Sub-effective Dose Research Articles

Enhancement of Ketone Supplements-Evoked Effect on Absence Epileptic Activity by Co-Administration of Uridine in Wistar Albino Glaxo Rijswijk Rats.

Both uridine and exogenous ketone supplements decreased the number of spike-wave discharges (SWDs) in a rat model of human absence epilepsy Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats. It has been suggested that alleviating influence of both uridine and ketone supplements on absence epileptic activity may be modulated by A1 type adenosine receptors (A1Rs). The first aim was to determine whether intraperitoneal (i.p.) administration of a specific A1R antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 0.2 mg/kg) and a selective adenosine A2A receptor antagonist (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo [1,5-c]pyrimidine) (SCH 58261; 0.5 mg/kg) have a modulatory influence on i.p. 1000 mg/kg uridine-evoked effects on SWD number in WAG/Rij rats. The second aim was to assess efficacy of a sub-effective dose of uridine (i.p. 250 mg/kg) combined with beta-hydroxybutyrate salt + medium chain triglyceride (KSMCT; 2.5 g/kg, gavage) on absence epilepsy. DPCPX completely abolished the i.p. 1000 mg/kg uridine-evoked alleviating effect on SWD number whereas SCH 58261 was ineffective, confirming the A1R mechanism. Moreover, the sub-effective dose of uridine markedly enhanced the effect of KSMCT (2.5 g/kg, gavage) on absence epileptic activity. These results demonstrate the anti-epilepsy benefits of co-administrating uridine and exogenous ketone supplements as a means to treat absence epilepsy.

Melatonin attenuated the behavioral despair induced by acute neurogenic stress through blockade of N-methyl D-aspartate receptors in mice

It has been well documented that administration of melatonin could reveal antidepressant-like effect in rodents. However, the protective effect of melatonin on stress-induced depression/anxiety and its underlying mechanism is yet to be understood. In this regard, in the current study, acute foot-shock stress (FSS) was used to evaluate the antidepressant-like effect of melatonin on neurogenic stress-induced depression in mice. Behavioral evaluation was done by using the forced swimming test (FST) and Open-field test (OFT). Melatonin, MK-801, and ketamine (NMDA receptor antagonists), and NMDA (NMDA receptor agonist) were used to elucidate any association between melatonin and NMDA pathway in behavioral despair induced by acute-FSS. Applying acute-FSS to mice significantly induced depressant-like behavior in FST without any significant impact on locomotor activity in the OFT. We observed that melatonin (dose-dependently) significantly improved the depressant-like effect of FSS, but it did not impact the locomotion in animals. Acute injection of MK-801 at sub-effective doses (0.01 mg/kg) or ketamine (0.1 mg/kg) potentiated the antidepressant-like effect of a sub-effective dose of melatonin. However, the sub-effective dose of NMDA (30 mg/kg) abolished the protective effect of melatonin on the behavioral profile of stressed animals. Our results could reflect the antidepressant-like effect of melatonin on neurogenic stress-induced depressive behaviors in mice. Also, our results showed that NMDA receptors could be involved in the antidepressant-like effect of melatonin.

Harmaline potentiates morphine-induced antinociception via affecting the ventral hippocampal GABA-A receptors in mice

Morphine is one of the most effective medications for treatment of pain, but its side effects limit its use. Therefore, identification of new strategies that can enhance morphine-induced antinociception and/or reduce its side effects will help to develop therapeutic approaches for pain relief. Considering antinociceptive efficacy of harmaline and also highlighted the important role of GABA-A receptors in the pain perception, this research aimed to determine whether the ventral hippocampal (vHip) GABA-A receptors are involved in the possible harmaline-induced enhancement of morphine antinociception. To achieve this, vHip regions of adult male mice were bilaterally cannulated and pain sensitivity was measured in a tail-flick apparatus. Intraperitoneally administration of morphine (0, 2, 4 and 6 mg/kg) or harmaline (0, 1.25, 5 and 10 mg/kg) increased the percentage of maximal possible effect (%MPE) and induced antinociception. Interestingly, co-administration of sub-effective doses of harmaline (5 mg/kg) and morphine (2 mg/kg) induced antinociception. Intra-vHip microinjection of muscimol (0, 200 and 300 ng/mice), a GABA-A receptor agonist, enhanced the anti-nociceptive effects of harmaline (2.5 mg/kg)+morphine (2 mg/kg) combination. Microinjection of the same doses of muscimol into the vHip by itself did not alter tail-flick latency. Intra-vHip microinjection of bicuculline (100 ng/mouse), a GABA-A receptor antagonist, did not cause a significant change in MPE%. Bicuculline (60 and 100 ng/mouse, intra-vHip) was administered with the harmaline (5 mg/kg)+morphine (2 mg/kg), and inhibited the potentiating effect of harmaline on morphine response. These findings favor the notion that GABAergic mechanisms in the vHip facilitate harmaline-induced potentiation of morphine response in the tail-flick test in part through GABA-A receptors. These findings shall provide insights and strategies into the development of pain suppressing drugs.

