Treadmill Exercise Improves Behavioral and Neurobiological Alterations in Restraint-Stressed Rats.

Stress is a state that is known to impact an organism's physiological and psychological balance as well as the morphology and functionality of certain brain areas. In the present work, chronic restraint stress (CRS) model rats treated with treadmill exercise were used to examine anomalies associated to emotion and mood as well as molecular changes in the brain. Forty male Sprague-Dawley rats were divided into control, stress, exercise, and stress+exercise groups. CRS were exposed to stress group rats and exercise group underwent a chronic treadmill exercise. Depressive-like behavior was evaluated with the forced swim test (FST) and tail suspension test (TST). For assessing anxiety-like behavior, the light-dark test (LDT) and the open field test (OFT) were used. The Morris water maze test (MWMT) was used for testing memory and learning. Brain's monoamine level and the expression of genes related to stress were measured. It was discovered that CRS lengthens latency in the MWMT, increases immobility in the FST and TST, decreases time in the light compartment, and causes hypoactivity in the OFT. CRS reduced the dopamine levels in the nucleus accumbens(NAc). Brain-derived neurotrophic factor (BDNF), dopamine receptors, and serotonin receptor (HTR2A) gene expression in the prefrontal cortex, corpus striatum, and hypothalamus were decreased by CRS. Exercise on a treadmill leads to increase NAc's dopamine and noradrenaline levels and prevented behavioral alterations. Exercise increased the alterations of BDNF expressions in the brain in addition to improving behavior. As a result, CRS-induced behavioral impairments were effectively reversed by chronic treadmill exercise with molecular alterations in the brain.

TRPM2 may be involved in high fructose corn syrup-induced anxiety-like behavior in adult male rats.

Excessive high fructose corn syrup (HFCS) consumption is known to cause oxidative stress, which induces transient receptor potential melastatin type 2 (TRPM2) channel gating. Oxidative stress-induced TRPM2 gating is suggested to play an important role in neurons, indicating a role for the TRPM2 channel in a variety of neuropsychiatric disorders including depression and anxiety. We investigated the effects of HFCS and chronic immobilization stress (CIS) on TRPM2 channel immunoreactivity, anxiety, and depressive-like behaviors in adult male rats. The male rats (n=8/group) were divided into 4 groups: Control, 20% HFCS (F20), 40% HFCS (F40), and stress. The control group received tap water, and F20 and F40 groups were exposed to HFCS 20% and 40% respectively for 14 consecutive days. Rats in the stress group were subjected to immobilization stress for 3 or 6 hours daily in the first and second weeks to induce CIS. Then, light/dark tests, open field tests (OFT), and tail suspension tests (TST) were performed, respectively. In the light/dark test, the time spent in the dark chamber significantly increased in all groups vs the control group (P<0.01). In support of this result, time spent in the light chamber significantly decreased in all groups vs the control group (P<0.01). Besides, CIS significantly increased depressive-like behavior in the stress group vs the control group (P<0.05). In serum hormone levels, corticosterone (CORT) levels significantly increased in the F40 and stress groups vs the control group (P<0.01). TRPM2 immunoreactivity significantly increased in the hippocampus, prefrontal cortex (PFC), nucleus accumbens (NaC), and amygdala regions by HFCS and CIS treatments. For the first time in the present study,  showed that f increased immunoreactivity of the TRPM2 cation channels may be linked to the anxiety-like behavior induced by HFCS.

Treadmill exercise has healing effects on obesity-induced sexual behavior disorder through kisspeptin and kiss1R expression in male rats.

