Pulsatile Release Pattern Research Articles

Study of estrogen, progesterone and glucose level in cows during overt and silent estrus

Significant economic losses result from increased days open because of failure to detect estrus. Studies (Callahan, et al., 1971; Kesler,et al., 1977) suggest that the endocrine balances required to support normal estrus cycles and for the reestablishment of fertility are restored gradually after calving. Silent estrus, or the lack of behavioral estrus expression, is a common occurrence (50 to 80%) at the first postpartum ovulation. However, the incidence of silent estrus decreases in the second and third estrous cycles (Bulman,1980 ; Labhsetwar, et al., 1946; Morrow, et al., 1966). Expression of estrus in cattle is dependent on sufficient secretion of endogenous E2 to provoke the standing response (Carrick and Shelton. 1969) Estrogen plays a key role in the regulation of endocrine and behavioral events associated with the estrus cycle. E2 plays a pivotal role in the induction of estrus behavior and ovulation (Pfaff, 2005). In many experiments that are performed to study the female reproduction, estrus is artificially induced by administering E2 (Fabre-Nys et al., 1993). Estrogen plays a central role in triggering the gonadotropin surge and ovulation as well as in facilitating the estrus behavior, and thus estrogen indirectly synchronizes mating and ovulation. The patterns of GnRH synthesis and pulsatile release from the hypothalamus are mainly regulated by E2 (Smith and Jennes, 2001) and progesterone (Richer et al., 2005 ;Zalanyi, 2001). E2 stimulates LH synthesis, but at levels below a certain threshold value it inhibits the release of LH. Above this threshold, the inhibitory effect on LH release switches to stimulatory effect (Reinecke and Deuflhard, 2007), which results in the LH surge. Progesterone plays an essential role in various reproductive functions, including regulating the length of the estrous cycle, maintaining pregnancy. Progesterone is high during the luteal phase and in pregnancy, however the levels decline if the animal fails to conceive. Progesterone has been implicated in inhibiting estrogen secretion and ovulation (Echternkamp et al. 1973 and Henricks, 1971). Blood glucose appear to be one of the key nutrients affecting cyclicity in farm animals and a minimum level of 40-60 mg/ml is required to maintain the physiological processes of the body (Duke, 1970). According to Dowine and Gelman (1976), low blood glucose may be associated with infertility. Estrus is negatively affected by alterations in blood metabolites and hormone profiles including glucose, insulin, IGF-I, non-esterified fatty acids, β-hydroxybutyrate (Wathes et al., 2007). Nadiu and Rao

Trimetazidine Dihydrochloride Pulsatile-Release Tablets for the Treatment of Morning Anginal Symptoms: Dual Optimization, Characterization and Pharmacokinetic Evaluation.

This research work aimed to target the early morning peak symptoms of chronic stable angina through formulating antianginal drug, Trimetazidine (TMZ) in a pulsatile-release tablet. The core formulae were optimized using 22 .31 factorial design to minimize disintegration time (DT) and maximize drug release after 5 minutes (Q5min). Different ratios of Eudragit S100 and Eudragit L100 were used as a coating mixture for the selected core with or without a second coating layer of hydroxypropyl methylcellulose (HPMC E50). The different formulation variables were statistically optimized for their effect on lag time and drug release after 7 hours (Q7h) using BoxBehnken design. The optimized formula (PO) was subjected to stability study and pharmacokinetic assessment on New Zealand rabbits. The optimal core (F8) was found to have 1.76 min disintegration time and 61.45% Q5min PO showed a lag time of 6.17 h with 94.80% Q7h and retained good stability over three months. The pharmacokinetics study confirmed the pulsatile-release pattern with Cmax of 206.19 ng/ml at 5.33 h (Tmax) and 95.85% relative bioavailability compared to TMZ solution. Overall pulsatile-release tablets of TMZ successfully released the drug after a desirable lag time, providing a promising approach for early morning anginal symptoms relief.