Pharmacological evaluation of NO/cGMP/KATP channels pathway in the antidepressant-like effect of carbamazepine in mice

Carbamazepine, an anticonvulsant drug, has shown antidepressant effects in clinical and experimental models. Nitric oxide (NO) is a neurotransmitter in the central nervous system and has been involved in a variety of diseases including depression. In the present study, the involvement of NO/cyclic GMP/KATP channels pathway in the antidepressant action of carbamazepine was investigated in mice. The antidepressant-like activity was assessed in the forced swim test (FST) behavioral paradigm. Carbamazepine reduced (40 mg/kg, intraperitoneal) immobility period. The antidepressant-like effect of carbamazepine (40 mg/kg, intraperitoneal) was prevented by pretreatment with L-arginine [substrate for NO synthase (NOS), 750 mg/kg, intraperitoneal], sildenafil (a PDE-5 inhibitor, 5 mg/kg, intraperitoneal) and diazoxide (K+ channels opener, 10 mg/kg). Pretreatment of mice with L-NAME (a non-selective NOS inhibitor, 10 mg/kg, intraperitoneal), methylene blue (direct inhibitor of both NOS and soluble guanylate cyclase, 10 mg/kg, intraperitoneal) and glibenclamide (an ATP-sensitive K+ channel blocker, 1 mg/kg, intraperitoneal) produced potentiation of the action of a sub-effective dose of carbamazepine (30 mg/kg, intraperitoneal). Also, carbamazepine (30 mg/kg) potentiated the antidepressant-like effect of fluoxetine through NO modulation. The various modulators used in the study did not produce any changes in locomotor activity per se. The results demonstrated that the antidepressant-like effect of carbamazepine in the FST involved an interaction with the NO/cGMP/KATP channels pathway.

Inhibition of PDE2 and PDE4 synergistically improves memory consolidation processes

Phosphodiesterases (PDE) are the only enzymes that degrade cAMP and cGMP which are second messengers crucial to memory consolidation. Different PDE inhibitors have been developed and tested for their memory-enhancing potential, but the occurrence of side effects has hampered clinical progression. As separate inhibition of the PDE2 and PDE4 enzyme family has been shown to enhance memory, we investigated whether concurrent treatment with a PDE2 and PDE4 inhibitor can have synergistic effects on memory consolidation processes. We found that combined administration of PF-999 (PDE2 inhibitor) and roflumilast (PDE4 inhibitor) increases the phosphorylation of the AMPA receptor subunit GluR1 and induces CRE-mediated gene expression. Moreover, when combined sub-effective and effective doses of PF-999 and roflumilast were administered after learning, time-dependent forgetting was abolished in an object location memory task. Pharmacokinetic assessment indicated that combined treatment does not alter exposure of the individual compounds. Taken together, these findings suggest that combined PDE2 and PDE4 inhibition has synergistic effects on memory consolidation processes at sub-effective doses, which could therefore provide a therapeutic strategy with an improved safety profile.

Regulation of Glutamatergic Activity via Bidirectional Activation of Two Select Receptors as a Novel Approach in Antipsychotic Drug Discovery

Schizophrenia is a mental disorder that affects approximately 1–2% of the population and develops in early adulthood. The disease is characterized by positive, negative, and cognitive symptoms. A large percentage of patients with schizophrenia have a treatment-resistant disease, and the risk of developing adverse effects is high. Many researchers have attempted to introduce new antipsychotic drugs to the clinic, but most of these treatments failed, and the diversity of schizophrenic symptoms is one of the causes of disappointing results. The present review summarizes the results of our latest papers, showing that the simultaneous activation of two receptors with sub-effective doses of their ligands induces similar effects as the highest dose of each compound alone. The treatments were focused on inhibiting the increased glutamate release responsible for schizophrenia arousal, without interacting with dopamine (D2) receptors. Ligands activating metabotropic receptors for glutamate, GABAB or muscarinic receptors were used, and the compounds were administered in several different combinations. Some combinations reversed all schizophrenia-related deficits in animal models, but others were active only in select models of schizophrenia symptoms (i.e., cognitive or negative symptoms).