The basic objective of this study was to examine the possible effects of treadmill exercise on obesity-related sexual behavior disorder in obese male rats and the role of kisspeptin in this effect. The rats were separated from their mothers at the age of 3 weeks, and classified into four groups as Control (C): normal diet-sedentary group, Exercise (E): normal diet-exercise group, Obese (O): high-fat diet-sedentary group, Obese + Exercise (O+E): high-fat diet-exercise grouSexual behavioral testing was conducted in the rats. At the end of the study, brain samples were taken from the animals for gene expression analyses. The treadmill exercise caused a significant increase in the O+E Group compared to the O Group in kisspeptin and kiss1R gene expression and in EF, ML, IL, MF, IF, III, EL, PEI, IR1, MFT, IFT, IRT sexual behavior parameters (p<0.05), and a significant decrease in ML, IL, III, EL sexual behavior parameters (p<0.05). Treadmill exercise caused a significant decrease in EF, ML, IL, MF, IF, III, EL, PEI, IR1, MFT, IFT, IRT sexual behavior parameters and kisspeptin and kiss1R gene expression in the hypothalamus, hippocampus, prefrontal cortex and corpus striatum in E Group compared to C Group (p<0.05), and a significant increase in ML, IL, III, EL sexual behavior parameters (p<0.05). Based on this effect, we believe that it is caused by an increase in kisspeptin and kiss1R expression in the hypothalamus, hippocampus, prefrontal cortex and corpus striatum. In conclusion, treadmill exercise-induced kisspeptin secretion may increase GnRH secretion and cause hypothalamo-pituitary gonadal axis activation and ameliorative effect on deteriorated sexual function.

The Effects of Chronic Asprosin Administration on Sense of Smell and Sexual Behavior in Female Rats.

INTRODUCTION: Asprosin is an adipokine released from white adipose tissue during fasting and acts through the olfactory receptor. It is known that adipokines play roles in reproductive physiology in mammals. However, there are very few studies conducted on role of asprosin in reproductive functions. There are no studies on its relationship with sexual motivation. It was shown in the literature that administration of asprosin to male mice improves olfaction. It is also known that there is a strong correlation between smell and sexual desire. In view of this, it was hypothesized that chronic administration of asprosin would improve olfactory performance and increase sexual incentive motivation in female rats for male partners. METHODS: This hypothesis was tested by applying the hidden cookie test, sexual incentive test, active research test, and sexual behavior test. The changes in serum hormone levels in female rats that chronically received asprosin were also measured and compared. RESULTS: Chronic asprosin exposure increased olfactory performance, male preference ratio, male investigation preference ratio, activity index, and anogenital investigation behavior. Also, serum oxytocin and estradiol levels increased following chronic administration of asprosin in female rats. CONCLUSION: These data suggest that chronic administration of asprosin can result in increased sexual incentive motivation for opposite sex in female rats over increased olfactory performance and changes in reproductive hormones.

Treadmill exercise training improves the high-fat diet-induced behavioral changes in the male rats.

The purpose of this study was to investigate the effects of treadmill exercise training on obesity-induced behavioral changes in high-fat diet (HFD)-induced male rats. In this study, 40 male Sprague-Dawley rats were divided into 4 groups after they were weaned: Control (C), Exercise (E), Obese (O) and Obese + Exercise (O + E). For the obesity model % 60 high-fat diet were applied. After obesity was induced, rats were either moderate aerobic exercise (treadmill running) trained or left untrained. Different tasks to assess spatial learning and memory (Morris water maze test (MWMT)), depressive-like behavior (forced swimming test(FST), tail suspension test (TST) and anxiety-like behavior (light-dark test (LDT) and open field test (OFT)) were conducted. Exercise caused a significant reduction in duration of immobility in the O group in FST and the decrease in immobility in the O + E rats in TST. The O + E rats demonstrated a significant increase in the time spent in the light box as compared to the O group in the LDT. The O + E rats did not show any behavioral alterations as compared to all the other groups in the OFT. In the O + E group, there was a significant increase in the time spent in the target quadrant compared to the O group in the MWMT. Our results support that treadmill exercise could improve cognitive, depressive-like, anxiety-like behavioral changes in the HFD-induced obese rats.

Crosstalk between kisspeptin and gonadotropin-inhibitory hormone in the silence of puberty: preclinical evidence from a calcium signaling study.

