Are the promnestic effects of neurokinin 3 receptor mediated by hippocampal neurogenesis in a Aß-induced rat model of Alzheimer's disease?

Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterised by cognitive dysfunction, memory loss and mood changes. Hippocampal neurogenesis has been suggested to play a role in learning and memory. Neurokinin 3 receptor (NK3R) has been shown to be prevalent in the hippocampus region. The aim of the project was to investigate the role of hippocampal neurogenesis in the promnestic effects of NK3R agonist administration in an amyloid beta-induced AD rat model. Wistar albino rats were divided into control, Alzheimer, NK3R agonist and Alzheimer + NK3R agonist groups. The open field (OF) test and Morris water maze (MWM) test were performed for locomotor activity and memory analysis. Peptide gene expression levels (Nestin, DCX, Neuritin, MASH1, Neun, BDNF) were analysed by quantitative reverse transcription polymerase chain reaction (RT-PCR). In the OF test, the group-time relationship was found to be statistically different in the parameters of distance travelled and percentage of movement (p < 0.05). In MWM, the time to reach the platform and the time spent in the target quadrant were statistically significant between the groups (p < 0.05). Statistically significant differences were observed in gene expression levels (Nestin, DCX, Neuritin, MASH1) in the hippocampal tissue of rats between the groups (p < 0.05). NK3 receptor agonism favourably affected hippocampal neurogenesis in AD model rats. It was concluded that NK3 receptor agonism in the hippocampus, which is the first affected region in the physiopathology of AD, may be effective in both the formation of neural precursor cells and the reduction of neuronal degeneration. The positive effect of NK3R on cognitive functions may be mediated by hippocampal neurogenesis.

Intracerebroventricular injection of kisspeptin in male rats activates hypothalamo-pituitary-gonadal axis, but not hypothalamo-pituitary-adrenal axis.

Kisspeptin is an important hormone involved in the stimulation of the hypothalamo-pituitary gonadal (HPG) axis. The HPG axis can be suppressed in certain conditions such as stress, which gives rise to the activation of the hypothalamo-pituitary-adrenal (HPA) axis. However, the physiological role of kisspeptin in the interaction of HPG and HPA axis is not fully understood yet. This study was conducted to investigate the possible effects of central kisspeptin injection on HPG axis as well as HPA axis activity. Adult male Wistar rats were randomly divided into seven groups as followed: sham (control), kisspeptin (50 pmol), P234 (1 nmol), kisspeptin + p234, kisspeptin + antalarmin (0.1 µg), kisspeptin + astressin 2B (1 µg), and kisspeptin + atosiban (300 ng/rat) (n = 10 each group). At the end of the experiments, the hypothalamus, pituitary, and serum samples of the rats were collected. There was no significant difference in corticotropic-releasing hormone immunoreactivity in the paraventricular nucleus of the hypothalamus, serum adrenocorticotropic hormone, and corticosterone levels among all groups. Moreover, no significant difference was detected in pituitary oxytocin level. Serum follicle-stimulating hormone and luteinizing hormone levels of the kisspeptin, kisspeptin + antalarmin, and kisspeptin + astressin 2B groups were significantly higher than the control group. Serum testosterone levels were significantly higher in the kisspeptin kisspeptin + antalarmin, kisspeptin + astressin 2B, and kisspeptin + atosiban groups compared to the control group. Our findings suggest that central kisspeptin injection causes activation in the HPG axis, but not the HPA axis in male rats.

How does neurokinin 3 receptor agonism affect pathological and cognitive impairments in an Alzheimer's disease-like rat model?

