Subchronic stress-induced depressive behavior in ovariectomized mice

AimsMood disorders including depression are more common in women than men, particularly in times of lower estradiol levels. In this study, we investigated the effect of estrogen on emotional behavior in mice in a stress environment.Main methodsFemale mice were divided into four groups: two groups were ovariectomized (OVX) and two were sham-operated. One group each of OVX and sham mice was kept in a normal environment and the other groups were assigned to a daily stress (1 h/day) for 7 days from 5 days after operation. On the 14th day after operation, subjects were measured to assess behavioral specificity, locomotor activity, elevated plus-maze (EPM) behavior, passive avoidance (PA) behavior and forced swimming behavior.Key findingsThe OVX plus stress (OVX + S) group showed a significant prolongation of immobility compared with the other groups. In all the groups there were no changes in locomotor activity, EPM behavior or PA behavior. We further examined the effect of estrogen against depressive behavior in the OVX + S group. The vehicle or 17β-estradiol (E2) was administered s.c. to OVX + S mice for 4 days beginning on post-operative day 11. Subchronic E2 treatment decreased the stress response and improved depressive behavior relative to the vehicle group.SignificanceThese data have important implications regarding the prevention of depression in postmenopausal women undergoing estrogen therapy.     

Kaempferol from the leaves of Apocynum venetum possesses anxiolytic activities in the elevated plus maze test in mice

The present work evaluated the anxiolytic activity of an aqueous extract of Apocynum venetum L. (Apocynaceae) and bioguided its fractionation using the elevated plus maze (EPM) in mice as a model of anxiety. A single treatment of AV extract markedly increased the percentage time spent on the open arms of the EPM in two distinct concentration ranges of 22.5–30 and 100–125 mg/kg p.o., respectively, indicating a putative anxiolytic-like activity. Fractions showing anxiolytic effects in concentrations equal to 30 or 125 mg/kg of whole extract were antagonized using the benzodiazepine antagonist flumazenil (3 mg/kg i.p.) or the 5-HT_1A receptor antagonist WAY-100635 (0.5 mg/kg i.p.). All active fractions in a concentration equal to 125 mg/kg were effectively blocked by the benzodiazepine antagonist flumazenil, while the anxiolytic activities of fractions in the lower dose equivalent to 30 mg/kg of whole extract were inhibited by the 5-HT_1A receptor antagonist WAY-100635. Through further separation of AV fractions it was possible to isolate and characterize the flavonol kaempferol which showed an anxiolytic-like activity in concentrations from 0.02 to 1.0 mg/kg p.o. The anxiolytic activity of kaempferol was partially antagonized by concomitant administration of flumazenil, but not by WAY-100635. In conclusion, our study clearly demonstrates that AV extract possesses anxiolytic-like activity and that at least one of its flavonoids, kaempferol, can elicit the same kind of neuropharmacological activity.     

Synthesis and pharmacological evaluation of new arylpiperazines N-{4-[4-(aryl) piperazine-1-yl]-phenyl}-amine derivatives: Putative role of 5-HT1A receptors

In an attempt to design novel 5-HT_1A agonists/partial agonists, based on an arylpiperazine nucleus, a series of N-{4-[4-(aryl)piperazine-1-yl]-phenyl}-amine derivatives were synthesized and biologically tested. The anxiolytic effect of the compounds was investigated employing the Elevated plus Maze (EPM) task. On the basis of in vivo functional test, compound 1c (3 mg/kg) and 4c (3 mg/kg) induced significant increments in open arm entries and time on EPM as compared to Buspirone. The anxiolytic effects of compounds 1c and 4c were effectively antagonized by WAY-100635, a 5-HT_1A receptor antagonist (0.5 mg/kg). Furthermore, we have also evaluated the concentration of 5-HT in the brain tissue using HPLC with fluorescent detection. Our result showed that serotonin levels were significantly decreased by ～38% (p < 0.001) and ～32% (p < 0.001) after acute administration of compounds 1c and 4c, respectively. These findings suggest that the anxiolytic like activity of these new arylpiperazines is mediated via 5-HT_1A receptors in the brain.     