Kisspeptin and gonadotropin-inhibitory hormone (GnIH) are among suggested neuroendocrine modulators of reproductive function. Intracellular calcium signaling is a critical component in the regulation of a variety of physiological and pathological processes including neurotransmitter release, and, therefore, can be used as signaling indicator for investigating the involvement of kisspeptin, GnIH, and gonadotropin-releasing hormone (GnRH) release. Hence, this study investigated the effects of kisspeptin and GnIH on calcium signaling using immortalized hypothalamic cells (rHypoE-8) as a model. Kisspeptin neurons were loaded with the ratiometric calcium dye (Fura-2 AM, 1 µmol) and intracellular free calcium ([Ca2+]i) responses were quantified using digital fluorescence imaging system. Kisspeptin-10 (100, 300, and 1000 nM) caused a significant increase in [Ca2+]i in rHypoE-8 cells (n = 58, n = 64, and n = 49, respectively, p < 0.001). The kisspeptin receptor antagonist, P234, inhibited the calcium responses to kisspeptin (p < 0.001, n = 32). GnIH (100 and 1000 nM), alone, did not cause any significant change in the mean basal [Ca2+]i levels in kisspeptin cells, but GnIH attenuated the kisspeptin-evoked [Ca2+]i transients (n = 47, p < 0.001). This novel findings of [Ca2+]i signaling in in vitro setting implicate that kisspeptin and GnIH may exert their effects on hypothalamus-pituitary-gonadal (HPG) axis by modulating kisspeptin neurons. These results also implicate that kisspeptin neurons may have an autocrine regulation.

Agomelatine modulates calcium signaling through protein kinase C and phospholipase C-mediated mechanisms in rat sensory neurons.

Agomelatine, a novel antidepressant exerting its effects through melatonergic and serotonergic systems, implicated to be effective against pain including neuropathic pain but without any knowledge of mechanism of action. To explore the possible role of agomelatine on nociceptive transmission at the peripheral level, the effects of agomelatine on intracellular calcium ([Ca2+ ]i ) signaling in peripheral neurons were investigated in cultured rat dorsal root ganglion (DRG) neurons. Using the fura-2-based calcium imaging technique, the effects of agomelatine on [Ca2+ ]i and roles of the second messenger-mediated pathways were assessed. Agomelatine caused [Ca2+ ]i signaling in a dose-dependent manner when tested at 10 and 100 µM concentration. Luzindole, a selective melatonin receptor antagonist, almost completely blocked the agomelatine-induced calcium signals. The agomelatine-induced calcium transients were also nearly abolished following pretreatment with the 100 ng/ml pertussis toxin, a Gi/o protein inhibitor. The stimulatory effects of agomelatine on [Ca2+ ]i transients were significantly reduced by applications of phospholipase C (PLC) and protein kinase C (PKC) blockers, 10 µM U73122, and 10 µM chelerythrine chloride, respectively. The obtained results of agomelatine-induced [Ca2+ ]i signals indicates that peripheral mechanisms are involved in analgesic effects of agomelatine. These mechanisms seems to involve G-protein-coupled receptor activation and PLC and PKC mediated mechanisms.

Agomelatine pretreatment prevents development of hyperglycemia and hypoinsulinemia in streptozotocin-induced diabetes in mice.