Alzheimer's disease (AD) is accepted as a form of progressive dementia. Cholinergic systems are commonly affected in AD. Neurokinin 3 receptor (NK3R) is involved in learning memory-related processes. It is known that the activation of NK3R affects the release of many neurotransmitters. The aim of this project was to investigate the effects of NK3R agonist senktide administration on neurobehavioral mechanisms in the experimental AD-like rat model. 50 male Wistar albino rats were divided into Control (C), AD, Control + NK3R agonist (CS), AD + NK3R agonist (ADS), AD + NK3Ragonist + antagonist groups (ADSO). We designed AD-like model by intrahippocampal administration of Aß1-42. After NK3R agonist + antagonist injections, open field (OF), Morris water maze (MWM) tests were applied. Cholinergic mechanism analysis from hippocampus-cortex tissues was performed by ELISA and catecholamine analysis from brain stem tissue were performed by HPLC method. The transitions from edge to center, rearing, grooming parameters were found to be reduced in final values of OF. While the group-time interaction was significant in the OF test findings, there was no significant difference between the groups. In MWM test, ADS group showed a learning level close to control group and animals in AD and ADSO groups could not learn target quadrant in MWM test. The brain stem NA and DA concentrations were not statistically significant. Hippocampal AChE-ChAT levels were supported by positive effects of senktide on learning via the cholinergic mechanisms. As a result, NK3R agonists were found to be effective in improving cognitive functions in rats with AD pathology. In the experimental AD model, positive effects of NK3R on learning memory may be mediated by cholinergic mechanisms.

Percentages of serum, liver and adipose tissue fatty acids and body weight are affected in female rats by long-term Central kisspeptin treatments.

This study was conducted to determine the possible effects of long-term exogenous kisspeptin and its antagonist P234 on serum, liver and adipose tissue fatty acids (FA) profiles, as well as body weight, in female rats. Kisspeptin (50 pmol) and P234 (1 nmol) were administrated to the weaned Sprague-Dawley female rats by an intracerebroventricular injection from the 26th postnatal day to the 60th postnatal day. Percentages of the serum total saturated FA (∑SFA) and total monounsaturated FA (∑MUFA) were lower in the kisspeptin group. In the adipose tissue, ∑SFA was lower and total unsaturated FA higher in the P234 group. Moreover, long-term central kisspeptin injection caused a decrease in the body weight. When compared to the kisspeptin group, the final body weights were higher in the P234 and kisspeptin + P234 groups. According to our results, we suggest that kisspeptin has a regulatory role in FA metabolism and regulation of body weight.

What is the protective effect of preischemic kisspeptin-10 administration against ischemia/reperfusion injury of striatum on mice?

BACKGROUND: Kisspeptin is a neuropeptide with a primary role on the onset of puberty and has beneficial effects on ischemia/ reperfusion (I/R) injury. In this study, we aimed to investigate the effect of kisspeptin administration on striatal I/R injury in mice. METHODS: Forty adult C57/BL6 mice were randomly divided into four groups: Sham, Kisspeptin, I/R, and I/R + Kisspeptin groups. The groups were administered with either physiological saline (Sham and I/R groups) or kisspeptin (Kisspeptin and I/R + Kisspeptin groups) intraperitoneally 40 min before the operation. A microdialysis probe was placed in the right striatum according to stereotaxic coordinates. During the experimental period, artificial cerebrospinal fluid was passed through the micropump. Then, transient cerebral ischemia was established by compressing both common carotid arteries with an aneurysm clip for 15 min and animals were reperfused for 2 h. Throughout the process of microdialysis (before, during and after I/R period), samples were collected to measure dopamine (DA), noradrenaline (NA), and 3,4-dihydroxyphenylglycine (DHPG) at intervals of 20 min continuously. At the end of the reperfusion period, the animals were decapitated, striatum was dissected, half of the animals were used for oxidative stress analyses (reduced glutathione, glutathione S-transferase (GST), superoxide dismutase (SOD), malondialdehyde (MDA), and the other half were used for histopathology analyses. RESULTS: Number of glial cells was significantly increased in kisspeptin-administered groups. DA levels in ischemic animals were decreased by kisspeptin administration (p < 0.0001). NA levels were reduced in animals administered with kisspeptin without I/R injury (p < 0.05). DHPG levels reduced during the reperfusion period in ischemic animals (p < 0.05). Kisspeptin did not exhibit a significant antioxidant activity in the ischemic animals, while GST and SOD levels were reduced in the I/R + kisspeptin group compared to the kisspeptin group (p < 0.05). DISCUSSION: Our results suggest that kisspeptin may be regulating the neurotransmitter release and metabolism, as well as inflammatory response in brain upon I/R injury.

