant effects in Flinders sensitive line rats.

A significant number of adolescents are considered insufficiently active. This is of concern considering the negative association between physical activity and major depressive disorder (MDD). There is a lack of approved pharmacological treatment options in this population partly due to limited information on the risks associated with lasting effects during early life. Therefore, interest in non-pharmacological strategies is gaining popularity with low- to moderate-intensity exercise being especially attractive for its antidepressant-like effects and augmentation properties in combination with antidepressants. Early-life development might present a unique "window of opportunity" to induce long-term beneficial effects in individuals treated with central acting drugs, such as antidepressants. Therefore, we investigated the bio-behavioural effects of pre-pubertal, low-intensity exercise (EXE) and/or venlafaxine (VEN) on depressive-like behaviour in juvenile (postnatal day 35 (PND35)) and young adult (PND60) stress-sensitive Flinders sensitive line (FSL) rats. Interventions were introduced during pre-pubertal development, that is PND21-34, followed by a 26-day washout/sedentary period, when bio-behavioural analyses were performed in the early adulthood group. VEN, alone or in combination with EXE, proved ineffective in inducing any bio-behavioural changes in either age group. EXE did not induce early-life antidepressant-like effects, despite increasing frontal serotonin (5-HT) and noradrenaline (NA) levels. Later in life (PND60), pre-pubertal exercise reduced immobility and increased coping behaviours, together with increased cortical 5-HT levels, despite a significant reduction in locomotor activity. These findings emphasize a strong serotonergic basis to the observed delayed antidepressant effects of EXE later in life.

Immediate and long-term antidepressive-like effects of pre-pubertal escitalopram and omega-3 supplementation combination in young adult stress-sensitive rats.

Major depressive disorder (MDD) affects a significant number of children and adolescents, yet treatment options for this population remain very limited. Escitalopram (ESC) is one of only two antidepressants approved as treatment for juvenile depression. Still, delayed onset of action, and immediate plus the risk of lasting side effects contribute to low patient adherence, and places the medical prescriber in a difficult situation weighing the potential long-term effects of juvenile treatment against the known consequences of untreated MDD. Research into alternative or augmentation strategies and their long-term effects are needed to improve clinical outcome and better our understanding of the long-term consequences of early-life treatment. We investigated the early-life (postnatal day 35 (PND35)) and lasting (PND60) bio-behavioural effects of pre-pubertal (PND21 to PND34) escitalopram (ESC) administration and/or ω-3 supplementation (OM3) in stress sensitive Flinders Sensitive Line rats. Only ESC treatment showed a strong trend to decrease depressive-like behaviour via significantly increased climbing behaviour on PND35. However, OM3 treatment reduced locomotor activity and increased hippocampal neuroplasticity on PND35, suggesting improved coping behaviour and masking of possible antidepressant-like effects. Reduced locomotor activity lasted into early-adulthood on PND60, despite a treatment-free period from PND35 to PND60. Regardless, early-adulthood antidepressive-like behaviour was only observed in the combination treatment (ESC + OM3) group, despite a significant increase in serotonin turnover, suggesting strong neurodevelopmental process to be involved. Taken together, the combination of ESC and OM3 might induce lasting beneficial neurodevelopmental effects in a stress-sensitive population, suggesting a possible role in current treatment strategies.

Long-lasting effects of fluoxetine and/or exercise augmentation on bio-behavioural markers of depression in pre-pubertal stress sensitive rats.