The main objective of this study was to investigate potential effectiveness of agomelatine pretreatment in the prevention of diabetes itself and encephalopathy, with a focus on brain tissue oxidative stress and inflammatory processes in streptozotocin (STZ)-induced diabetic mice. Interleukine-1ß (IL-1ß) and TACR1 (NK1), which is a tachykinine receptor, were used for the investigation of inflammation in the brain regions including raphe nucleus, periaqueductal gyrus (PAG), amygdala, and nucleus accumbens. The effects of agomelatine on total antioxidant capacity were also evaluated. In the in vitro part of the study, the effects of agomelatine on cell viability were investigated in dorsal root ganglion (DRG) neurons. Fasting blood glucose levels were measured 72 h after STZ injection to determine the diabetic condition. Agomelatine pretreatment prevented both hyperglycemia and hypoinsulinemia in STZ-treated mice. When STZ was injected to induce diabetes in mice, neither hyperglycemia nor hypoinsulinemia was developed in agomelatine pretreated mice and 6 weeks after development of diabetes, agomelatine treatment significantly decreased levels of IL-1ß mRNA in raphe nucleus and nucleus accumbens. TACR1 mRNA levels were lower in raphe nucleus, PAG, and amygdala of agomelatine-treated diabetic mice. The increase in total antioxidant capacity after agomelatine administration may responsible for its beneficial effect in the prevention of diabetes. We showed that agomelatine reversed high glucose-induced cell viability decreases in DRG neurons. Both the antihyperglycemic and antioxidant effects of agomelatine might have contributed to the DRG neuron viability improvement. In conclusion, agomelatine seems to both prevent development of diabetes and reverse the encephalopathic changes caused by diabetes.

The effects of apelin on myometrium contractions in pregnant rats.

Apelin, which is a new hormone, is secreted especially in the brain by hypothalamus as well as by many other organs like the stomach, fat tissue, and the heart. For apelin, whose possible effects on many bodily functions like the endocrine system, cardiovascular system and metabolic activities are still under investigation, the reproductive system is also an important target area. The purpose of the present study was to investigate the effects of plasma apelin levels in rats that were in diestrus, pregnancy and lactation periods, and to examine its possible effects on myometrium contractions of pregnant rats and non-pregnant rats that were in diestrus period.  The plasma apelin concentrations in female adult Wistar rats were determined with the ELISA method in the diestrus period, and on the 12th, 18th, and 21st days of the pregnancy, and on the 2nd and 10th days of lactation (n=7 for each group). In addition, the effect of apelin at 0.01, 0.1, 1 and 10 µM doses on isometric contractions in the rat uterus on the 21st day of pregnancy and in diestrus period was tested by using isolated organ bath. This protocol was repeated under conditions that were pre-treated with protein kinase C inhibitor in calcium-free medium, and the possible effect of apelin on contractions and the mechanisms that might mediate this effect were investigated. When plasma apelin levels were compared with the rats in diestrus period, the apelin concentrations in the 21-day pregnancy group were high at a significant level (p<0.05); and low at a significant level in the 2-day lactation group (p<0.05). In myometrium contraction trials, it was observed that apelin induced the contractions. Apelin increased the frequency of the myometrium contractions at a significant level when applied at 1 µM and 10 µM concentrations (p<0.05 and p<0.001). Only after the apelin application at 10 µM concentration did the amplitude of the contractions increase at a significant level (p<0.01). In the myometrium of the rats that were on the 21st day of pregnancy, the frequency of the contractions was statistically significant at 0.1 µM, 1 µM and 10 µM doses (p<0.01). In addition, the amplitude of the contractions increased at a statistically significant level at 1 µM and 10 µM dose application (p<0.05 and p<0.01, respectively). The apelin application induced the contractions in calcium-free medium. When apelin was applied after the pre-application with protein kinase C inhibitor, no contractions were observed. The present study showed that apelin levels were increased at the end of pregnancy in rats, and the hormone induced the uterus contractions. This effect does not occur with protein kinase C inhibitor and in calcium-free medium, which shows that protein kinase C pathway might play a role in these mechanism.  These findings show that apelin might be an endogenous peptide that plays a role on uterine contractions at birth in rats.

Kisspeptin increases intracellular calcium concentration by protein kinase C-mediated signaling in the primary cultured neurons from rat hippocampus.