Does Sertraline Affect Hypothalamic Food Intake Peptides in the Rat Experimental Model of Chronic Mild Stress-Induced Depression?

Depression is a chronic, recurrent and life-threatening disease affecting approximately 15% of the world population. Depression is responsible for neuropathologies like decreased neurogenesis and increased dendritic atrophy. Antidepressant treatments increase hippocampal neurogenesis and neurotrophic factor expression. Based on this information, it was aimed to investigate effect of sertraline on depression in rats with chronic mild stress (CMS) model and to determine how it affects cell proliferation and hypothalamic peptide levels in hypothalamus. 56 adult male Wistar albino; control, depression(D), depression + sertraline, sertraline were divided into groups. Various stressors were applied to D for 30 days. Open field test (OFT) and forced swimming test (FST) were conducted to check whether the animals were depressed. On the 16th day osmotic minipump was placed subcutaneously and sertraline (10 mg/kg/day) was administered for 15 days. Behavior tests were done. Hypothalamic peptide gene expression levels were analyzed by quantitative RT-PCR. Statistical evaluations were made using ANOVA. It caused a decrease in the percentage of movement in the D and control groups in the OFT, an increase in the immobility time in the D group in the FST, and an increase in the swimming behavior in the DS group. Animals did not show any anxiological behavior based on the elevated plus maze test results. CMS caused a decrease in GLUT2 and NPY gene expression in the hypothalamus of animals, an increase in POMC and FGFR2, and an increase in IGFIR and GLUT2 gene expression in the DS group. Sertraline has been shown to ameliorate the effects of CMS-induced depression. Sertraline is thought to have a positive regulatory effect on both the formation of neural precursor cells and the survival of newly formed neurons in the hypothalamus. Newly formed neurons in the hypothalamus express food intake-related NPY, POMC, GLUT2 neurons, and thus hypothalamic tanycytes may play a key role in the control of energy metabolism.

Chronic immobilization stress induces anxiety-related behaviors and affects brain essential minerals in male rats.

Alterations of essential elements in the brain are associated with the pathophysiology of many neuropsychiatric disorders. It is known that chronic/overwhelming stress may cause some anxiety and/or depression. We aimed to investigate the effects of two different chronic immobilization stress protocols on anxiety-related behaviors and brain minerals. Adult male Wistar rats were divided into 3 groups as follows (n = 10/group): control, immobilization stress-1 (45 minutes daily for 7-day) and immobilization stress-2 (45 minutes twice a day for 7-day). Stress-related behaviors were evaluated by open field test and forced swimming test. In the immobilization stress-1 and immobilization stress-2 groups, percentage of time spent in the central area (6.38 ± 0.41% and 6.28 ± 1.03% respectively, p < 0.05) and rearing frequency (2.75 ± 0.41 and 3.85 ± 0.46, p < 0.01 and p < 0.05, respectively) were lower, latency to center area (49.11 ± 5.87 s and 44.92 ± 8.04 s, p < 0.01 and p < 0.01, respectively), were higher than the control group (8.65 ± 0.49%, 5.37 ± 0.44 and 15.3 ± 3.32 s, respectively). In the immobilization stress-1 group, zinc (12.65 ± 0.1 ppm, p < 0.001), magnesium (170.4 ± 1.7 ppm, p < 0.005) and phosphate (2.76 ± 0.1 ppm, p < 0.05) levels were lower than the control group (13.87 ± 0.16 ppm, 179.31 ± 1.87 ppm and 3.11 ± 0.06 ppm, respectively). In the immobilization stress-2 group, magnesium (171.56 ± 1.87 ppm, p < 0.05), phosphate (2.44 ± 0.07 ppm, p < 0.001) levels were lower, and manganese (373.68 ± 5.76 ppb, p < 0.001) and copper (2.79 ± 0.15 ppm, p < 0.05) levels were higher than the control group (179.31 ± 1.87 ppm, 3.11 ± 0.06 ppm, 327.25 ± 8.35 ppb and 2.45 ± 0.05 ppm, respectively). Our results indicated that 7-day chronic immobilization stress increased anxiety-related behaviors in both stress groups. Zinc, magnesium, phosphate, copper and manganese levels were affected in the brain.