Preparation and Pulsatile Release Evaluation of Teriparatide-Loaded Multilayer Implant Composed of Polyanhydride-Hydrogel Layers Using Spin Coating for the Treatment of Osteoporosis

Teriparatide (PTH(1–34)), as one of the FDA-approved anabolic medications for postmenopausal osteoporosis treatment, shows anabolic effects in intermittent administration. In the current study, a multilayer implantable device was developed by spin-coating and dip-coating techniques for providing pulsatile pattern release of teriparatide, composed of polymeric and hydrogel layers. Copolymers of poly [1,3-bis (p-carboxyphenoxy) propane-sebasic acid] (CPP-SA) with molar ratios of 20:80 and 10:90 were synthesized and characterized by GPC, 1HNMR, and FTIR. Swelling property and in vitro drug release from hyaluronic acid hydrogel were evaluated. The degradation behavior of the polymer layer and the morphology of the fabricated implant was evaluated by SEM images. An in vitro release study was done for evaluating the pulsatile release pattern. Histology assessment aimed to determine the biocompatibility of implants in rats. MTT assay was applied for cell cytotoxicity study. Plasma drug concentration and pharmacokinetic data were achieved by in vivo study. According to in vivo and in vitro data, the best formulation was CPP-SA (10:90) with a concentration of 20%. Histology assessment showed mild inflammation. MTT assay showed no significant toxicity compared with control in normal doses of the drug. The pulsatile delivery of peptide the drug from the multilayer device with hyaluronic acid hydrogel and CPP-SA polymer alternative layers can be achieved.

Novel Biology of Tachykinins in Gonadotropin-Releasing Hormone Secretion.

The tachykinin family of peptides, composed of the neurokinins A and B (NKA, NKB) and substance P are involved in the central control of gonadotropin-releasing hormone (GnRH) release through a variety of neuronal circuitries that mediate the activation of Kiss1 neurons and the synchronization of their activity within the arcuate nucleus. The major outcome of this role is the precise regulation of the pulsatile pattern of GnRH release. In addition, tachykinins are involved in the maturation of the reproductive axis by determining the optimal timing of puberty onset, as well as in the timing of the preovulatory luteinizing hormone surge in females. Therefore, the action of tachykinins in reproduction appears to extend to all the critical aspects required for the successful attainment and maintenance of fertility. In this review, we summarize the latest advances in our understanding of the biology of tachykinins in the control of GnRH release, addressing the existing controversies, open questions, and future perspectives.

SUN-483 High-Fat Diet Suppresses Basal Lh Pulsatile Levels in Female Mice at Estrus

Pubertal timing and reproductive maturation in females are known to be susceptible to obesogenic diets. Metabolic energy balance and reproduction are homeostatic processes that are tightly regulated, in part, by hypothalamic neurons in the arcuate nucleus (ARC) and high-fat diet is reported to steadily increase estradiol levels in rodents compared to standard chow. Estradiol regulates the hypothalamic-pituitary-gonadal axis via negative feedback mechanisms in estrogen-sensitive ARC neurons by modulating the pulsatile release of the gonadotropin luteinizing hormone (LH). However, it is unclear how exactly the circulating estradiol milieu of adult females interacts with a state of high caloric fat intake to alter the parameters of LH release, such as pulse frequency, pulse amplitude, and basal set points of pulsatile release. The objective of this study was to use a non-invasive method of measuring pulsatile LH release patterns at different stages of the estrous cycle in mice fed a high-fat diet, using an ultrasensitive mouse LH assay (1). Starting at weaning age (21 days old), female C57BL/6J mice were fed ad libitum with either regular chow diet (RD) or 60% kcal high-fat diet (HFD), and free access to water. After 12 weeks on either RD or HFD, estrous cyclicity was determined via examination of vaginal cytological smears. Blood samples were collected when mice were at diestrus, followed by one full estrous cycle, and then collected again at estrus. To examine LH pulsatile release during both estrous cycle stages, mice had tail tips snipped and whole blood samples were collected and immediately frozen in assay buffer at 10-second intervals for 3 hours (between 0900 and 1200 hours). Pulsatile LH release was analyzed for pulse frequency, basal mean LH levels, and pulse amplitude using the DynPeak algorithm program. Compared to RD controls, mice fed a HFD displayed significantly lower basal LH levels during estrus, but not diestrus. However, there were no differences in pulse amplitude between HFD and RD fed mice. Mice on HFD (at both diestrus and estrus) also showed a trend towards increased pulse frequency. These results suggest that endogenous circulating estradiol influences the impact that HFD has on regular pulsatile release of LH by changing the set point for basal LH levels, thereby modulating when obesogenic diet-induced changes may be most detrimental. This has implications on how diet may put women at risk of failing reproductive health when ovarian estradiol levels drastically change, for example during menopause or with hormone replacement therapy. 1) Steyn FJ et al. Development of a methodology for and assessment of pulsatile luteinizing hormone secretion in juvenile and adult male mice. Endocrinology 2013;154(12):4939-4945. doi:10.1210/en.2013-1502.

