Antidepressant activity of pharmacological and genetic deactivation of the small-conductance calcium-activated potassium channel subtype-3.

RATIONALE: The voltage-insensitive, small-conductance calcium-activated potassium (SK) channel is a key regulator of neuronal depolarization and is implicated in the pathophysiology of depressive disorders. OBJECTIVE: We ascertained whether the SK channel is impaired in the chronic unpredictable stress (CUS) model and whether it can serve as a molecular target of antidepressant action. METHODS: We assessed the depressive-like behavioral phenotype of CUS-exposed rats and performed post-mortem SK channel binding and activity-dependent zif268 mRNA analyses on their brains. To begin an assessment of SK channel subtypes involved, we examined the effects of genetic and pharmacological inhibition of the SK3 channel using conditional knockout mice and selective SK3 channel negative allosteric modulators (NAMs). RESULTS: We found that [125I]apamin binding to SK channels is increased in the prefrontal cortex and decreased in the hippocampus, an effect that was associated with reciprocal levels of zif268 mRNA transcripts indicating abnormal regional cell activity in this model. We found that genetic and pharmacological manipulations significantly decreased immobility in the forced swim test without altering general locomotor activity, a hallmark of antidepressant-like activity. CONCLUSIONS: Taken together, these findings link depression-related neural and behavioral pathophysiology with abnormal SK channel functioning and suggest that this can be reversed by the selective inhibition of SK3 channels.

Antidepressant action of transcranial direct current stimulation in olfactory bulbectomised adolescent rats.

BACKGROUND: Antidepressant drugs in adolescent depression are sometimes mired by efficacy issues and paradoxical effects. Transcranial direct current stimulation (tDCS) could represent an alternative. AIMS/METHODS: We tested the antidepressant action of prefrontal tDCS and paroxetine (20 mg/kg, intraperitoneal) in olfactory bulbectomised (OBX) adolescent rats. Using enzyme-linked immunosorbent assays and in situ hybridisation, we examined treatment-induced changes in plasma brain-derived neurotrophic factor (BDNF) and brain serotonin transporter (SERT) and 5-HT-1A mRNA. RESULTS: OBX-induced anhedonia-like reductions in sucrose preference (SP) correlated with open field (OF) hyperactivity. These were accompanied by decreased zif268 mRNA in the piriform/amygdalopiriform transition area, and increased zif268 mRNA in the hypothalamus. Acute paroxetine (2 days) led to a profound SP reduction, an effect blocked by combined tDCS-paroxetine administration. Chronic (14 days) tDCS attenuated hyperlocomotion and its combination with paroxetine blocked OBX-induced SP reduction. Correlations among BDNF, SP and hyperlocomotion scores were altered by OBX but were normalised by tDCS-paroxetine co-treatment. In the brain, paroxetine increased zif268 mRNA in the hippocampal CA1 subregion and decreased it in the claustrum. This effect was blocked by tDCS co-administration, which also increased zif268 in CA2. tDCS-paroxetine co-treatment had variable effects on 5-HT1A receptors and SERT mRNA. 5-HT1A receptor changes were found exclusively within depression-related parahippocampal/hippocampal subregions, and SERT changes within fear/defensive response-modulating brainstem circuits. CONCLUSION: These findings point towards potential synergistic efficacies of tDCS and paroxetine in the OBX model of adolescent depression via mechanisms associated with altered expression of BDNF, 5-HT1A, SERT and zif268 in discrete corticolimbic areas.

Neonatal Piglets Can Synthesize Adequate Creatine, but Only with Sufficient Dietary Arginine and Methionine, or with Guanidinoacetate and Excess Methionine.

BACKGROUND: Suckling piglets synthesize most of their creatine requirement, which consumes substantial amounts of arginine in order to synthesize guanidinoacetic acid (GAA) and methionine in order to transmethylate GAA to creatine. OBJECTIVES: To determine whether supplemental GAA or creatine spare arginine and/or methionine for protein synthesis and, if GAA is supplemented, whether excess methionine is needed for conversion to creatine. METHODS: Yucatan miniature piglets (9-11 days old; both sexes) were fed 1 of 5 elemental diets for 5 days: 1) low arginine (0.3 g·kg-1·d-1) and low methionine (0.20 g·kg-1·d-1; Base); 2) Base plus GAA (0.093 g·kg-1·d-1; +GAA); 3) Base plus GAA plus excess methionine (0.5 g·kg-1·d-1; +GAA/Met); 4) Base plus creatine (0.12 g·kg-1·d-1; +Cre); or 5) excess arginine (1.8 g·kg-1·d-1) and excess methionine (+Arg/Met). Isotope tracers were infused to determine whole-body GAA, creatine, and protein synthesis; tissues were analyzed for creatine synthesis enzymes and metabolite concentrations. Data were analyzed by 1-way ANOVA. RESULTS: : GAA and creatine syntheses were 115% and 32% higher, respectively, with the +Arg/Met diet (P < 0.0001), in spite of 33% lower renal L-arginine: glycine amidinotransferase activity (P < 0.0001) compared to Base, suggesting substrate availability dictates synthesis rather than enzyme capacity. GAA or creatine supplementation reduced arginine conversion to creatine by 46% and 43%, respectively (P < 0.01), but did not spare amino acids for whole-body protein synthesis, suggesting that limited amino acids were diverted to protein at the expense of creatine synthesis. The +GAA/Met diet led to higher creatine concentrations in the kidney (2.6-fold) and liver (7.6-fold) than the +GAA diet (P < 0.01), suggesting excess methionine is needed for GAA conversion to creatine. CONCLUSIONS: Piglets are capable of synthesizing sufficient creatine from the precursor amino acids arginine and methionine, or from GAA plus methionine.