Gonadotropin levels reduced in seven days immobilization stress-induced depressive-like behavior in female rats.

OBJECTIVES: Reproduction is one of the physiological functions that are often negatively affected by chronic stress. We aimed to examine effects of two distinct 7-day chronic immobilization stress (IMO) models on gonadotropins levels and depression-like behaviors in female rats. METHODS: Adult Wistar albino female rats were divided into three groups as follows (n=7 for each group): control, IMO-1 (45 min daily for 7-day) and IMO-2 (45 min twice a day for 7-day). Neuropsychiatric behaviors were determined by using forced swimming test (FST) and open field test (OFT). Gonadotropins were analyzed using ELISA tests. RESULTS: In FST, swimming was lower, and immobility was higher in the IMO-1 group and IMO--2 group. Climbing score of the IMO-2 group was higher compared to the control group. In OFT, there was no significant alteration in the mean velocity, total distance, duration of time spent in the central area and duration of latency in the central area between the stress groups and the control group. Final body weight and percentage of body weight change were lower in both stress groups. The follicle-stimulating hormone level was lower only in the IMO-2 group, and the luteinizing hormone concentrations were significantly lower in the IMO-1 group and IMO-2 group. CONCLUSIONS: Our results indicated that depression-like behaviors increased, and gonadotropins decreased in the female rats exposed to 7-day chronic IMO.

Normobaric oxygen treatment improves neuronal survival functional recovery and axonal plasticity after newborn hypoxia-ischemia.

BACKGROUND: Newborn hypoxia ischemia (HI) is one of the most prevalent cases in the emergency and can result from fetal hypoxia during delivery. In HI, restricted blood supply to the fetal brain may cause epilepsy or mental disorders. METHODS: In the present study, seven-day-old pups were subjected HI and treated with different normobaric oxygen (NBO) concentrations (21%, 70% or 100%). In the acute phase, we analyzed infarct area, disseminate neuronal injury and surviving neurons. In addition, we studied the regulation of PTEN and MMP-9 proteins which were suggested to be activated by HI in the ischemic tissue. Moreover, long-term effects of NBO treatments were evaluated with open field, rotarod and Barnes maze tests. We also examined axonal plasticity with EGFP-AAV injection. RESULTS: Here, we demonstrate that hyperoxic NBO concentration causes an increase in cellular survival and a decrease in the number of apoptotic cells, meanwhile inhibiting the proteins involved in cellular death mechanisms. Moreover, we found that hyperoxia decreases anxiety, promotes motor coordination and improve spatial learning and memory. Notably that axonal sprouting was promoted by hyperoxia. CONCLUSION: Our data suggest that NBO is a promising approach for the treatment of newborn HI, which encourage proof-of-concept studies in newborn.

Acute and Post-acute Neuromodulation Induces Stroke Recovery by Promoting Survival Signaling, Neurogenesis, and Pyramidal Tract Plasticity.

Repetitive transcranial magnetic stimulation (rTMS) has gained interest as a non-invasive treatment for stroke based on the data promoting its effects on functional recovery. However, the exact action mechanisms by which the rTMS exert beneficial effects in cellular and molecular aspect are largely unknown. To elucidate the effects of high- and low-frequency rTMS in the acute-ischemic brain, we examined how rTMS influences injury development, cerebral blood flow (CBF), DNA fragmentation, neuronal survival, pro- and anti-apoptotic protein activations after 30 and 90 min of focal cerebral ischemia. In addition, inflammation, angiogenesis, growth factors and axonal outgrowth related gene expressions, were analyzed. Furthermore, we have investigated the effects of rTMS on post-acute ischemic brain, particularly on spontaneous locomotor activity, perilesional tissue remodeling, axonal sprouting of corticobulbar tracts, glial scar formation and cell proliferation, in which rTMS was applied starting 3 days after the stroke onset for 28 days. In the high-frequency rTMS received animals reduced DNA fragmentation, infarct volume and improved CBF were observed, which were associated with increased Bcl-xL activity and reduced Bax, caspase-1, and caspase-3 activations. Moreover, increased angiogenesis, growth factors; and reduced inflammation and axonal sprouting related gene expressions were observed. These results correlated with reduced microglial activation, neuronal degeneration, glial scar formation and improved functional recovery, tissue remodeling, contralesional pyramidal tract plasticity and neurogenesis in the subacute rTMS treated animals. Overall, we propose that high-frequency rTMS in stroke patients can be used to promote functional recovery by inducing the endogenous repair and recovery mechanisms of the brain.