Novel Positive Allosteric Modulators of µ Opioid Receptor-Insight from In Silico and In Vivo Studies.

Opioids are the drugs of choice in severe pain management. Unfortunately, their use involves serious, potentially lethal side effects. Therefore, efforts in opioid drug design turn toward safer and more effective mechanisms, including allosteric modulation. In this study, molecular dynamics simulations in silico and ‘writhing’ tests in vivo were used to characterize potential allosteric mechanism of two previously reported compounds. The results suggest that investigated compounds bind to μ opioid receptor in an allosteric site, augmenting action of morphine at subeffective doses, and exerting antinociceptive effect alone at higher doses. Detailed analysis of in silico calculations suggests that first of the compounds behaves more like allosteric agonist, while the second compound acts mainly as a positive allosteric modulator.

Protective effect of modafinil on skin flap survival in the experimental random-pattern skin flap model in rats: The role of ATP-sensitive potassium channels and nitric oxide pathway

Background The brain-stimulating agent modafinil acts through nitric oxide (NO) and adenosine triphosphate (ATP)-sensitive potassium (KATP) channels, involved in the skin flap survival (SFS). The main aim of this study was to investigate the efficacy of modafinil on SFS in rats through the involvement of NO pathway and KATP channels. Methods Using controlled experiment study design, we enrolled a sample of Wistar male rats. Different doses of modafinil (10, 25, 50, and 100 mg/kg) were injected intraperitoneally (i.p.) before the surgery. L-NAME (non-selective nitric oxide synthase [NOS] inhibitor), aminoguanidine (inducible NOS inhibitor), and 7-nitroindazole (neuronal NOS inhibitor) were administered prior to modafinil. The role of KATP channels was determined by coadministering glibenclamide (KATP channel blocker) or cromakalim (KATP channel opener) with modafinil. The predictor variables were administration of different doses of modafinil, and the coadministration of modafinil with L-NAME, aminoguanidine, 7-nitroindazole, glibenclamide, and cromakalim. The main outcome variables included the percentage of necrotic area (PNA) in flap tissues, histopathological results, vascular endothelial growth factor (VEGF) immunohistochemical (IHC) staining, and nitrite concentrations. Appropriate statistics were computed considering p-value ≤ 0.05 significant. Results Modafinil 25 mg/kg was the most effective dose (PNA: 26 [95% CI: 19–33]) vs. control (PNA: 81 [95% CI: 71–92]) (p< 0.001). All NOS inhibitors significantly reversed the protective effect of modafinil (p< 0.001). Non-effective dose of cromakalim had a synergistic effect with the sub-effective dose of modafinil (10 mg/kg), while glibenclamide reversed the effect of modafinil 25 mg/kg (p< 0.001). Conclusions Modafinil increases SFS mediated by NO pathway and KATP channels, which could therefore be a target to improve SFS. The brain-stimulating agent modafinil acts through nitric oxide (NO) and adenosine triphosphate (ATP)-sensitive potassium (KATP) channels, involved in the skin flap survival (SFS). The main aim of this study was to investigate the efficacy of modafinil on SFS in rats through the involvement of NO pathway and KATP channels. Using controlled experiment study design, we enrolled a sample of Wistar male rats. Different doses of modafinil (10, 25, 50, and 100 mg/kg) were injected intraperitoneally (i.p.) before the surgery. L-NAME (non-selective nitric oxide synthase [NOS] inhibitor), aminoguanidine (inducible NOS inhibitor), and 7-nitroindazole (neuronal NOS inhibitor) were administered prior to modafinil. The role of KATP channels was determined by coadministering glibenclamide (KATP channel blocker) or cromakalim (KATP channel opener) with modafinil. The predictor variables were administration of different doses of modafinil, and the coadministration of modafinil with L-NAME, aminoguanidine, 7-nitroindazole, glibenclamide, and cromakalim. The main outcome variables included the percentage of necrotic area (PNA) in flap tissues, histopathological results, vascular endothelial growth factor (VEGF) immunohistochemical (IHC) staining, and nitrite concentrations. Appropriate statistics were computed considering p-value ≤ 0.05 significant. Modafinil 25 mg/kg was the most effective dose (PNA: 26 [95% CI: 19–33]) vs. control (PNA: 81 [95% CI: 71–92]) (p< 0.001). All NOS inhibitors significantly reversed the protective effect of modafinil (p< 0.001). Non-effective dose of cromakalim had a synergistic effect with the sub-effective dose of modafinil (10 mg/kg), while glibenclamide reversed the effect of modafinil 25 mg/kg (p< 0.001). Modafinil increases SFS mediated by NO pathway and KATP channels, which could therefore be a target to improve SFS.