The oxytocin receptor, luteolysis and the maintenance of pregnancy

During luteal regression episodic pulses of oxytocin secretion become coupled to the release of prostaglandin F2 alpha (PGF2 alpha) following synthesis of endometrial oxytocin receptors, but in early pregnancy the inhibition of oxytocin receptor formation by the conceptus prevents the development of the pulsatile pattern of PGF2 alpha release needed to achieve luteolysis. Oxytocin receptors are present on the luminal epithelium in ovariectomized and anoestrous ewes, in pregnant animals throughout most of gestation (day 21 to term) and in explants of endometrial tissue cultured in vitro. These receptors can be downregulated for a brief period by progesterone (10-12 days in sheep, 12-14 days in cattle). This period of inhibition can be extended by infusion of interferon tau (IFN-tau) (which probably inhibits oxytocin receptor gene transcription) or of oxytocin into the systemic circulation (which may act further downstream, possibly at the level of translation). Oxytocin receptors also develop on the caruncular stroma and deep glands at oestrus, but these need positive upregulation and appear dependent on an oestrogenic environment. Only epithelial receptors are needed to achieve a maximal PGF2 alpha response to an oxytocin challenge, but the presence of oxytocin receptors does not necessarily confer responsiveness as other factors may influence intracellular coupling mechanisms and precursor availability. The duration of the luteal phase is regulated by the time of the initial post-ovulatory rise in progesterone and the duration of exposure to progesterone.(ABSTRACT TRUNCATED AT 250 WORDS)

Reducing Glucokinase Activity Restores Endogenous Pulsatility and Enhances Insulin Secretion in Islets From db/db Mice.

An early sign of islet failure in type 2 diabetes (T2D) is the loss of normal patterns of pulsatile insulin release. Disruptions in pulsatility are associated with a left shift in glucose sensing that can cause excessive insulin release in low glucose (relative hyperinsulinemia, a hallmark of early T2D) and β-cell exhaustion, leading to inadequate insulin release during hyperglycemia. Our hypothesis was that reducing excessive glucokinase activity in diabetic islets would improve their function. Isolated mouse islets were exposed to glucose and varying concentrations of the glucokinase inhibitor d-mannoheptulose (MH) to examine changes in intracellular calcium ([Ca2+]i) and insulin secretion. Acutely exposing islets from control CD-1 mice to MH in high glucose (20 mM) dose dependently reduced the size of [Ca2+]i oscillations detected by fura-2 acetoxymethyl. Glucokinase activation in low glucose (3 mM) had the opposite effect. We then treated islets from male and female db/db mice (age, 4 to 8 weeks) and heterozygous controls overnight with 0 to 10 mM MH to determine that 1 mM MH produced optimal oscillations. We then used 1 mM MH overnight to measure [Ca2+]i and insulin simultaneously in db/db islets. MH restored oscillations and increased insulin secretion. Insulin secretion rates correlated with MH-induced increases in amplitude of [Ca2+]i oscillations (R2 = 0.57, P < 0.01, n = 10) but not with mean [Ca2+]i levels in islets (R2 = 0.05, not significant). Our findings show that correcting glucose sensing can restore proper pulsatility to diabetic islets and improved pulsatility correlates with enhanced insulin secretion.

A long-lasting oral preformulation of the angiotensin II AT1 receptor antagonist losartan

Losartan (Los), a non-peptidic orally active agent, reduces arterial pressure through specific and selective blockade of angiotensin II receptor AT1. However, this widely used AT1 antagonist presents low bioavailability and needs once or twice a day dosage. In order to improve its bioavailability, we used the host: guest strategy based on β-cyclodextrin (βCD). The results suggest that Los included in βCD showed a typical pulsatile release pattern after oral administration to rats, with increasing the levels of plasma of Los. In addition, the inclusion compound presented oral efficacy for 72 h, in contrast to Los alone, which shows antagonist effect for only 6 h. In transgenic (mREN2)L27 rats, the Los/βCD complex reduced blood pressure for about 6 d, whereas Los alone reduced blood pressure for only 2 d. More importantly, using this host: guest strategy, sustained release of Los for over a week via the oral route can be achieved without the need for encapsulation in a polymeric carrier. The proposed preformulation increased the efficacy reducing the dose or spacing between each dose intake.