The inhibitory effects of corncob bedding on sexual behavior in the ovariectomized Long–Evans rat treated with estradiol benzoate are overcome by male cues

The mechanisms underlying the sensitization of sexual behaviors by repeated administration of estradiol benzoate (EB) to ovariectomized (OVX) rats are not well understood. Here we tested whether two housing conditions play a role. Sexual behavior in the female rat is dependent on the activation of ERα (estrogen receptor alpha) by estradiol. Corncob (CC) bedding has been reported to have adverse effects on the reproductive behavior and physiology of rats, and to disrupt ERα signaling in mice. In addition, some rodent behaviors are stimulated by olfactory stimuli and enhanced in the presence of estradiol. Upon arrival to the facilities OVX Long–Evans rats were housed on either Sani-Chips (SC) or CC in a room that housed only females (F) or males and females (M). Females were first given four sexual training sessions with 10 μg EB + 500 μg progesterone (P; administered 48 h and 4 h prior to training, respectively), followed by a 2-week hormone washout period. Next, 10 μg EB was administered s.c. every 4 days, 48 h prior to each of 8 test sessions in a unilevel 4-hole pacing chamber. On the final training day (i.e., when primed with EB + P), no inhibitory effects of corncob bedding were found, however a facilitation of the lordosis quality occurred in SC/F. Although all groups appear to have sensitized to the repeated administration of EB, CC/F animals displayed fewer high quality lordosis magnitudes and hop/darts, and received fewer mounts and intromissions overall. They also had a lower lordosis quotient (LQ) on tests 2–4 although this effect disappeared by test 5. These results suggest that although CC may inhibit some components of female sexual behavior when primed with EB alone, cues from sexually vigorous males can overcome that inhibition. Moreover, they suggest that male cues can facilitate mechanisms of estradiol sensitization. We recommend that quality control studies be conducted at individual institutions to assess any impact of corncob bedding on animal physiology and behavior.     

Effect of corticosterone on growth and welfare of broiler chickens showing long or short tonic immobility

Tonic immobility (TI) test is commonly used to assess fear. Animals showing different TI durations demonstrate distinct behavior and biochemical responses to stress. However, less is known about how TI phenotype affects growth and welfare of domestic fowl. In this study, broiler chickens (Gallus gallus) were classified into short and long TI duration (STI and LTI) phenotypes and treated chronically with vehicle (CON) or corticosterone (CORT). STI broilers demonstrated significantly higher growth rate with higher breast muscle yield (P < 0.05) and liver weight relative to BW tended to be lower (P = 0.053), which was accompanied by higher serum concentration of CORT (P < 0.05) and uric acid (P < 0.01), but lower serum level of T4 (P = 0.01). CORT severely reduced body weight, as well as the relative weight of muscle, bursa of Fabricius and spleen (P < 0.001), but relative liver weight was increased (P < 0.001). CORT-treated chickens had reduced serum CORT, elevated heterophile/lymphocyte ratio, and increased serum levels of total and free T3. STI broilers displayed more preening behavior (P < 0.05), yet CORT elicited more walking behavior (P < 0.05). No difference was observed in the welfare assessment scores between STI and LTI phenotypes under basal situation, while LTI chickens showed significantly increased incidence of pad dermatitis compared to STI under CORT exposure. The results suggest that STI broilers demonstrate better growth performance and higher adaptability to stress compared to LTI chickens.     

Acute effects of corticosterone injection on paternal behavior in California mouse (Peromyscus californicus) fathers

Glucocorticoids are thought to mediate the disruption of parental behavior in response to acute and chronic stress. Previous research supports their role in chronic stress; however, no study has experimentally tested the effects of acute glucocorticoid elevation on paternal behavior. We tested the prediction that acute corticosterone (CORT) increases would decrease paternal behavior in California mouse fathers and would lead to longer-term effects on reproductive success, as even short-term increases in CORT have been shown to produce lasting effects on the hypothalamic-pituitary-adrenal axis. First-time fathers were injected with 30 mg/kg CORT, 60 mg/kg CORT or vehicle, or left unmanipulated. Interactions between the male and its pup(s) were recorded 1.5–2 h after injection and scored for paternal and non-paternal behavior. Treatment groups were combined into control (unmanipulated + vehicle, n = 15) and CORT (30 mg/kg + 60 mg/kg, n = 16) for analysis based on resulting plasma CORT concentrations. CORT treatment did not alter paternal or non-paternal behaviors or any long-term measures (male body mass or temperature, pup growth rate, pup survival, interbirth interval, number or mass of pups born in the second litter). Fathers showed a significant rise in body mass at day 30 postpartum, followed by a decrease in body mass after the birth of the second litter; however, this pattern did not differ between the CORT and control groups. In summary, acute elevation of plasma CORT did not alter direct paternal behavior, body mass, or reproductive outcomes, suggesting that acute CORT elevation alone does not overtly disrupt paternal care in this biparental mammal.     