Juvenile depression is of great concern with only limited treatment currently approved. Delayed onset of action, low remission and high relapse rates, and potential long-lasting consequences further complicates treatment and highlights the need for new treatment options. Studies reporting on long-lasting effects of early-life treatment have reported conflicting results, with the pre-adolescent period mostly overlooked. The anti-depressive effect of exercise, as a possible treatment option or augmentation strategy, is dependent on age and exercise intensity. We investigated the immediate (i.e. postnatal day 35 (PND35)) and lasting (PND60 to PND61) effects of pre-pubertal (PND21 to PND34) fluoxetine and/or exercise on bio-behavioural markers of depression and oxidative stress in stress sensitive Flinders Sensitive Line rats. Low, but not moderate, intensity exercise or 5, but not 10, mg/kg/day fluoxetine displayed anti-depressant-like properties at PND35. Pre-pubertal treatment with 5mg/kg/day fluoxetine or low intensity exercise exerted lasting anti-depressive-like effects into adulthood, whereas the combination of these two treatments did not. Furthermore, the combination of fluoxetine plus exercise reduced hippocampal BDNF levels as compared to exercise alone, which may explain the latter findings. In all treatment groups hippocampal SOD activity was significantly increased at PND61, suggesting an increased anti-oxidant capacity in adulthood. In conclusion, the data confirm the anti-depressant-like properties of both early-life fluoxetine and exercise in a genetic animal model of depression. However, optimal lasting effects of early-life interventions may require adjustment of antidepressant dose and/or exercise intensity to developmental age, and that a combination of antidepressant and exercise may not necessarily be augmentative.

The long-term effects of methamphetamine exposure during pre-adolescence on depressive-like behaviour in a genetic animal model of depression.

Methamphetamine (METH) is a psychostimulant and drug of abuse, commonly used early in life, including in childhood and adolescence. Adverse effects include psychosis, anxiety and mood disorders, as well as increased risk of developing a mental disorder later in life. The current study investigated the long-term effects of chronic METH exposure during pre-adolescence in stress-sensitive Flinders Sensitive Line (FSL) rats (genetic model of depression) and control Flinders Resistant Line (FRL) rats. METH or vehicle control was administered twice daily from post-natal day 19 (PostND19) to PostND34, followed by behavioural testing at either PostND35 (early effects) or long-lasting after withdrawal at PostND60 (early adulthood). Animals were evaluated for depressive-like behaviour, locomotor activity, social interaction and object recognition memory. METH reduced depressive-like behaviour in both FSL and FRL rats at PostND35, but enhanced this behaviour at PostND60. METH also reduced locomotor activity on PostND35 in both FSL and FRL rats, but without effect at PostND60. Furthermore, METH significantly lowered social interaction behaviour (staying together) in both FRL and FSL rats at PostND35 and PostND60, whereas self-grooming time was significantly reduced only at PostND35. METH treatment enhanced exploration of the familiar vs. novel object in the novel object recognition test (nORT) in FSL and FRL rats on PostND35 and PostND60, indicative of reduced cognitive performance. Thus, early-life METH exposure induce social and cognitive deficits. Lastly, early-life exposure to METH may result in acute antidepressant-like effects immediately after chronic exposure, whereas long-term effects after withdrawal are depressogenic. Data also supports a role for genetic predisposition as with FSL rats.

The impact of HIV/AIDS on compliance with antidepressant treatment in major depressive disorder: A prospective study in a South African private healthcare cohort.

BACKGROUND: MDD and HIV/AIDS have a high prevalence worldwide with severe consequences for patients. In both conditions, compliance with treatment is key to successfully treat these disorders. In the current study, we examine the effect of MDD on the compliance with ADs in patients diagnosed with co-morbid HIV/AIDS and how different classes of ADs influence compliance in this group of patients. METHODS: A prospective, cohort study design was used to analyse nationally representative medicine claims data submitted to a privately-owned South African Pharmaceutical Benefit Management (PBM) company. Two groups were distinguished in the database, namely patients with only MDD and patients with both MDD and HIV/AIDS, over a six-year study period. The study population was determined by the following inclusion criteria: patients older than 18 years, MDD should be diagnosed by a psychiatrist supported by an appropriate ICD-10 code, and all patients have to be on combination antiretroviral treatment (cARV) treatment. The medicine possession ratio (MPR) was used as proxy to determine patient compliance with AD medication. RESULTS: 127 patients (i.e. 0.24%) met the criteria of co-morbid MDD and HIV/AIDS. Females have a significantly higher prevalence of MDD and HIV/AIDS when compared to males. Patients diagnosed with both HIV/AIDS and MDD (74.43. ± 32.03, 95% Cl: 71.51-77.34) have a statistical significantly (p < 0.0001) lower compliance with AD treatment vs. MDD patients (80.94% ± 29.44, 95% Cl: 80.56-81.33), but the practical significance thereof, is low (Cohen's d = 0.2255). In this group only 26.83% of TCA had acceptable compliance compared to the 58.57% of SNRIs. Noteworthy observations were that 75% (p < 0.0217; Cramer's V = 0.0388) of venlafaxine and 28.6% (p < 0.0197; Cramer's V = -0.0705) of the paroxetine items were compliant in patients diagnosed with both HIV/AIDS and MDD. CONCLUSIONS: AD compliance is statistical significantly lower in depressed HIV/AIDS vs. depressed non-HIV/AIDS patients. However, these differences is of low practical or clinical significance, meaning that depressed HIV/AIDS patients would have missed approximately two AD doses (6.5% of a 30-day treatment period) more than the non-HIV/AIDS depressed patient over the same treatment period.