Zaleplon loaded bi-layered chronopatch: A novel buccal chronodelivery approach to overcome circadian rhythm related sleep disorder

The aim of this study was to develop a novel buccal bi-layered chronopatch capable of eliciting pulsatile release pattern of drugs treating diseases with circadian rhythm related manifestation. Zaleplon (ZLP) was used as a model drug intended to induce sleep and to treat middle of night insomnia. The chronopatch was prepared adopting double casting technique. The first layer was composed of a controlled release patch containing ZLP-Precirol melt granules intended to release ZLP in a sustained manner to maintain sleep and to prevent early morning awakening. The second layer was composed of a fast release lyophilized buccal disc containing ZLP loaded SNEDDS (Z-SNEDDS) intended for rapid sleep induction. Pharmacokinetic parameters of ZLP from the chronopatch were compared to those of the immediate release capsule, Siesta®, as reference in Mongrel dogs using a randomized crossover design. The appearance of two peaks having two Cmax and Tmax proved the pulsatile release pattern. The increase in relative bioavailability of ZLP from the chronopatch was 2.63 folds. The results revealed the ability of the developed ZLP loaded bi-layered chronopatch to be a candidate for overcoming early morning awakening without middle of night dose administration.

Development of Pulsatile Drug Delivery for Chronotherapeutics of Hypertension

The goal of present work was to investigate the effects of rupturable material ethyl cellulose combines with erodible material Klucel EXF on the pulsatile release pattern of Candesartan cilexetil in order to prevent morning rise in blood pressure. A tablet prepared by compression coating method contains core and coat components. Core consists of active ingredients with its various superdisintegrants where as coat contains different grades of ethyl cellulose and Klucel HXF in various combinations. All these tablets were evaluated for its micromeritics, weight variations, hardness, friability and in vitro dissolution testing. Drug-excipients interactions were carried out by FTIR. Dissolution studies were carried out in simulated gastric fluid followed by phosphate buffer of pH 6.5. The optimized formulation PT6 which give lag time of 6 hrs and released 99.10 % within 7 hr, as well as found to be stable in ICH stability testing guidelines.

Development and Characterization Pulsatile Microspheres of Nifedipine for Hypertension.

To develop pulsatile microspheres of Nifedipine blend of polymers Carbopol 971P and Eudragit RS100 for antihypertensive to release the drug at a particular lag time. Pulsatile nifedipine microspheres were prepared using the blend of Carbopol 971P and Eudragit RS100 at different ratios in a mixture of solvents. Formulations were prepared and optimized on the basis of particle size, percentage yield and drug entrapment efficiency. Optimized formulations were further analyzed at different resolutions per minute (rpm) by optical microscopy, scanning electron microscopy (SEM), in-vitro dissolution studies and kinetics model. The most promising formulation was compared with the marketed products. The recent patents on microsphere used for hypertension for drug used Liensinine (CN104757561). Dopamine antagonist (US20030069232A1), Human Serum Albumin (EP19990304616) and Omeprazole (IN2107/DEL/2014) helped in selecting the drug and polymers. The formulated microspheres had mean particle size ranging from 177.31 to 277.48 &amp;micro;m. Formulation F83 showed slow release of drug initially for 4 h and drastically release of drug enhanced at a particular lag time (Showed pulsatile release) after 6 h. However, conventional marketed products showed maximum release initially at 1 h and decreased gradually while controlled released marketed product showed sustained pattern. On the basis of data formulation F83 showed slow release and at a particular lag time followed by pulsatile release pattern and followed first order kinetics model. It was concluded that the formulated nifedipine pulsatile dosage form could be useful in the treatment of hypertension.

Development of timely controlled-release systems for chronotherapy of propranolol with minimization of the pH effect in the simulated gastrointestinal medium

A timely controlled release dosage form with minimization of transit time and pH effects of gastrointestinal tract for chronotherapy of propranolol was developed. Ethylcellulose (EC) and Eudragit RS selected as the controlling membrane for drug-layering non-pareil seeds (NP) or extrusion-spheronized pellets incorporated with matrix materials or osmogents were characterized in the simulated gastric fluid (SGF) and pH change medium. Results demonstrated that when propranolol was layered on NP, Eudragit RS plasticized with 30% triethylcitrate (TEC) coated at 15% level could adjust a lag time close to the reference but with a slower release rate after then, while EC plasticized with HPMC could not delay drug release at a lag time close to the reference. Furthermore, EC incorporated with lactose coated on the extrusion-spheronized pellets could adjust the lag time, but a release rate correspondingly decreased with increasing lag time. Nevertheless, coating Eudragit RS on matrix pellets as controlling membrane at different levels could adjust a desired lag time with a pulsatile release pattern. However, the lag time adjusted was only within a 5 h range. Finally, coating Eudragit RS at different level on matrix pellets containing various ratio of osmogent, NaCl or lactose, could effectively adjust a desired lag time as long as 15 h with a pulsatile release pattern to accomplish the timely controlled release with minimization of the pH effects of gastrointestinal tract that could meet the clinical need for chronotherapy of propranolol.