The Benefit of Passion Fruit as an Anti-ulcerogenic Diet: Scientific Evidence by In vitro and In silico H+/K+ATPase Inhibitory Activity Assessment.

H+/K+ ATPase a protein present in the gastric parietal cells is a better target for the prevention and treatment of gastric ulcer. Plant flavonoids have been reported to elicit anti-ulcer activity by inhibiting the proton pump as well as by antioxidant defense mechanism. Chloroform fraction of hydro-alcoholic extract of passion fruit was screened for proton pump inhibitory assay using goat parietal cell. In-silico computational docking studies were carried out using Glide program in order to validate the inhibitory action of selected constituents. The flavonoid rich fruit possess a promising radical scavenging activity against DPPH. 10.41μg/mL is sufficient to inhibit 50% of ATPase enzyme activity. A synergistic activity was also achieved by the fruit with sub-effective doses of lansoprazole. Fenton's oxidation induced by H2O2 was also blunted by the fruit extract. The in-vitro and in-silico findings indicated that, passion fruit can be a good dietary supplement for the prevention and management of ulcer.

Ivermectin Increases Random-Pattern Skin Flap Survival in Rats: The Novel Role of GABAergic System

Ivermectin (IVM) was first used as an antiparasitic agent; however, the role of this drug evolved into a broad spectrum. Many mechanisms have been proposed, including interaction with the GABAergic system. Considering the presence of GABA receptor in the skin tissue and its role in ischemia-reperfusion I/R injury, we aimed to evaluate the effect of IVM through GABA receptors on random-pattern skin flap survival. Sixty Wistar male rats were used. Multiple doses of IVM (0.01, 0.05, 0.2, and 0.5mg/kg) were injected intraperitoneally before the surgery. Baclofen (selective GABAB agonist) and bicuculline (selective GABAA antagonist) were administered in combination with IVM to assess the role of the GABAergic system. Histopathological evaluations, immunohistochemical staining, quantitative assessment of IL-1β and TNFα, and the expression of GABAA α1 subunit and GABAB R1 receptors were evaluated in the skin tissue. IVM 0.05mg/kg could significantly increase flap survival compared with the control group (P<0.001). Subeffective dose of baclofen (0.1mg/kg) had synergistic effect with the subeffective dose of IVM (0.01mg/kg) (P<0.001), whereas bicuculline 1mg/kg reversed the effect of IVM (0.05mg/kg) (P<0.001). IVM 0.05mg/kg could also decrease the IL-1β and TNFα levels and increase the expression of GABAA α1 subunit and GABAB R1 receptors in the flap tissue compared with the control group. IVM could improve skin flap survival, probably mediated by the GABAergic pathway. Both GABAA and GABAB receptors are involved in this process. This finding may repurpose the use of old drug, "Ivermectin."

Nitric oxide mediates the antidepressant-like effect of modafinil in mouse forced swimming and tail suspension tests.