Some like it hot: Mouse temperature preferences in laboratory housing

In standard laboratory environments mice are housed at 20–24 °C. However, their thermoneutral zone ranges between 26 °C and 34 °C. This challenge to homeostasis is by definition stressful, and could therefore affect many aspects of physiology and behavior. We tested the hypothesis that mice under standard laboratory conditions are not housed at a preferred temperature, and predicted that this would be evident in thermotaxis and other behavioral responses to ambient cage temperature. We assessed the temperature preferences of C57BL/6J mice in standard laboratory housing from 4 to 11 weeks of age. Forty-eight mice (24 male and 24 female in groups of three) all born on the same day were randomly assigned to one of eight age treatments. One cage of males and one cage of females were tested each consecutive week. Mice were tested in a set of three connected cages with each cage's temperature set using a water bath. On days 1–3 each group of mice was acclimated to each of the three temperatures (20 °C, 25 °C, or 30 °C) in a random order. Then each group was given free access to all temperatures on days 4–6, and video taped continuously. The location of each mouse and the occurrence of three behavioral categories (Active, Inactive, and Maintenance) were recorded by instantaneous scan samples every 10 min over the 3 days, and time budgets calculated. While both sexes chose warmer temperatures overall (P < 0.001), they preferred warmer temperatures only for maintenance and inactive behavior (P < 0.001). This effect was most pronounced in females (P = 0.017). As temperature selection varied with time of day (P < 0.001), these behavioral differences cannot be due to ambient temperature dictating behavior. We conclude that C57BL/6J mice at 20–24 °C are not housed at their preferred temperature for all behaviors or genders, and that it may not be possible to select a single preferred temperature for all mice.     

Antidepressant- and anxiolytic-like activities of an oil extract of propolis in rats

PurposePropolis biological effects are mainly attributed to its polyphenolic constituents such as flavonoids and phenolic acids that were recently described in the chemical composition of an extract of propolis obtained with edible vegetal oil (OEP) by our group. The aim of this study was to evaluate the effect of OEP on the behavior of rats.Materials and methodsAn in vivo open field (OF), elevated Plus-maze (EPM), and forced swimming (FS) tests were performed to evaluate locomotor activity, anxiolytic- and antidepressant effects of the extract. Besides, oxidative stress levels were measured in rat blood samples after the behavioral assays by evaluation of the Trolox equivalent antioxidant capacity (TEAC) and nitric oxide levels.ResultsOEP increased locomotion in the OF test (50 mg/kg) and central locomotion and open arm entries in the OF and EPM tests (10–50 mg/kg) and decreased the immobility time in the FS test (10–50 mg/kg). Moreover, OEP reduced nitric oxide levels in response to swim stress induced in rats.ConclusionOEP exerted stimulant, anxiolytic and antidepressant effects on the Central Nervous System and antioxidant activity in rats, highlighting propolis as a potential therapeutic compound for behavior impairment of anxiety and depression.     

Corticosterone suppresses vasotocin-enhanced clasping behavior in male rough-skinned newts by novel mechanisms interfering with V1a receptor availability and receptor-mediated endocytosis

In rough-skinned newts, Taricha granulosa, exposure to an acute stressor results in the rapid release of corticosterone (CORT), which suppresses the ability of vasotocin (VT) to enhance clasping behavior. CORT also suppresses VT-induced spontaneous activity and sensory responsiveness of clasp-controlling neurons in the rostromedial reticular formation (Rf). The cellular mechanisms underlying this interaction remain unclear. We hypothesized that CORT blocks VT-enhanced clasping by interfering with V1a receptor availability and/or VT-induced endocytosis. We administered a physiologically active fluorescent VT conjugated to Oregon Green (VT–OG) to the fourth ventricle 9 min after an intraperitoneal injection of CORT (0, 10, 40 μg/0.1 mL amphibian Ringers). The brains were collected 30 min post-VT–OG, fixed, and imaged with confocal microscopy. CORT diminished the number of endocytosed vesicles, percent area containing VT–OG, sum intensity of VT–OG, and the amount of VT-V1a within each vesicle; indicating that CORT was interfering with V1a receptor availability and VT-V1a receptor-mediated endocytosis. CORT actions were brain location-specific and season-dependent in a manner that is consistent with the natural and context-dependent expression of clasping behavior. Furthermore, the sensitivity of the Rf to CORT was much higher in animals during the breeding season, arguing for ethologically appropriate seasonal variation in CORT's ability to prevent VT-induced endocytosis. Our data are consistent with the time course and interaction effects of CORT and VT on clasping behavior and neurophysiology. CORT interference with VT-induced endocytosis may be a common mechanism employed by hormones across taxa for mediating rapid context- and season-specific behavioral responses.     