The Kidneys Are Quantitatively More Important than Pancreas and Gut as a Source of Guanidinoacetic Acid for Hepatic Creatine Synthesis in Sow-Reared Yucatan Miniature Piglets.

BACKGROUND: Arginine:glycine amidinotransferase, necessary for the conversion of arginine (Arg) to guanidinoacetic acid (GAA), is expressed mainly in kidney and pancreas. The methylation of GAA to creatine (Cre) primarily occurs in the liver. The role of the gut in Cre homeostasis has not been characterized. OBJECTIVE: We aimed to quantify the contribution of kidney, pancreas, and gut as sources of GAA for Cre synthesis. METHODS: Sow-reared, feed-deprived Yucatan miniature piglets (17-21 d old) were randomly assigned to acute intravenous treatments (expressed in µmol/kg/min) of: 1) Arg (4.8) + methionine (1.4) (Arg/Met), 2) Cre (0.6) with Arg/Met (Cre/Arg/Met), 3) citrulline (4.8) + methionine (1.4) (Cit/Met), or 4) alanine (6.2) (Ala). Suckling piglets were also studied. RESULTS: Renal GAA release was higher during Cit/Met compared with all other treatments (53-360% higher; P < 0.01), suggesting that Cit is a better precursor than Arg for renal GAA synthesis. Kidneys contributed higher (P < 0.01) proportions of the total GAA with Cit/Met (89%) and Arg/Met (68%) treatments compared with pancreas and gut. In the suckling pigs, kidneys contributed 88% of the GAA, with the remainder released by pancreas. None of the treatments resulted in a net flux of Cre across the kidney or pancreas. In the gut, Arg/Met and Cre/Arg/Met, but not Cit/Met, resulted in a net release of Cre. Cre/Arg/Met resulted in a higher net GAA release from the gut (P < 0.0001) and pancreas (P < 0.001) (68% of total GAA produced) compared with all other treatments (<19% from both organs), perhaps because GAA not needed for creatine synthesis was subsequently released. CONCLUSIONS: Cit is a better precursor than Arg for renal GAA synthesis, and kidney is the major source of GAA for Cre synthesis in neonatal piglets, but the gut also has the capacity to synthesize GAA and Cre when Arg and Met are available.

Creatine supplementation to total parenteral nutrition improves creatine status and supports greater liver and kidney protein synthesis in neonatal piglets.

BackgroundCreatine is not included in commercial pediatric parenteral products; the entire creatine requirement must be met by de novo synthesis from arginine during parenteral nutrition (PN). Poor arginine status is common during PN in neonates, which may compromise creatine accretion. We hypothesized that creatine supplementation will improve creatine status and spare arginine in PN-fed piglets.MethodsPiglets (3-5-day (d) old) were provided PN with or without creatine for 14 d. Tissue concentrations of creatine metabolites and activities of creatine-synthesizing enzymes, as well as tissue protein synthesis rates and liver lipid parameters, were measured.ResultsCreatine provision lowered kidney and pancreas L-arginine:glycine amidinotransferase (AGAT, EC number 2.1.4.1) activities and plasma guanidinoacetic acid (GAA) concentration, suggesting the downregulation of de novo creatine synthesis. Creatine increased plasma creatine concentrations to sow-fed reference levels and increased the creatine concentrations in most tissues, but not in the brain. PN creatine resulted in greater protein synthesis in the liver and the kidney, but not in the pancreas, skeletal muscle, or gut. Creatine supplementation also reduced liver cholesterol concentrations, but not triglyceride or total fat.ConclusionThe addition of creatine to PN may optimize the accretion of creatine and reduce the metabolic burden of creatine synthesis in rapidly growing neonates.

Enteral arginine partially ameliorates parenteral nutrition-induced small intestinal atrophy and stimulates hepatic protein synthesis in neonatal piglets.

BACKGROUND: Arginine is an indispensable amino acid in neonates; de novo synthesis of arginine occurs in the small intestine (SI) but is reduced during parenteral nutrition (PN), limiting the arginine available to the mucosa. We assessed the effects of route of intake and dietary concentration of arginine on protein synthesis, superior mesenteric artery (SMA) blood flow, and SI morphology. METHODS: Piglets (n = 18, 14-17 days old) were given complete PN for 3 days to induce SI atrophy, then switched to 1 of 3 treatments: arginine-free PN plus an intragastric (IG) infusion of high arginine (1.6 g · kg(-1)· d(-1), IG-H Arg) or low arginine (0.6 g · kg(-1)· d(-1), IG-L Arg) or complete high-arginine PN (1.6 g · kg(-1)· d(-1), IV-H Arg). RESULTS: Enteral arginine, irrespective of amount provided, stimulated hepatic protein synthesis compared with intravenous delivery of arginine (P = .01). SMA blood flow declined for all groups following the initiation of PN. After 48 hours on the test diets, all groups reached low constant levels, but the IV-H group was significantly higher than both IG groups (P < .05). Despite greater blood flow, the SI morphological characteristics in IV-H Arg pigs were not significantly improved over the other groups. IV-H Arg pigs had higher plasma concentrations of indispensable amino acids (tyrosine, isoleucine, and valine) compared with IG-H Arg, despite identical amino acid intakes. CONCLUSIONS: Intravenous delivery of arginine sustained the best SMA blood flow, whereas even a moderate amount of enteral arginine stimulated liver protein synthesis and maintained SI growth, independent of blood flow.