Previous studies have suggested antidepressant properties for modafinil; however, the underlying mechanisms mediating the antidepressant effect of modafinil have not been well recognized in clinical and animal studies. Nitric oxide (NO) is involved in the pathophysiology of depression. We attempted to investigate the possible role of NO in the antidepressant-like effect of modafinil in mouse forced swimming test (FST) and tail suspension test (TST). The antidepressant-like effect of modafinil (25, 50 and 75mg/kg), alone and in combination with l-arginine, l-arg, (100mg/kg) and NG-l-arginine methyl ester, l-NAME (5mg/kg), was evaluated using FST and TST. Following behavioral tests, the hippocampi were dissected out to measure nitrite levels. Findings suggested that administration of modafinil at doses of 50 and 75mg/kg significantly reduced immobility time in the FST and TST. Furthermore, administration of l-arg and l-NAME increased and decreased, respectively, the immobility time in the FST and TST. We showed that co-administration of a sub-effective dose of modafinil (25mg/kg) plus l-NAME potentiated the antidepressant-like effect of the sub-effective dose of modafinil. In addition, co-treatment of an effective dose of modafinil (75mg/kg) with l-arg attenuated the antidepressant-like effect of the effective dose of modafinil. We showed that the antidepressant-like effect of modafinil is associated with decreased nitrite levels in the hippocampus. Our findings for the first time support that the modulation of NO, partially at least, is involved in the antidepressant-like effect of modafinil in mouse FST and TST.

Indomethacin, a nonselective cyclooxygenase inhibitor, does not interact with MTEP in antidepressant-like activity, as opposed to imipramine in CD-1 mice

The contribution of metabotropic glutamate receptors (mGlu receptors) in depression is well known and tested worldwide. Our previous study showed the involvement of the cyclooxygenase-2 (COX-2) pathway in behavioral changes mediated by an antagonist of metabotropic glutamate receptor subtype 5 (mGlu5 receptor) 3-[(2-methyl-1,3-tiazol-4-yl)ethynyl]-pyridine (MTEP). Among others, we have found that chronic concomitant administration of a COX-2 inhibitor and sub-effective dose of MTEP accelerates antidepressant-like activity of MTEP. This paper seeks to explore whether the same effect would be observed with the use of a non-selective COX inhibitor 2-[1-(4-chlorobenzoyl)-5-methoxy-2-methylindol-3-yl]acetic acid (indomethacin). To that end, we have employed experimental procedure implemented in the earlier research. MTEP and indomethacin or MTEP + indomethacin were used chronically for 7 or 14 days. Then, the Porsolt test, tail suspension test and locomotor activity test were performed. Imipramine was used as a reference compound, as its action is connected with mGlu5 receptor. We found that, in contrast to COX-2 inhibition, indomethacin – acting both through COX-1 and COX-2 – did not release antidepressant-like potential of MTEP. The opposite effect was shown when imipramine was used.

Diazepam and SL-327 synergistically attenuate anxiety-like behaviours in mice – Possible hippocampal MAPKs specificity

Intracellular signalling pathways have been extensively studied as therapeutic targets for the treatment of mental diseases. Our attention has been caught by two kinases potentially involved in anxiety, ERK1/2 and CaMKII. The study aimed to examine changes in the activation of ERK1/2 and CaMKII concerning anxiolytic-like behaviours in mice.To evaluate anxiety-related response in mice, we used the open field test and the elevated plus maze test. Behavioural studies were complemented with the immunoblotting analysis to identify proteins of interest in the cortex, hippocampus, and striatum. We analysed the phosphorylation status of ERK1/2 and CaMKII in mice treated with a well-known anxiolytic drug – diazepam. Next, the blockade of ERK1/2 pathway by SL-327, a selective MEK1/2 inhibitor, was checked for anxiolytic action. Finally, the co-administration of subeffective doses of diazepam and SL-327 was investigated for a potential synergistic anxiolytic effect.Anxiolytic effects of acute diazepam are accompanied by decreased p-ERK1/2 and upregulation of p-CaMKII. Subchronic treatment with SL-327 leads to the manifestation of anxiolytic-like behaviours and changes in the phosphorylation status of both kinases in a diazepam-like manner. Co-administration of subeffective doses of SL-327 and diazepam induces anxiolysis, which is CaMKII-independent and correlates to selectively decreased phosphoactive ERK1/2 in the hippocampus.The MEK-ERK pathway is significantly involved in anxiolytic action of diazepam and its prolonged inhibition produces anxiolytic-like phenotype in mice. ERK inhibition could be used to manage anxiety symptoms in a benzodiazepine-sparing regimen for treatment of anxiety.