Ozone exposure of Flinders Sensitive Line rats is a rodent translational model of neurobiological oxidative stress with relevance for depression and antidepressant response.

RATIONALE: Major depression has been associated with higher levels of air pollution that in turn leads to neurodegeneration via increased oxidative stress. There is a need for suitable translational animal models to study the role of oxidative stress in depression and antidepressant action. OBJECTIVE: Considering the gene X environment hypothesis of depression, the present study investigated the effect of chronic ozone inhalation on depression and anxiety-related behavior, cognition, and brain markers of oxidative stress in the Flinders Sensitive Line (FSL) rat. In addition, response to the antioxidant melatonin, and the antidepressants desipramine or escitalopram, was assessed. METHODS: Rats were exposed to ozone (0.0 or 0.3 parts per million (ppm)) per inhalation for 4 h daily for a period of 15 days, while simultaneously receiving saline or the above-mentioned drugs. RESULTS: The data indicate that chronic ozone inhalation induced memory impairment, anxiety and depression-like effects, reduced cortical and hippocampal superoxide dismutase and catalase activity, and compromised central monoamine levels similar to that noted in depression. Moreover, the behavioral and neurochemical effects of melatonin, desipramine, and escitalopram were mostly attenuated in the presence of ozone. CONCLUSION: Thus, genetically susceptible individuals exposed to high levels of oxidative stress are at higher risk of developing mood and/or an anxiety disorders, showing greater redox imbalance and altered behavior. These animals are also more resistant to contemporary antidepressant treatment. The presented model provides robust face, construct, and predictive validity, suitable for studying neuronal oxidative stress in depression, antidepressant action and mechanisms to prevent neuronal oxidative stress.

Late-life effects of chronic methamphetamine exposure during puberty on behaviour and corticostriatal mono-amines in social isolation-reared rats.

Chronic methamphetamine (MA) abuse results in an acute psychosis indistinguishable from paranoid schizophrenia. However, less is known of the interaction between MA use and environmental insults, and how this contributes to late-onset psychopathology. Using social isolation rearing (SIR), a neurodevelopmental animal model of schizophrenia, we investigated the association between changes in corticostriatal mono-amines and putative behaviours related to MA-induced psychosis in isolation and group-housed rats following chronic MA or saline exposure. Weaned male offspring of MA-naive female Wistar rats, either group- or isolation-housed from postnatal day (PND) +21, received saline (2 ml/kg s.c. b.i.d.) or an escalating dose of MA (0.2-6 mg/kg s.c. b.i.d.) for 16 days from PND +35 to +50. On PND +78, offspring were tested for deficits in social interactive behaviour (SIB) and prepulse inhibition (PPI) of startle, with frontal cortex and striatum harvested for the assessment of mono-amine concentrations. SIR significantly reduced rearing time, staying together, approaching and anogenital sniffing (outward-directed SIB), but increased self-grooming and locomotor activity (self-directed SIB), and also induced profound deficits in PPI. Pubertal MA exposure in group-housed animals also induced similar alterations in outward- and self-directed SIB and reduced PPI. Combined MA+SIR exposure evoked a similarly intense behavioural response as SIR or MA separately, with no exacerbation evident. Neither treatment separately nor together affected corticostriatal serotonin or noradrenaline levels, although frontal cortical dopamine (DA) levels were significantly increased in SIR and MA+group-housed animals. A trend towards further elevated frontal cortical DA was noted in the MA+SIR treatment group. Striatal DA was unaltered by all treatments. This study provides the first evidence that chronic pubertal MA exposure evokes postpubertal psychosis-like behaviours in rats of similar intensity to that induced by a neurodevelopmental animal model of schizophrenia (SIR). Moreover, the study is unique in that these behavioural changes occur together with associated changes in frontal cortical but not striatal DA, without affecting other mono-amines, and strongly implicates frontal cortical DA changes in the psychotogenic effects of early-life MA exposure or environmental insult. Although MA exposure in animals with a history of environmental insult (i.e. MA+SIR) has similar effects, combined exposure was not additive with regard to behavioural or neurochemical changes. We conclude that a ceiling effect or compensatory mechanisms prevent more pronounced neurobehavioural deficits occurring following MA+SIR treatment, at least under the current study conditions.