Formulation and process optimization of multiparticulate pulsatile system delivered by osmotic pressure-activated rupturable membrane

In this study, a multiparticulate pulsatile drug delivery system activated by a rupturable controlled-release membrane (Eudragit® RS) via osmotic pressure (with NaCl as the osmogent) was developed and characterized for omeprazole, omeprazole sodium, and propranolol HCl which have different water solubilities. Multiparticulates in pellet form for incorporation with or without the osmogent were manufactured by three methods and then used to coat a polymeric membrane. Results demonstrated that drug/osmogent-containing pellets manufactured by the extrusion/spheronization method with incorporation of the osmogent were optimal. The lag time (tL) to initiate pulsatile release is regulated by tL=l2/(6×D), which is dependent on the coating levels (l2) and plasticizer content (D). The pulsatile release pattern was found to be dependent on the osmotic pressure (osmogent), drug solubility, and mechanical properties of the polymeric membrane (elasticity and toughness). Omeprazole with lower water solubility could not generate sufficient osmotic pressure to create a crack in the membrane to activate pulsatile release, whereas the two other model drugs with higher solubilities could. But adsorption of omeprazole sodium on Eudragit® RS via charge–charge interactions led the its incomplete release. Finally, with 4% osmogent of NaCl added, a lag time in a range from 0 to 12h proportionally regulated by varying both the membrane thickness and plasticizer level initiated the complete pulsatile release of propranolol HCl. In conclusion, a multiparticulate pulsatile drug delivery system activated by a rupturable controlled-release membrane via osmotic pressure was successfully developed, and clinical applications of chronotherapy with drugs like propranolol HCl are expected.

Novel time and site specific “tablets in capsule” system for nocturnal asthma treatment

The objective of present work was to develop a “tablets in capsule” system for facilitating both immediate and pulsatile drug deliveries of theophylline to mimic the circadian rhythm of nocturnal asthma. The system comprised of capsule filled with two tablets, first pulse and second pulse tablet prepared by wet granulation method. First pulse tablet was not coated and was responsible for providing loading dose whereas; second pulse tablet was coated with Eudragit L100 and Eudragit S100 to release drug in colon after specific lag time. Two independent variables, amount of polymers and coating thickness, were optimized by 32 full factorial design. The optimum formulation consisted of Eudragit L100: Eudragit S100 in 1:1.5 ratio and coating thickness of 20 % (w/w). In vitro drug release of “tablets in capsule” system in three different media (pH 1.2, pH 6.8, and pH 7.4) revealed immediate and pulsatile release patterns.

Formulating SLMs as oral pulsatile system for potential delivery of melatonin to pediatric population.

The formulation development of melatonin (MLT) for infants and children with neurodevelopmental difficulties was fully investigated. This population have a higher prevalence of sleep disorders and present special challenges for drug administration and swallowing. To solve these issues, solid lipid microparticles (SLMs) were designed to obtain an oral flexible dosage form constituted by GRAS excipients and a free flow pulsatile delivery system for MLT, able to maintain its release through 8h. Three groups of SLMs were produced by spray congealing and characterized as regards particle size, morphology, flowability, solid state, drug content and release behavior. The SLMs manipulation with milk and yogurt and the MLT stability in these foods were also investigated. Microparticles with different excipient composition were selected to obtain a pulsatile release pattern over 8h. The final delivery platform displayed a prompt release from group I SLMs together with a lag phase of groups II and III SLMs, followed by a repeated MLT release from group II and a prolonged MLT release related to the last group. Finally, MLT was compatible and stable in milk and yogurt suggesting that microparticles sprinkled into food is acceptable for MLT administration to children unable to swallow capsules or tablets.