Digoxin enhances the effect of antiepileptic drugs with different mechanism of action in the pentylenetetrazole-induced seizures in mice

The worldwide prevalence of epilepsy with high percentage of multidrug-resistant patients make it urgent to find new approaches to treating, including the use of combinations of classic anticonvulsants with drugs that have an exclusively original mechanism of action, in particular digoxin. The aim of this work was to investigate the influence of low-dose digoxin on the anticonvulsant effect of sodium valproate, topiramate, levetiracetam, phenobarbital and clonazepam. A basic model of pentylenetetrazole-induced seizures in mice was used. Antiepileptic drugs were administered intragastrically in conditionally effective (ED50) and sub-effective (½ ED50) doses at 30 min, digoxin – subcutaneously at a dose of 0.8 mg/kg (1/10 LD50) at 10−15 min before seizures induction. Pentylenetetrazole at a dose of 80 mg/kg was administered subcutaneously. Experimental data demonstrates that cardiac glycoside digoxin enhances the anticonvulsant activity of sodium valproate, topiramate, levetiracetam, phenobarbital and clonazepam in the model of pentylenetetrazole-induced seizures, providing a clear protective effect of their sub-effective doses. Digoxin may be a valuable component of adjuvant pharmacotherapy for epilepsy, as it reduces the doses of the classic AEDs without compromising the effectiveness of treatment.

Possible involvement of NMDA receptor in the anxiolytic-like effect of caffeic acid in mice model of maternal separation stress

Background and aimAnxiety disorders are one of the most common psychiatric disorders worldwide. Common anti-anxiety medications are associated with several side effects. Caffeic acid (CA) is a phenolic compound with several pharmacological effects. The aim of this study was to investigate the anxiolytic-like effect of CA in maternally separated (MS) mice focusing on the possible involvement of the NMDA receptor. Materials and methodsIn this study, we used the MS paradigm (as a valid animal model of anxiety) in male mice and examined their anxiety-like behavior in postnatal day (PND) 45. The animals were divided into 12 experimental groups. Mice treated with CA alone and in combination with the NMDA receptor agonist/antagonist and then using open field (OFT) and elevated plus maze (EPM) anxiety-like behavior was assessed. Finally, the expression of NMDA receptor subtypes was assessed in the hippocampus using RT- PCR. ResultsFinding showed that CA exerted anxiolytic –like effects in the OFT and EPM tests. We showed that administration of effective dose of NMDA significantly reversed the anxiolytic-like effect of effective dose of CA and co-administration of ketamine (a NMDA receptor antagonist) significantly potentiated the effect of sub-effective dose of CA. Furthermore, ketamine enhanced the CA-reducing effect on NMDA receptors in the MS mice. ConclusionOur finding demonstrated that, probably at least, NMDA receptors are involved in the anxiety-like properties of CA in MS mice.

The involvement of PI3K/Akt/mTOR/GSK3β signaling pathways in the antidepressant-like effect of AZD6765

AZD6765 (lanicemine) is a non-competitive NMDA receptor antagonist that induces a fast-acting antidepressant effect without presenting psychotomimetic effects. However, the mechanisms underlying its effects remain to be established. In this context, we demonstrated that a single administration of AZD6765 (1 mg/kg, i.p.) was able to induce an antidepressant-like effect in mice submitted to tail suspension test (TST), an effect reversed by LY294002 (a reversible PI3K inhibitor, 10 nmol/site, i.c.v.), wortmannin (an irreversible PI3K inhibitor, 0.1 μg/site, i.c.v.) and rapamycin (a selective mTOR inhibitor, 0.2 nmol/site, i.c.v.). In addition, the administration of sub-effective doses of AZD6765 (0.1 mg/kg, i.p.) in combination with lithium chloride (non-selective GSK-3β inhibitor, 10 mg/kg, p.o.) or AR-A014418 (selective GSK-3β inhibitor, (0.01 μg/site, i.c.v.) caused a synergistic antidepressant-like effect. These results suggest the involvement of PI3K/Akt/mTOR/GSK3β signaling in the AZD6765 antidepressant-like effect. In addition, western blotting analysis showed an increased immunocontent of synapsin in the prefrontal cortex and a tendency to an increased immunocontent of this protein in the hippocampus 30 min after AZD6765 administration, but no significant effect of AZD6765 was observed in P70S6K (Thr389) phosphorylation and GluA1 immunocontent. A single dose of AZD6765 (3 mg/kg, i.p.), similarly to ketamine (1 mg/kg, i.p.), decreased the latency to feed in the novelty suppressed feeding (NSF) test, a behavioral paradigm that evaluates depression/anxiety-related behavior. This effect was reversed by rapamycin administration, suggesting the activation of mTOR signaling in the effect of AZD in the NSF test. In addition, a single administration of AZD6765 (1 mg/kg, i.p.) or ketamine (1 mg/kg, i.p.) reversed the depressive-like behavior induced by chronic unpredictable stress (CUS). Altogether, the results provide evidence for the fast-acting antidepressant profile of AZD6765, by a mechanism likely dependent on PI3K/Akt/mTOR/GSK3β.