Behaviorally mediated,warm adaptation: A physiological strategy when mice behaviorally thermoregulate

Laboratory mice housed under standard vivarium conditions with an ambient temperature (T_a) of ~22 °C are likely to be cold stressed because this T_a is below their thermoneutral zone (TNZ). Mice raised at T_as within the TNZ adapt to the warmer temperatures, developing smaller internal organs and longer tails compared to mice raised at 22 °C. Since mice prefer T_as equal to their TNZ when housed in a thermocline, we hypothesized that mice reared for long periods (e.g., months) in a thermocline would undergo significant changes in organ development and tail length as a result of their thermoregulatory behavior. Groups of three female BALB/c mice at an age of 37 days were housed together in a thermocline consisting of a 90 cm long aluminum runway with a floor temperature ranging from 23 to 39 °C. Two side-by-side thermoclines allowed for a total of 6 mice to be tested simultaneously. Control mice were tested in isothermal runways maintained at a T_a of 22 °C. All groups were given cotton pads for bedding/nest building. Mass of heart, lung, liver, kidney, brain, and tail length were assessed after 73 days of treatment. Mice in the thermocline and control (isothermal) runways were compared to cage control mice housed 3/cage with bedding under standard vivarium conditions. Mice in the thermocline generally remained in the warm end throughout the daytime with little evidence of nest building, suggesting a state of thermal comfort. Mice in the isothermal runway built elaborate nests and huddled together in the daytime. Mice housed in the thermocline had significantly smaller livers and kidneys and an increase in tail length compared to mice in the isothermal runway as well as when compared to the cage controls. These patterns of organ growth and tail length of mice in the thermocline are akin to warm adaptation. Thus, thermoregulatory behavior altered organ development, a process we term behaviorally mediated, warm adaptation. Moreover, the data suggest that the standard vivarium conditions are likely a cold stress that alters normal organ development relative to mice allowed to select their thermal preferendum.     

Does sex matter? Differential responses to corticosterone administration in the zebra finch

Because of potentially deleterious effects of chronic stress, physiological measurements of stress hormones (in birds, corticosterone (CORT)) are often used to determine the consequences of natural or human-induced change. Often, it is assumed that CORT levels will be similar between the sexes and the results are pooled. However, recent studies have reported sex differences in CORT concentrations in avian species. As zebra finches (Taeniopygia guttata) are one of the most widely used bird species in laboratory studies worldwide, potential sex-specific differences in hormone metabolism, as well as the clearance rate of oral doses of exogenous CORT, are highly relevant. The results of this study show that female zebra finches have a significantly higher baseline CORT than males, which could partially be a product of differential responses to semi-isolation. In addition, a single dose of exogenous CORT resulted in different blood profiles between the sexes over time, though exogenous CORT was cleared from blood within 90 min following treatment in both sexes. Interestingly, exposure to multiple doses of exogenous CORT resulted in elevated CORT levels 24 h after treatment in both sexes. These results highlight the need for further investigations into potential sex differences in hormone metabolism, as well as possible cumulative effects of repeated stress.     

Apigenin 7-glucoside from Stachys tibetica Vatke and its anxiolytic effect in rats