Long-term metabolic cage housing increases anxiety/depression-related behaviours in adult male rats.

There are several reports on unfavourable effects of metabolic cage housing on animal welfare mainly due to the characteristic structures of these cages such as single housing and grid flooring. This study was aimed to compare the effects of long-term metabolic cage housing and conventional housing (normal grouped housing in standard cages) on the anxiety/depression-like behaviours in male rats. Anxiety/depression-related behaviours were evaluated by use of forced swimming test and open field test. Swimming and climbing were significantly lower and immobility duration higher in the metabolic cage group. In the open field test, total distance, mean velocity, time spent in the central area, zone transition, grooming, and rearing scores were significantly lower in the metabolic cage. Moreover, serum corticosterone level was higher in the metabolic cage group. The results of the study indicate that long-term metabolic cage housing may cause an increase in the anxiety- and depression-related behaviours in male rats.

Pinealectomy alters IFN-γ and IL-10 levels in primary thymocyte culture of rats.

Melatonin, produced mainly by the pineal gland, has an immunomodulatory role. However, the effects of the pineal gland and/or melatonin on thymus cytokine levels such as interferon-gamma (IFN-γ), interleukin (IL)-4, and IL-10 are not well known. Twenty-one male Wistar rats (220-250 gr) were randomly divided into three groups (n=7): intact control, sham, and pinealectomy. Primary thymocyte cultures were prepared from each group and dispensed into well plates as Control, DMSO (or vehicle), Sham-pinealectomy, Pinealectomy, Pinealectomy+10µM melatonin, and Pinealectomy+100µM melatonin. IFN-γ, IL-4, and IL-10 concentrations were measured in the thymocytes (as nonstimulated and Concanavalin A-stimulated) after 24 h. IFN-γ levels significantly increased and IL-10 levels significantly decreased in both media prepared from pinealectomized rats. There was no significant difference between the groups in terms of IL-4. In the pinealectomy+10µM melatonin group, IFN-γ and IL-10 levels did not differ from the pinealectomy group. However, the dose of 100µM melatonin caused a decrease in levels of IFN-γ in both thymocyte media and an increase in the concentration of IL-10 in Concanavalin A-stimulated thymocytes. In conclusion, pineal gland and/or melatonin affect IFN-γ and IL-10 levels in the thymus gland.

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.

Time-of-Day Dependent Neuronal Injury After Ischemic Stroke: Implication of Circadian Clock Transcriptional Factor Bmal1 and Survival Kinase AKT.

Occurrence of stroke cases displays a time-of-day variation in human. However, the mechanism linking circadian rhythm to the internal response mechanisms against pathophysiological events after ischemic stroke remained largely unknown. To this end, temporal changes in the susceptibility to ischemia/reperfusion (I/R) injury were investigated in mice in which the ischemic stroke induced at four different Zeitgeber time points with 6-h intervals (ZT0, ZT6, ZT12, and ZT18). Besides infarct volume and brain swelling, neuronal survival, apoptosis, ischemia, and circadian rhythm related proteins were examined using immunohistochemistry, Western blot, planar surface immune assay, and liquid chromatography-mass spectrometry tools. Here, we present evidence that midnight (ZT18; 24:00) I/R injury in mice resulted in significantly improved infarct volume, brain swelling, neurological deficit score, neuronal survival, and decreased apoptotic cell death compared with ischemia induced at other time points, which were associated with increased expressions of circadian proteins Bmal1, PerI, and Clock proteins and survival kinases AKT and Erk-1/2. Moreover, ribosomal protein S6, mTOR, and Bad were also significantly increased, while the levels of PRAS40, negative regulator of AKT and mTOR, and phosphorylated p53 were decreased at this time point compared to ZT0 (06:00). Furthermore, detailed proteomic analysis revealed significantly decreased CSKP, HBB-1/2, and HBA levels, while increased GNAZ, NEGR1, IMPCT, and PDE1B at midnight as compared with early morning. Our results indicate that nighttime I/R injury results in less severe neuronal damage, with increased neuronal survival, increased levels of survival kinases and circadian clock proteins, and also alters the circadian-related proteins.