Chronic treatment with the phosphodiesterase type 5 inhibitors sildenafil and tadalafil display anxiolytic effects in Flinders Sensitive Line rats.

There are conflicting results from behavioural studies regarding whether the activation or inhibition of the cGMP-nitric oxide (NO) pathway induces anxiolytic-like behaviour. Sildenafil, an inhibitor of cGMP-selective phosphodiesterase-5, increases anxiety acutely, but previous evidence suggests that its chronic administration may be anxiolytic, and could involve a cholinergic interaction. We used the Flinders Sensitive Line (FSL) rat, a genetic model of depression that presents with increased anxiety- and depression-like behaviour, to investigate the action of chronic treatment with the PDE5 inhibitors sildenafil or tadalafil, with/without atropine on social interaction behaviour, a correlate for anxiety. Fluoxetine was used as positive control, with validation performed using Flinders Resistant Line (FRL) rats. In order to relate behavioural changes to brain penetration, we determined the concentration of sildenafil in cortex and hippocampus of rats following the schedule above using LC-MS/MS. FSL rats displayed significantly reduced social interactive behaviour than FRL rats, while sildenafil, tadalafil, and fluoxetine significantly reversed these deficits. Atropine did not exert effects on social interactive behaviour, nor did it modulate the effects of sildenafil or tadalafil. Sildenafil was present in cortex and hippocampus regions in lower nanomolar concentrations after chronic treatment, in agreement with the binding to PDE5 required for pharmacological effects. This study emphasizes the complicated regulation of anxiety by the cGMP-NO system, and provides supporting evidence for an anxiolytic action after the chronic activation of this pathway. As far as we know this is also the first report to formally demonstrate that sildenafil effectively crosses the blood-brain barrier to elicit central effects.

An inhibitor of cAMP-dependent protein kinase induces behavioural and neurological antidepressant-like effects in rats.

Although it is well established that cyclic adenosine monophosphate (cAMP) signalling via cAMP-dependent protein kinase (PKA)within neurons plays an important role in depression and antidepressant treatment, the importance of several newly discovered targets that function independently from PKA, such as exchange protein activated by cAMP (Epac), remains unexplored in this regard. In this study we used a cAMP analogue that inhibits PKA but not Epac (Rp-8-Br-cAMP), to explore the modifying actions of these two targets on immobility in the forced swim test (FST) and cerebellar cAMP response element binding protein (CREB) phosphorylation in rats. In addition, we assessed central cAMP and cGMP levels and investigated the involvement of cGMP-dependent protein kinase (PKG) on any observed effects by using a selective PKG inhibitor (Rp-8-Br-PET-cGMPS).Interestingly, Rp-8-Br-cAMPS strongly reduced immobility in the FST and induced an increase in the phosphorylation of CREB in the cerebellum, effects that were unaltered by the co-administration of Rp-8-Br-PET-cGMPS. Furthermore, Rp-8-Br-cAMPS increased the accumulation of cAMP and cGMP in the hippocampus, frontal cortex and cerebellum of these rats. Together, these results suggest that in addition to activating PKA, elevated cAMP may also stimulate other targets that mediate antidepressant activity. According to the pharmacodynamic profile of Rp-8-Br-cAMPS and taking into consideration what has recently been discovered regarding the cAMP signalling system, a likely candidate is the guanine nucleotide exchange factor, Epac.

Role of monoamine oxidase, nitric oxide synthase and regional brain monoamines in the antidepressant-like effects of methylene blue and selected structural analogues.