BackgroundStachys tibetica Vatke (Himalayan or mountain tea) grows abundantly in the tropical and subtropical locations of the world including India, Tibet and China. The traditional healers of Kargil and adjoining areas in Ladakh, Jammu and Kashmir in India use the drug to treat fever, cough, phobias and various mental disorders etc. in the form of a decoction or as a tea. Flavonoids are important components in most herbal teas and play an important role in the management of various brain disorders via mimicking the action of benzodiazepines or through benzodiazepine receptors.Aim of the studyThe present study aimed to isolate flavonoids from S. tibetica and to evaluate their anxiolytic potential in comparison to reference synthetic (diazepam) and natural (apigenin) molecules.Materials and methodsS. tibetica root powder was extracted with 95% methanol for about 72 h using a soxhlet apparatus and the resultant extract was subjected to isolation procedures, resulting in the isolation of apigenin 7-glucoside and characterisation by various physical and spectrometric analyses. Apigenin 7-glucoside was evaluated for anxiolytic activity in rats in comparison with the reference compounds diazepam and apigenin using the elevated plus maze (EPM) model.ResultsPhytochemical investigations of S. tibetica revealed the presence of tannins, phenolics, flavonoids, saponins, glycosides and carbohydrates. A flavonoid glucoside, apigenin 7-glucoside was isolated for the first time from the roots of S. tibetica Vatke. The percentage of time spent and arm entries in the open arms was increased while the arms entries and duration of time spent in closed arms were decreased in the groups treated with apigenin 7-glucoside (which dose). In a similar fashion, diazepam and apigenin also exhibited anxiolytic activity (*p < 0.05, **p < 0.01). Apigenin 7-glucoside significantly decreased the percentage of head dips in EPM. Apigenin 7-glucoside showed anxiolytic potential comparable to the reference drugs apigenin and diazepam.ConclusionApigenin 7-glucoside could be an important molecule for the treatment of anxiety and further studies are required to elucidate its possible mechanism of action.     

Growth performance and carcass traits of forage-fed hair sheep wethers

The objective of the present study was to compare live animal performance and carcass characteristics of 3/4 or 7/8 Dorper (DO; n = 30), purebred Katahdin (KA; n = 20), purebred St. Croix (SC; n = 17) and purebred Suffolk (SU; n = 10) lambs born in the spring and fall of 2001. After weaning, lambs were supplemented with up to 680 g of a corn-soybean meal supplement while grazing bermudagrass pastures overseeded with ryegrass. Lambs were slaughtered at approximately 210 d of age. From birth to weaning, DO lambs gained faster (P < 0.001) than KA or SC lambs, whereas KA lambs had higher (P < 0.001) ADG than SC lambs. Additionally, DO and SU wethers had greater (P < 0.02) ADG from weaning to slaughter than SC or KA wethers. Suffolk lambs were heavier (P < 0.001) at slaughter and produced heavier (P < 0.001) carcasses than lambs from hair-sheep breeds. Carcasses of KA lambs were fatter (actual fat thickness; P < 0.02) resulting in higher yield grades (P < 0.03) than carcasses of DO, SC, or SU lambs. Carcasses of DO and SU had larger (P < 0.001) longissimus muscle (LM) areas than those of KA or SC carcasses, whereas kidney fat weight and percentage were greater (P < 0.001) in carcasses from KA and SC than DO and SU lambs. Lean maturity was similar (P = 0.32) among breed-types. However, skeletal maturity was greater (P < 0.001) in SU than hair-sheep carcasses. Flank-streaking scores were similar (P = 0.19) among the breed-types, but conformation scores were higher (P < 0.001) for DO and SU carcasses and resulted in higher (P < 0.001) quality grades than SC carcasses. The LM of SU lambs was lighter (higher L^* values; P < 0.05) than that of KA and SC lambs, whereas the LM from DO lambs was redder (higher a^* values; P < 0.001) than SC and SU and more (P < 0.001) yellow than that of the other breed-types. Chops from SU lambs were tougher (higher shear force values; P < 0.007) than chops from the hair-sheep breeds. Results of this study indicate that ADG, carcass muscularity and meat quality were similar between DO and SU lambs, and, although fatter, carcass muscularity of KA was similar to that of DO lambs.     

Chronic corticosterone treatment enhances extinction-induced depression in aged rats

Withdrawal and avoidance behavior are common symptoms of depression and can appear as a consequence of absence of reward, i.e. extinction-induced depression (EID). This is particularly relevant for the aged organism subjected to pronounced loss of former rewards. Avoidance of the former site of reward and increased withdrawal into a distant compartment accompany extinction of food-rewarded behavior in rodent models. During extinction, behavioral markers for re-learning dissociate from indicators of extinction-induced depression. Here we examined the effect of a chronic treatment with corticosterone (CORT), a well-known inducer of depression-related behavior, on EID in adult and aged rats. Adult (3–4 months) and aged (18 months) male rats were treated with CORT via drinking water for 3 weeks prior to extinction of a cued food-reward task. CORT treatment increased the distance from the site of reward and decreased goal tracking behavior during extinction, especially in the aged rats. Plasma hormone levels measured before and after restraint stress showed a decline in basal ACTH- and CORT-levels after chronic CORT treatment in aged animals. The treatment significantly impaired the HPA-axis activation after acute stress in both, adult and aged animals, alike. Altogether, these findings show an enhancement of EID after chronic CORT treatment in the aged organism, which may be mediated by an impaired HPA-axis sensitivity. These findings may have special relevance for the investigation of human geriatric depression.     