Abstract 494: Effect of Aloysia polystachya in cancer stem cells of colorectal cancer

Abstract Colorectal cancer (CRC) is one of the most commonly diagnosed cancers and the Cancer Stem Cells (CSC) are the responsible of CRC persistence. Aloysia polystachya (AP) is an aromatic native plant of Verbenaceae family which is widely distributed and consumed in infusions in subtropical regions of South America. Its effects include diuretic, sedative, antispasmodic activities. We have previously demonstrated that AP extract has a cytotoxic effect in several human tumor cell lines, including apoptosis. The aim of this work was to investigate the cytotoxic effects of AP in vitro and in vivo in a CRC model. Therefore, CRC cell lines CT26 (murine) and HCT116 (human), were stimulated with AP extract or vehicle, with or without 5-fluorouracyle (5-FURA) and the survival and CSC properties were determined. The survival was measured after crystal violet staining at 570nm and CSC phenotype was determined by measuring the plurytpotency markers (CD133, OCT4 and Nanog by PCR and IFI), colony formation by clonogenic assay and HOESCHT efflux capacity (chemotherapeutic drugs efflux via ABCG2-transporters). In vivo experiments involved male BALB/c mice that were subcutaneously inoculated with murine CRC CT26 cell line. Once the tumor was detected, animals were treated twice a week (days 14, 17, 21, 24 and 28) with an intraperitoneal injection with AP and the sub-effective dose of 5-FURA (5mg/kg). Normal animals, not inoculated with CT26 cells were also used as control for histological analysis.The tumor size was determined twice a week. After 30 days, the mice were sacrificed and necropsied and the histopathology of tumors and the liver sections was performed by staining with hematoxylin and eosin. We found that AP extract decreased significantly the expression level of pluripotency markers, the colony formation and the efflux capacity of chemotherapeutic drugs respect to control cells and increased the cell death induced by 5-FURA treatment respect to cells stimulated with 5-FURA alone (P&amp;lt;0.05; Tukey test). We found that tumor growth was significantly inhibited by AP or AP plus 5-FURA respect to control animals without treatment or treated with 5-FURA alone (p&amp;lt;0.05; Tukey test). Histopathological analysis of tumor sections shown some necrosis area only in animals treated with AP or AP plus 5-FURA while the hepatic parenchyma shown a preserved architecture, confirming non-toxic effects of AP in vivo. Our results demonstrate that AP is capable to induce the cell death of CRC, resulted effective in vivo, non-toxic and has the ability to reduce the CSC side population. Being CSC the most resistant to chemotherapeutic drugs, responsible of cancer progression and perpetuation, the autochthonous plant derivatives could be attractive tools to be investigated as future oncologic therapeutics. Citation Format: Mileni Soares-Machado, Alejandra Palma, Laura Panelo, Leonardo Paz, Francisco Rosa, Cecilia Lira, Pablo Azurmendi, Fernanda Rubio, Guido Lenz, Alejandro Urtreger, Monica Costas. Effect of Aloysia polystachya in cancer stem cells of colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 494.

Involvement of adenosine A1 and A2A receptors on guanosine-mediated anti-tremor effects in reserpinized mice.