Dual action antidepressants have important therapeutic implications. Methylene blue (MB), a charged compound structurally related to tricyclic antidepressants, acts on both monoamine oxidase (MAO) and the nitric oxide (NO)-cGMP pathway, and has demonstrated antidepressant activity in rodents. We investigated the antidepressant properties of MB and selected structural analogues and whether their actions involve MAO, NO synthase (NOS) and regional brain monoamines. Acute imipramine (IMI, 15 mg/kg), saline, MB, acriflavine (ACR), methylene green (MG), methylene violet (MV), thionine (THI) and tacrine (TAC) (1-60 mg/kg i.p.) were tested for antidepressant activity in the forced swim test (FST), as well as MAO-A/B inhibitory activity. Active antidepressant compounds were subsequently studied at their most effective dose during sub-chronic treatment, followed by behavioural sampling in the FST and assay of cortico-limbic monoamines and hippocampal nitrate (for NOS activity). Only IMI, MB (15, 30, 60 mg/kg) and MG (7.5, 25, 40 mg/kg) reduced immobility in the acute FST. MB, MG and ACR were potent inhibitors of especially MAO-A. Following sub-chronic treatment, IMI (15 mg/kg) increased noradrenergic behaviour in the FST, while MB (15 mg/kg) and MG (15 mg/kg) enhanced serotonergic behaviour. MB and MG bolstered cortico-limbic serotonin (5HT) levels and to a lesser extent l-norepinephrine (l-NE), but did not significantly alter regional dopamine (DA) levels. MB, and to lesser degree MG, reduced hippocampal nitrate levels. MB and MG present with structure-specific antidepressant-like effects following acute and sub-chronic treatment, possibly involving NOS and MAO-A inhibition and cortico-limbic 5HT and l-NE release. A role for MAO-B and DA appears minimal.

Antidepressant-like properties of phosphodiesterase type 5 inhibitors and cholinergic dependency in a genetic rat model of depression.

We explored the antidepressant-like properties of two phosphodiesterase type 5 (PDE5) inhibitors in a genetic animal model of depression, namely Flinders sensitive line rats. We investigated the dose-dependency of the antidepressant-like action of sildenafil, and its interaction with the cholinergic system and behavioural correlates of monoaminergic neurotransmission, in the forced swim test. Antidepressant-like properties of tadalafil (a structurally distinct PDE5 inhibitor) were also evaluated. Flinders sensitive line rats were treated for 14 days with vehicle, fluoxetine, atropine or PDE5 inhibitors+/-atropine. Immobility, swimming and climbing behaviours were assessed in the forced swim test. In combination with atropine (1 mg/kg), both sildenafil (10, 20 mg/kg) and tadalafil (10 mg/kg) decreased immobility while increasing swimming (serotonergic) and climbing (noradrenergic) behaviours. Interestingly, sildenafil (3 mg/kg) decreased immobility while selectively increasing climbing behaviour in the absence of atropine. These results suggest that the antidepressant-like activity of PDE5 inhibitors involve alterations in monoaminergic neurotransmission, but involve a dependence on inherent cholinergic tone so that the final response is determined by the relative extent of activation of these systems. Furthermore, the behavioural profile of sildenafil alone, and its observed antidepressant-like properties, shows strict dose-dependency, with only higher doses showing an interaction with the cholinergic system.

Ozone modulates the effects of imipramine on immobility in the forced swim test, and nonspecific parameters of hippocampal oxidative stress in the rat.

Depression has been associated with oxidative stress. There is increased awareness of the role of environmental toxins in the development of mood disorders. Ozone, a pro-oxidant and environmental pollutant, has been noted to have central nervous system effects. We investigated the effects of acute and chronic ozone inhalation on the response of imipramine in the forced-swim test (FST) and on biomarkers of oxidative stress in rat hippocampus. Sprague Dawley rats were exposed to 0, 0.25 or 0.7 ppm ozone per inhalation 4 h daily for either 30 days (chronic) or once (acute). Animals were then injected intraperitoneally with imipramine (10 mg/kg) or saline 24, 5 and 1 h before the forced-swim test. Hippocampal superoxide accumulation and lipid peroxidation were measured. Imipramine evoked an antidepressant-like effect independent of acute or chronic ozone exposure. However, 0.7 ppm acute ozone and 0.25 ppm chronic ozone attenuated the antidepressant-like effects of imipramine. The ozone exposures also elevated hippocampal superoxide accumulation and lipid peroxidation. Importantly, imipramine reversed the lipid peroxidation induced by chronic ozone, thereby preventing cellular damage induced by oxidative stress. Ozone exposure presents a feasible model with etiological validity to investigate oxidative stress in depression and antidepressant action.