Evaluation of total and non-fatty ether extract in feeds and cattle feces using two analytical methods

The objective of this study was to evaluate ether extract (EE) concentrations, pigments, and wax in forages (n = 14), concentrates (n = 23), and cattle feces (n = 100) using extraction methods recommended by Association of Official Analytical Chemists (AOAC; method 920.39) and American Oil Chemist's Society (AOCS; method Am 5-04). The EE contents were compared by adjusting a linear regression model for each sample type. For the feces and forage samples, the EE contents produced by the AOCS method were greater (P<0.05) compared to those obtained using the AOAC method. No differences between methods were observed in EE content of concentrates (P>0.05). Concentration of vegetable pigments and wax were evaluated by using analysis for variance. Vegetable pigments were lower (P<0.05) in the post-extraction residues using the AOCS method, than the AOAC method, indicating greater participation of vegetable pigments in the EE. No differences were observed between the methods in wax concentration of the post-extraction residues (P>0.05). The quantification method of the EE content that is recommended by AOCS is not suggested for analyses of forage and feces of ruminants because it possibly increases the removal of non-fatty material, mostly pigments, in comparison to the method recommended by AOAC.     

Genetic differences in the elevated plus-maze persist after first exposure of inbred rats to the test apparatus

The elevated plus-maze (EPM) is an anxiety model thought to assess different types of emotional states depending on whether or not the animals have been previously exposed to the test apparatus. Accordingly, benzodiazepine-treated rodents generally differ from controls in the first but not in the second EPM trial. Inbred Lewis and SHR rats of both sexes (N = 10) were submitted twice (test and retest) to the EPM with a 24 h interval between trials. Overall strain differences (Lewis < SHR) were observed in both males and females concerning anxiety-related measures (time spent and percent of entries in the open arms) regardless of previous maze experience. Moreover, prior exposure to the test apparatus produced an overall decrease in the approach towards the open arms in both strains and sexes. The fact that genetic differences did not diminish or disappear in the second trial, suggests that test and retest in the EPM are likely to share some common emotional components and that differences between naïve LEW and SHR rats are not similar to those observed between control and benzodiazepine-treated animals.     

Hyperuricemia influences tryptophan metabolism via inhibition of multidrug resistance protein 4 (MRP4) and breast cancer resistance protein (BCRP)

Hyperuricemia is related to a variety of pathologies, including chronic kidney disease (CKD). However, the pathophysiological mechanisms underlying disease development are not yet fully elucidated. Here, we studied the effect of hyperuricemia on tryptophan metabolism and the potential role herein of two important uric acid efflux transporters, multidrug resistance protein 4 (MRP4) and breast cancer resistance protein (BCRP). Hyperuricemia was induced in mice by treatment with the uricase inhibitor oxonic acid, confirmed by the presence of urate crystals in the urine of treated animals. A transport assay, using membrane vesicles of cells overexpressing the transporters, revealed that uric acid inhibited substrate-specific transport by BCRP at clinically relevant concentrations (calculated IC₅₀ value: 365 ± 13 μM), as was previously reported for MRP4. Moreover, we identified kynurenic acid as a novel substrate for MRP4 and BCRP. This finding was corroborated by increased plasma levels of kynurenic acid observed in Mrp4^?/? (107 ± 19 nM; P = 0.145) and Bcrp^?/? mice (133 ± 10 nM; P = 0.0007) compared to wild type animals (71 ± 11 nM). Hyperuricemia was associated with > 1.5 fold increase in plasma kynurenine levels in all strains. Moreover, hyperuricemia led to elevated plasma kynurenic acid levels (128 ± 13 nM, P = 0.005) in wild type mice but did not further increase kynurenic acid levels in knockout mice. Based on our results, we postulate that elevated uric acid levels hamper MRP4 and BCRP functioning, thereby promoting the retention of other potentially toxic substrates, including kynurenic acid, which could contribute to the development of CKD.     