Oxytocin activates calcium signaling in rat sensory neurons through a protein kinase C-dependent mechanism

In addition to its well-known effects on parturition and lactation, oxytocin (OT) plays an important role in modulation of pain and nociceptive transmission. But, the mechanism of this effect is unclear. To address the possible role of OT on pain modulation at the peripheral level, the effects of OT on intracellular calcium levels ([Ca2+]i) in rat dorsal root ganglion (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 OT on [Ca2+]i and role of the protein kinase C (PKC)-mediated pathway in OT effect were assessed. OT caused a significant increase in basal levels of [Ca2+]i after application at the doses of 30 nM (n = 34, p < 0.01), 100 nM (n = 41, p < 0.001) and 300 nM (n = 46, p < 0.001). The stimulatory effect of OT (300 nM) on [Ca2+]i was persistent in Ca2+-free conditions (n = 56, p < 0.01). Chelerythrine chloride, a PKC inhibitor, significantly reduced the OT-induced increase in [Ca2+]i (n = 28, p < 0.001). We demonstrated that OT activates intracellular calcium signaling in cultured rat primary sensory neurons in a dose- and PKC-dependent mechanism. The finding of the role of OT in peripheral pain modification may serve as a novel target for the development of new pharmacological strategies for the management of pain

Oxytocin activates calcium signaling in rat sensory neurons through a protein kinase C-dependent mechanism.

In addition to its well-known effects on parturition and lactation, oxytocin (OT) plays an important role in modulation of pain and nociceptive transmission. But, the mechanism of this effect is unclear. To address the possible role of OT on pain modulation at the peripheral level, the effects of OT on intracellular calcium levels ([Ca(2+)](i)) in rat dorsal root ganglion (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 OT on [Ca(2+)](i) and role of the protein kinase C (PKC)-mediated pathway in OT effect were assessed. OT caused a significant increase in basal levels of [Ca(2+)](i) after application at the doses of 30 nM (n = 34, p < 0.01), 100 nM (n = 41, p < 0.001) and 300 nM (n = 46, p < 0.001). The stimulatory effect of OT (300 nM) on [Ca(2+)](i) was persistent in Ca(2+)-free conditions (n = 56, p < 0.01). Chelerythrine chloride, a PKC inhibitor, significantly reduced the OT-induced increase in [Ca(2+)](i) (n = 28, p < 0.001). We demonstrated that OT activates intracellular calcium signaling in cultured rat primary sensory neurons in a dose- and PKC-dependent mechanism. The finding of the role of OT in peripheral pain modification may serve as a novel target for the development of new pharmacological strategies for the management of pain.

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.

Effects of levobupivacaine and bupivacaine on intracellular calcium signaling in cultured rat dorsal root ganglion neurons.