Studies on cellular resilience and adaptation following acute and repetitive exposure to ozone in cultured human epithelial (HeLa) cells.

Ozone is used to treat several medical conditions, while the underlying mechanisms of action are sometimes poorly understood. In the current study, we exposed cultured human epithelial (HeLa) cells acutely and repeatedly to ozone and investigated the effects thereof on cell viability. The involvement of anti-apoptotic pathways in observed adaptive responses to ozone were investigated by employing the Akt inhibitor (-)-deguelin. Cells were exposed to an ozone-saturated physiological solution using various dosing regimens, including acute exposure and various repetitive exposures. Cell viability was determined with Trypan Blue or MTT tests, or by a DNA-fragmentation (comet) assay. Acute ozone exposure compromised cell membrane integrity severely, while adaptation to reverse an initial reduction in mitochondrial activity was observed. Repetitive, short-duration exposures followed by a single long-duration exposure to ozone furnished a protective adaptation that was reversed by Akt inhibition. Extracellular and intracellular damage (and adaptation) occurs differentially. While acute ozone may decrease cell viability, multiple preexposures up-regulates cellular plasticity via induction of anti-apoptotic pathways in a treatment regimen-specific manner.

Tianeptine: a novel atypical antidepressant that may provide new insights into the biomolecular basis of depression.

Tianeptine, an atypical antidepressant patented and developed by Servier, enhances the synaptic reuptake of serotonin, without affecting norepinephrine and dopamine uptake, while it lacks affinity for neurotransmitter receptors. This mechanism for an antidepressant is apparently paradoxical, since the currently employed antidepressants enhance serotonin by inhibiting its breakdown or by inhibiting monoaminergic reuptake. Although tianeptine has been shown to reduce central 5HT availability and to indirecty modulate central adrenergic and dopaminergic systems and to indirectly inhibit cholinergic hyperactivity, its antidepressant action is believed to be more directly related to central neuronal remodeling and restoration of neuronal plasticity. In reliable animal models of depression tianeptine has been shown to prevent neurodegeneration and decreases in hippocampal volume in response to chronic stress. These effects on neuroplasticity are suspected to involve the normalization of the hypothalamic-pituitary-adrenal axis and modulatory effects on excitatory amino acids and N-methyl-D-aspartate receptors. Together with a body of related studies, these data provide further support for the hypothesis that depression may involve dysregulation of pathways controlling cellular resilience and that treatment should be directed towards the reversal thereof. Importantly, tianeptine is not anxiogenic and has also been shown to be effective in treatment-resistant depression, which may lead the way to a major breakthrough in the treatment of depression.

Phenoxybenzamine and benextramine, but not 4-diphenylacetoxy-N-[2-chloroethyl]piperidine hydrochloride, display irreversible noncompetitive antagonism at G protein-coupled receptors.