In addition to the fact that kisspeptin and its receptor GPR54 are well known to be abundantly expressed in the hypothalamus with suggestive roles in the initiation of puberty and similar reproductive system properties, there is also proof showing that kisspeptin might have influences on hippocampal functions. In our previous study, it was shown that kisspeptin increased free intracellular Ca2+ values ([Ca2+]i) through protein kinase C (PKC) activation in GT1-7 cells. For this reason, we examined the influences of kisspeptin on [Ca2+]i in hippocampal neurons to determine if kisspeptin shows its effects on hippocampus through the same mechanism. Hippocampal neurons were excised from the brains of fetuses on 17th embryonic day from maternal rats. The influences of kisspeptin on [Ca2+]i in hippocampal neurons were examined through in vitro calcium imaging system. The responses of [Ca2+]i to kisspeptin were quantified by the changes in 340nm/380nm ratio.  Kisspeptin-10 caused [Ca2+]i transients in hippocampal neurons. The change in [Ca2+]i by 100 nM kisspeptin was prevented by pre-treating the cells in PKC inhibitor chelerythrine chloride. According to the results, kisspeptin activates intracellular calcium signaling in hippocampal neurons via the pathway that depends on PKC. The results of this study suggest that kisspeptin may have a role in hippocampal neuron functions.

Nesfatin-1 increases intracellular calcium concentration by protein kinase C activation in cultured rat dorsal root ganglion neurons.

Nesfatin-1 is a recently identified anorexigenic hypothalamic polypeptide derived from the posttranslational processing of nucleobindin 2 (NUCB2). Several studies have indicated that this neuropeptide may be participated in somatosensory and visceral transmission including pain signals in addition to energy metabolism. The aim of this study was to explore the possible role of nesfatin-1 in the transmission of peripheral neural signals by investigating the effects of nesfatin-1 on intracellular free calcium levels ([Ca(2+)]i) in cultured neonatal rat dorsal root ganglion (DRG) neurons. The effects of nesfatin-1 on [Ca(2+)]i in DRG neurons were investigated by using an in vitro calcium imaging system. DRG neurons were grown in primary culture following enzymatic and mechanical dissociation of ganglia from 1-or 2-day-old neonatal Wistar rats. Using the fura-2-based calcium imaging technique, the effects of nesfatin-1 on [Ca(2+)]i and role of the protein kinase C (PKC)-mediated pathway in nesfatin-1 effect were assessed. Nesfatin-1 elevated [Ca(2+)]i in cultured DRG neurons. The response was prevented by pretreating the cells with pertussis toxin. The protein kinase C inhibitor chelerythrine chloride suppressed nesfatin-1-induced rise in [Ca(2+)]i. The result shows that nesfatin-1 interacts with a G protein-coupled receptor, leading to an increase of [Ca(2+)]i, which is linked to protein kinase C activation in cultured rat DRG neurons.

Studies on the reproductive effects of chronic treatment with agomelatine in the rat.

Agomelatine is an antidepressant with a novel mechanism of action. It is a melatonergic agonist for MT1 and MT2 receptors and a serotonin (5-HT2C) receptor antagonist. Agomelatine has been suggested not to have adverse effects on sexual functions. However, the effects of chronic agomelatine administration on reproductive functions have not been sufficiently studied in animal models. We mainly aimed to explore the effects of agomelatine on reproductive functions in the male and female rats. For the experimental studies, Sprague Dawley rats were used. The animals started to receive daily oral agomelatine (10mg/kg) on post-natal day 21. Agomelatine advanced vaginal opening in the female rats whereas it delayed puberty onset in the male rats. Agomelatine treatment significantly decreased intromission frequencies, which indicates a facilitator role of this antidepressant on male sexual behavior. In the forced swimming test (FST) used for assessing antidepressant efficacy, agomelatine induced a significant decrease in duration of immobility, and an increase in the swimming time, respectively, which confirms the antidepressant-like activity of agomelatine. The present findings suggest that agomelatine shows a strong antidepressant effect in the male rats without any adverse influences on sexual behavior, and its effects on pubertal maturation seem to show sex-dependent differences.

In vitro effects of sodium nitroprusside and leptin on norepinephrine-induced vasoconstriction in human internal mammary artery.