Bupivacaine and levobupivacaine have been shown to be effective in the treatment of pain as local anesthetics, although the mechanisms mediating their antinociceptive actions are still not well understood. The aim of this study was to investigate the effects of bupivacaine and levobupivacaine on intracellular calcium ([Ca(2+)](i)) signaling in cultured rat dorsal root ganglion (DRG) neurons. DRG neuronal cultures loaded with 5 microM Fura-2/AM and [Ca(2+)](i) transients for stimulation with 30 mM KCl (Hi K(+)) were assessed by using fluorescent ratiometry. DRGs were excited at 340 and 380 nm, emission was recorded at 510 nm, and responses were determined from the change in the 340/380 ratio (basal-peak) for individual DRG neurons. Data were analyzed by using Student's t-test. Levobupivacaine and bupivacaine attenuated the KCl-evoked [Ca(2+)](i) transients in a reversible manner. [Ca(2+)](i) increase evoked by Hi K(+) was significantly reduced to 99.9 +/- 5.1% (n = 18) and 62.5 +/- 4.2% (n = 15, P < 0.05) after the application of 5 and 50 microM levobupivacaine, respectively. Bupivacaine also inhibited Hi K(+)-induced [Ca(2+)](i) responses, reduced to 98.7 +/- 4.8% (n = 10) and 69.5 +/- 4.5% (n = 9, P < 0.05) inhibition of fluorescence ratio values of Hi K(+)-induced responses at 5 and 50 microM, respectively. Our results indicate that bupivacaine and levobupivacaine, with no significant differences between both agents, attenuated KCl-evoked calcium transients in a reversible manner. The inhibition of calcium signals in DRG neurons by levobupivacaine and bupivacaine might contribute to the antinociceptive effects of these local anesthetics.

Leptin modulates noradrenaline release in the paraventricular nucleus and plasma oxytocin levels in female rats: a microdialysis study.

The neural control and mutual interrelationships among individual factors involved in the regulation of food intake and simultaneously related to reproduction are far from being understood. We have suggested that at least some of the effects of orexigenic and anorexigenic peptides might be mediated via noradrenaline release in the paraventricular nucleus (PVN). The main hypothesis was that leptin has an inhibitory action on oxytocin secretion and hypothalamic release of noradrenaline. Non-pregnant female rats in their diestrus were subjected to cannulation of the carotid artery and a microdialysis procedure with the probes in the hypothalamic PVN. Intra-arterial administration of cholecystokinin-8 (CCK) at the dose of 50 mg/kg was used to induce oxytocin and noradrenaline release. Leptin (10 mg/5 ml) was intracerebroventricularly injected in addition to CCK. Blood and microdialysis samples were collected at 20-min intervals for 80 min. Central administration of leptin significantly reduced both plasma oxytocin and hypothalamic noradrenaline responses to CCK at 20 min following the treatments. In conclusion, leptin may inhibit oxytocin secretion by lowering noradrenergic neurotransmission in the PVN. The modulator effect of leptin on noradrenaline release in the PVN may be related to feeding behavior.

Combined use of melatonin and terazosin restores bladder contractility in rabbits with partial outlet obstruction.

OBJECTIVES: To investigate the effects of terazosin and melatonin on isolated rabbit bladder strips after partial bladder outlet obstruction and determine responses to agonist-induced contractility and changes in oxidant-antioxidant system. METHODS: We created partial bladder outlet obstruction in 5 groups of rabbits, each containing 8. Rabbits with sham operation (group 1) received no drug treatment. Similarly, animals in group 2 underwent partial bladder outlet obstruction and received no drug treatment. Rabbits in groups 3 were administered 5 mg/day oral terazosin, and rabbits in group 4 received 10 mg/kg/day melatonin intraperitoneally. Animals in group 5 received both terazosin and melatonin. We removed their bladders and performed histopathological and biochemical measurements. We assessed tissue malondialdehyde and antioxidant enzyme activity levels and recorded in vitro contractility response to KCl in isolated organ baths. RESULTS: The thickness of muscularis propria was significantly increased in group 2 compared with all other groups. KCl-evoked contractions after partial outlet obstruction were significantly impaired in group 3 and 4 animals receiving terazosin and melatonin, respectively. However, combined use of melatonin and terazosin in group 5 showed contractility responses similar to sham-operated animals (P <0.05). Melatonin administration to groups 4 and 5 showed decreased levels of lipid peroxidation. Similarly, animals receiving melatonin and melatonin plus terazosin showed statistically significant increase in antioxidant enzyme activities. CONCLUSIONS: In the present study, we showed that oxidative stress induced by partial bladder outlet obstruction can be successfully antagonized by the potent antioxidant melatonin, and its combined use with an alpha-antagonist such as terazosin may restore in vitro contractility.