Many irreversible antagonists have been shown to inactivate G protein-coupled receptors (GPCRs) and used to study agonists and spare receptors. Presumably, they bind to primary (agonist) binding sites on the GPCR, although noncompetitive mechanisms of antagonism have been demonstrated but not thoroughly investigated. We studied noncompetitive antagonism by phenoxybenzamine and benextramine at alpha(2A)-adrenoceptors in stably transfected Chinese hamster ovary cells, benextramine and 4-diphenylacetoxy-N-[2-chloroethyl]piperidine hydrochloride (4-DAMP mustard) at endogenous muscarinic acetylcholine (mACh) receptors in human neuroblastoma SH-SY5Y cells, and benextramine at serotonin 5-HT(2A) receptors in stably transfected SH-SY5Y cells. Primary binding sites were protected by reversible competitive antagonists during pretreatment with irreversible antagonists. We conducted appropriate radioligand binding assays by measuring remaining primary binding sites and agonist affinity, functional assays to evaluate agonist-induced responses, and constitutive guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS)-Galpha(o) binding assays to determine remaining G protein activity. Phenoxybenzamine (100 microM; 20 min) and benextramine (10 or 100 microM; 20 min) at alpha(2A)-adrenoceptors, but not 4-DAMP mustard (100 nM; 120 min) at mACh receptors, displayed irreversible noncompetitive antagonism in addition to their known irreversible competitive antagonism. Although agonist binding affinity is not influenced, signal transduction is modulated in a G protein-dependent manner via allotopic interactions. Benextramine noncompetitively inhibits agonist-induced responses at three different GPCR types (alpha(2A), mACh, and 5-HT(2A) receptors) that signal via three families of G proteins (G(i/o), G(s), and G(q/11)). We conclude that, where irreversible antagonists are utilized to study drug-receptor interaction mechanisms, the presence of significant irreversible noncompetitive antagonism may influence the interpretation of results under the experimental conditions used.

Effects of myo-inositol versus fluoxetine and imipramine pretreatments on serotonin 5HT2A and muscarinic acetylcholine receptors in human neuroblastoma cells.

myo-Inositol (mI) is a key metabolic precursor to the phospoinositide (PI) metabolic pathway as a key component of central G-protein coupled receptor signaling systems, including several subtypes of adrenergic, cholinergic, serotonergic and metabotropic glutamatergic receptors. High dose mI has also been shown to be clinically effective in the treatment of obsessive-compulsive disorder, as well as panic and depression, although its mechanism of action remains elusive. The current study aimed to investigate the possible modulatory role of mI versus fluoxetine or imipramine pretreatments on serotonin-2A receptor (5HT2A-R) and muscarinic acetylcholine receptor (mAChR) function and binding in in vitro systems. After pretreating human neuroblastoma cells with different concentrations of mI, fluoxetine, or imipramine, receptor function was measured by second messenger [3H]-IPx accumulation and [35S]-GTPgammaS binding to G alpha(q) protein. Total [3H]-mI uptake into cells was measured, as well as specific receptor binding to determine receptor binding after the pretreatments. Results suggest that mI reduces 5HT2A-R function at the receptor-G protein level. While fluoxetine also reduced 5HT2A-R function, but to a lesser degree, imipramine increased 5HT2A-R function, which may explain why mI seems to be effective exclusively in selective serotonin reuptake inhibitor-sensitive disorders. In addition mI, and at high concentrations fluoxetine and imipramine, also reduces mAChR function. Furthermore the results suggest that the attenuating effect of mI on mAChRs is partially dependent on the PI metabolic pathway. The data provide novel information on understanding the mechanism of action of mI in depression and related anxiety disorders and added to the evidence suggesting a role for the cholinergic system in the pathophysiology of depression.

Defining the neuromolecular action of myo-inositol: application to obsessive-compulsive disorder.

Dietary inositol is incorporated into neuronal cell membranes as inositol phospholipids where it serves as a key metabolic precursor in G protein-coupled receptors. In the brain, several subtypes of adrenergic, cholinergic, serotonergic and metabotropic glutamatergic receptors are coupled to the hydrolysis of phosphoinositides (PI) with myo-inositol (MI) crucial to the resynthesis of PI and the maintenance and effectiveness of signalling. Despite a mode of action that remains illusive, MI has demonstrated therapeutic efficacy in obsessive-compulsive disorder (OCD), putative OCD-spectrum disorders, as well as panic and depression. Behavioural and biochemical studies indicate that this efficacy does not involve simply the replenishing of the membrane PI pool. In addition to its precursory role in cell signalling, inositol lipids alter receptor sensitivity, can direct membrane trafficking events, and have been found to modulate an increasing array of signalling proteins. These effects may afford MI an ability to modulate the interaction between neurotransmitters, drugs, receptors and signalling proteins. This paper reviews the neuromolecular and genetic aspects of OCD in terms of the PI-linked 5HT receptor subtypes and relates these to the behavioural and therapeutic effects of MI. Since OCD often is poorly responsive to current drug treatment, understanding the neuropharmacology of MI holds great promise for understanding the neuropathology of this and other MI-responsive disorders.