Cocaine-induced sensitization correlates with testosterone in male Japanese quail but not with estradiol in female Japanese quail

Research has indicated that gonadal hormones may mediate behavioral and biological responses to cocaine. Estrogen, in particular, has been shown to increase behavioral responding to cocaine in female rats relative to male rats. The current study investigated the effect of cocaine on locomotor activity and hormonal correlates in male and female Japanese quail (Coturnix japonica). In Japanese quail, circulating hormone levels can be manipulated without surgical alterations via modifying the photoperiod. Male and female quail were housed on either 8L:16D (light:dark) or 16L:8D (light:dark) cycle for 21 days. Blood samples were taken prior to the beginning of the experiment and assays were performed to determine the levels of testosterone (T) and estradiol (E2). Quail were given injections of saline or cocaine (10 or 20 mg/kg) once a day for 10 days. Immediately after each injection, birds were placed in open field arenas and distance traveled was measured for 30 min. Results showed that male quail housed under long-light conditions exhibited cocaine-induced sensitization to 10 mg/kg cocaine which was correlated with the high levels of plasma T. Female quail housed under short-light conditions demonstrated sensitization to 10 mg/kg cocaine, but this was not correlated with the levels of plasma E2. The current findings suggest that cocaine-induced locomotor activity was associated with T in males but not with E2 in females.     

Orexin A-induced anxiety-like behavior is mediated through GABA-ergic, α- and β-adrenergic neurotransmissions in mice

Orexins are hypothalamic neuropeptides, which are involved in several physiological functions of the central nervous system, including anxiety and stress. Several studies provide biochemical and behavioral evidence about the anxiogenic action of orexin A. However, we have little evidence about the underlying neuromodulation. Therefore, the aim of the present study was to investigate the involvement of neurotransmitters in the orexin A-induced anxiety-like behavior in elevated plus maze (EPM) test in mice. Accordingly, mice were pretreated with a non-selective muscarinic cholinergic antagonist, atropine; a γ-aminobutyric acid subunit A (GABA-A) receptor antagonist, bicuculline; a D2, D3, D4 dopamine receptor antagonist, haloperidol; a non-specific nitric oxide synthase (NOS) inhibitor, nitro-l-arginine; a nonselective α-adrenergic receptor antagonist, phenoxybenzamine and a β-adrenergic receptor antagonist, propranolol 30 min prior to the intracerebroventricular administration of orexin A. The EPM test started 30 min after the i.c.v. injection of the neuropeptide. Our results show that orexin A decreases significantly the time spent in the arms (open/open + closed) and this action is reversed by bicuculline, phenoxybenzamine and propranolol, but not by atropine, haloperidol or nitro-l-arginine. Our results provide evidence for the first time that the orexin A-induced anxiety-like behavior is mediated through GABA-A-ergic, α- and β-adrenergic neurotransmissions, whereas muscarinic cholinergic, dopaminergic and nitrergic neurotransmissions may not be implicated.     

Improved cryopreservation protocol for Blanca-Celtibérica buck semen collected by electroejaculation

The collection of sperm samples by electroejaculation (EE) leads to an increase of the production of seminal plasma which could modify the tolerance of spermatozoa to the cryopreservation procedure. This study aims to compare a standard sperm cryopreservation protocol for samples collected by artificial vagina (AV) with the same protocol and modifications to this for samples obtained by EE. Semen from six males of Blanca-Celtibérica goat breed was collected by AV (control) and EE, and three experiments were conducted. In Experiment 1, it was examined the effects of egg yolk concentration contained in freezing extender (0%, 1.5%, 10% and 20% of egg yolk); in Experiment 2, it was evaluated the cooling rate from 30 to 5 °C (fast: 10 min and slow: 90 min) and the temperature of glycerol addition (30 and 5 °C); and in Experiment 3, it was examined the time of equilibration at 5 °C (0, 1, 2 or 3 h). A heterologous in vitro fertilization test was carried out in order to compare the fertility of control samples with that resulting from the EE protocol which showed the highest sperm quality. Results showed greater sperm motility parameters after thawing for control samples cryopreserved in standard conditions in the three experiments. For samples collected by EE, extender with 20% egg yolk, a slow cooling rate and a longer equilibration time (3 h) provided higher sperm quality, and no differences were observed between temperatures of glycerol addition. Samples collected by EE and cryopreserved with the protocol which yielded the best sperm quality after thawing showed higher fertility compared to AV.