AIM: The biological and pharmacological properties of vessels used in coronary artery bypass graft (CABG) surgery are as important as their mechanical properties. The aim of this study was to investigate the possible role of protein kinase C (PKC)-dependent mechanisms in leptin-induced relaxation in the human internal mammary artery (IMA). METHODS: IMA rings, obtained from patients undergoing CABG surgery, were suspended in isolated tissue baths containing Krebs-Henseleit solution, which were continuously gassed with 95% O2 and 5% CO2 at 37(°)C. RESULTS: The IMA rings were pre-contracted with increasing concentrations of norepinephrine (NE 10(-9)-10(-4) mol/l) and the relaxation responses to sodium nitroprusside (SNP), a nitrosovasodilator, and leptin were studied in the presence and absence of a PKC inhibitor. Leptin (1 µM) caused a dose-dependent relaxation in NE pre-contracted IMA rings. Pre-treatment with a PKC inhibitor significantly attenuated this vasorelaxatory response to leptin in human isolated IMA. CONCLUSION: It was found that SNP and leptin caused significant relaxation of the NE pre-contracted human IMA rings, and PKC was probably the sub-cellular mediator for this effect. Our findings may have clinical or pharmacological importance as it could be hypothesised that obese subjects who have a left IMA bypass graft would have better myocardial perfusion.

Arg-Phe-amide-related peptides influence gonadotropin-releasing hormone neurons.

The hypothalamic Arg-Phe-amide-related peptides, gonadotropin-inhibitory hormone and orthologous mammalian peptides of Arg-Phe-amide, may be important regulators of the hypothalamus-pituitary-gonadal reproductive axis. These peptides may modulate the effects of kisspeptins because they are presently recognized as the most potent activators of the hypothalamus-pituitary-gonadal axis. However, their effects on gonadotropin-releasing hormone neurons have not been investigated. In the current study, the GT1-7 cell line-expressing gonadotropin-releasing hormone was used as a model to explore the effects of Arg-Pheamide-related peptides on kisspeptin activation. Intracellular calcium concentration was quantified using the calcium-sensitive dye, fura-2 acetoxymethyl ester. Gonadotropin-releasing hormone released into the medium was detected via enzyme-linked immunosorbent assay. Results showed that 100 nmol/L kisspeptin-10 significantly increased gonadotropin-releasing hormone levels (at 120 minutes of exposure) and intracellular calcium concentrations. Co-treatment of kisspeptin with 1 µmol/L gonadotropin-inhibitory hormone or 1 µmol/L Arg-Phe-amide-related peptide-1 significantly attenuated levels of kisspeptin-induced gonadotropin-releasing hormone but did not affect kisspeptin-induced elevations of intracellular calcium concentration. Overall, the results suggest that gonadotropin-inhibitory hormone and Arg-Phe-amide-related peptide-1 may have inhibitory effects on kisspeptin-activated gonadotropin-releasing hormone neurons independent of the calcium signaling pathway.

Melatonin elicits protein kinase C-mediated calcium response in immortalized GT1-7 GnRH neurons.

Melatonin is suggested to have effects on hypothalamic-pituitary-gonadal (HPG) axis. The pulsatile pattern of GnRH release, which results in the intermittent release of gonadotropic hormones from the pituitary, has a critical importance for reproductive function but the factors responsible from this release pattern are not known. Calcium is a second messenger involved in hormone release. Therefore, investigation of the effects of melatonin on intracellular free calcium levels ([Ca(2+)](i)) would provide critical information on hormone release in immortalized GnRH neurons. The pattern of melatonin-induced intracellular calcium signaling was investigated by fluorescence calcium imaging using the immortalized GnRH-secreting GT1-7 hypothalamic neurons. Melatonin caused a significant increase in [Ca(2+)](i,) which was greatly blocked by luzindole, a melatonin antagonist, or attenuated by pre-treatment with protein kinase C inhibitor. This study suggests that melatonin seems to have a direct effect on GnRH neurons.