Parkinson's disease (PD) signs and symptoms regularly include tremor. Interestingly, the nucleoside guanosine (GUO) has already proven to be effective in reducing reserpine-induced tremulous jaw movements (TJMs) in rodent models, thus becoming a promising antiparkinsonian drug. Here, we aimed at revealing the mechanism behind GUO antiparkinsonian efficacy by assessing the role of adenosine A1 and A2A receptors (A1R and A2AR) on GUO-mediated anti-tremor effects in the reserpinized mouse model of PD. Reserpinized mice showed elevated reactive oxygen species (ROS) production and cellular membrane damage in striatal slices assessed ex vivo and GUO treatment reversed ROS production. Interestingly, while the simultaneous administration of sub-effective doses of GUO (5mg/kg) and SCH58261 (0.01mg/kg), an A2AR antagonist, precluded reserpine-induced TJMs, these were ineffective on reverting ROS production in ex vivo experiments. Importantly, GUO was able to reduce TJM and ROS production in reserpinized mouse lacking the A2AR, thus suggesting an A2AR-independent mechanism of GUO-mediated effects. Conversely, the administration of DPCPX (0.75mg/kg), an A1R antagonist, completely abolished both GUO-mediated anti-tremor effects and blockade of ROS production. Overall, these results indicated that GUO anti-tremor and antioxidant effects in reserpinized mice were A1R dependent but A2AR independent, thus suggesting a differential participation of adenosine receptors in GUO-mediated effects.

Multiple cellular targets involved in the antidepressant-like effect of glutathione

A previous study demonstrated that glutathione (GSH) produces specific antidepressant-like effect in the forced swimming test (FST), a predictive test of antidepressant activity. The present study investigated the involvement of multiple cellular targets implicated in the antidepressant-like effect of GSH in the FST. The antidepressant-like effect of GSH (300 nmol/site, icv) lasted up to 3 h when mice were submitted to FST. The central administration of oxidized GSH (GSSG, 3–300 nmol/site) did not alter the behavior of mice submitted to the FST. Furthermore, the combined treatment of sub-effective doses of GSH (100 nmol/site, icv) with a sub-effective dose of classical antidepressants (fluoxetine 10 mg/kg, and imipramine 5 mg/kg, ip) presented synergistic effect by decreasing the immobility time in the FST. The antidepressant-like effect of GSH was abolished by prazosin (1 mg/kg, ip, α1-adrenoceptor antagonist), baclofen (1 mg/kg, ip, GABAB receptor agonist), bicuculline (1 mg/kg, ip, GABAA receptor antagonist), l-arginine (750 mg/kg, ip, NO precursor), SNAP (25 μg/site, icv, NO donor), but not by yohimbine (1 mg/kg, ip, α2-adrenoceptor antagonist). The NMDA receptor antagonists, MK-801(0.001 mg/kg, ip) or GMP (0.5 mg/kg, ip), potentiated the effect of a sub-effective dose of GSH in the FST. These results suggest that the antidepressant-like effect induced by GSH is connected to the activation of α1 adrenergic and GABAA receptors, as well as the inhibition of GABAB and NMDA receptors and NO biosyntesis. We speculate that redox-mediated signaling on the extracelular portion of cell membrane receptors would be a common mechanism of action of GSH.

Diosgenin via NMDA Receptor Exerted Anxiolytic-like Effect on Maternally Separated Mice.

Anxiety is one of the most common psychiatric disorders that lead to the disruption of daily life and also the quality of life. Routine medications have many side effects and cause physical dependence and psychosocial addiction. Diosgenin is a phytosteroid found in a number of herbs. The present study aimed to investigate the anxiolytic-like effect of diosgenin in the maternal separation model in male mice focusing on the role of NMDA receptors. Maternal separation (MS) paradigm was performed daily (3 h) from postnatal day (PND) 2-14. Male mice were treated with different doses of diosgenin to find effective and sub-effective doses. In the next step, mice were treated with an effective dose of diosgenin plus NMDA and or a sub-effective dose of diosgenin plus ketamine (NMDA antagonist). Valid behavioral tests for the evaluation of anxiety-like behavior were performed. Then, mice were euthanized, the hippocampus was dissected out and gene expression of NMDA receptors (NR2a and NR2b subunits) was assessed. MS provokes anxiety-like behaviors in the open field test (OFT) and elevated plus maze (EPM) test. Diosgenin significantly mitigated the negative effects of MS. Co-administration of NMDA attenuated anxiolyticlike effect of the effective dose of diosgenin, while ketamine potentiated the anxiolytic effect of sub-effective dose of diosgenin. Furthermore, MS increased the expression of the NMDA receptor in the hippocampus which to some extent modulated with diosgenin. Diosignin has an anxiolytic-like effect on MS mice which at least, in part, mediated through NMDA receptors.