The central CLOCK system and the stress axis in health and disease

Address for correspondence: Dr E. Kassi, Department of Biological Chemistry, Medical School, Athens University, 75 Mikras Asias Str., Goudi 11527, Athens, Greece. E-mail: ekassi@med.uoa.gr Received 22-11-2012, Accepted 12-02-2013 are secreted in the portal system in a circadian and highly concordant pulsatile fashion. The activity of the HPA axis is in fact characterized not only by a classic circadian rhythm, but also by an ultradian pattern of discrete pulsatile release of glucocorticoids. Glucocorticoids are the final effectors of the HPA axis. These hormones are pleiotropic and exert their effects through their ubiquitously distributed intracellular glucocorticoid receptors alpha and beta (GRα and GRβ, respectively), both members of the nuclear receptor superfamily. The circadian release of ACTH/cortisol in their characteristic pulsatile manner appears to be controlled by one or more pace makers, whose exact location in the brain has not as yet been fully explored in humans. These diurnal variations are perturbed during daily activities like eating, sleeping, etc., while they are more greatly disrupted under stress conditions.

Improved blood glucose disposal and altered insulin secretion patterns in adenosine A1receptor knockout mice

Type 2 diabetes mellitus (T2DM) is characterized by the inability of the pancreatic β-cells to secrete enough insulin to meet the demands of the body. Therefore, research of potential therapeutic approaches to treat T2DM has focused on increasing insulin output from β-cells or improving systemic sensitivity to circulating insulin. In this study, we examined the role of the A(1) receptor in glucose homeostasis with the use of A(1) receptor knockout mice (A(1)R(-/-)). A(1)R(-/-) mice exhibited superior glucose tolerance compared with wild-type controls. However, glucose-stimulated insulin release, insulin sensitivity, weight gain, and food intake were comparable between the two genotypes. Following a glucose challenge, plasma glucagon levels in wild-type controls decreased, but this was not observed in A(1)R(-/-) mice. In addition, pancreas perfusion with oscillatory glucose levels of 10-min intervals produced a regular pattern of pulsatile insulin release with a 10-min cycling period in wild-type controls and 5 min in A(1)R(-/-) mice. When the mice were fed a high-fat diet (HFD), both genotypes exhibited impaired glucose tolerance and insulin resistance. Increased insulin release was observed in HFD-fed mice in both genotypes, but increased glucagon release was observed only in HFD-fed A(1)R(-/-) mice. In addition, the regular patterns of insulin release following oscillatory glucose perfusion were abolished in HFD-fed mice in both genotypes. In conclusion, A(1) receptors in the pancreas are involved in regulating the temporal patterns of insulin release, which could have implications in the development of glucose intolerance seen in T2DM.

Understanding calcium homeostasis in postnatal gonadotropin-releasing hormone neurons using cell-specific Pericam transgenics

The gonadotropin-releasing hormone (GnRH) neurons are the key output cells of a complex neuronal network controlling fertility in mammals. To examine calcium homeostasis in postnatal GnRH neurons, we generated a transgenic mouse line in which the genetically encodable calcium indicator ratiometric Pericam (rPericam) was targeted to the GnRH neurons. This mouse model enabled real-time imaging of calcium concentrations in GnRH neurons in the acute brain slice preparation. Investigations in GnRH-rPericam mice revealed that GnRH neurons exhibited spontaneous, long-duration (∼8s) calcium transients. Dual electrical-calcium recordings revealed that the calcium transients were correlated perfectly with burst firing in GnRH neurons and that calcium transients in GnRH neurons regulated two calcium-activated potassium channels that, in turn, determined burst firing dynamics in these cells. Curiously, the occurrence of calcium transients in GnRH neurons across puberty or through the estrous cycle did not correlate well with the assumption that GnRH neuron burst firing was contributory to changing patterns of pulsatile GnRH release at these times. The GnRH-rPericam mouse was also valuable in determining differential mechanisms of GABA and glutamate control of calcium levels in GnRH neurons as well as effects of G-protein-coupled receptors for GnRH and kisspeptin. The simultaneous measurement of calcium levels in multiple GnRH neurons was hampered by variable rPericam fluorescence in different GnRH neurons. Nevertheless, in the multiple recordings that were achieved no evidence was found for synchronous calcium transients. Together, these observations show the great utility of transgenic targeting strategies for investigating the roles of calcium with specified neuronal cell types.

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 ([Ca2+]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 [Ca2+]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.

Kisspeptin-10 elicits triphasic cytosolic calcium responses in immortalized GT1-7 GnRH neurones

Kisspeptins, which are alternatively called as metastin since they were originally identified as products of metastasis suppressor gene KiSS-1, are the natural ligands for the G protein-coupled receptor 54 (GPR54). Kisspeptins are the most potent activators of hypothalamic–pituitary–gonadal (HPG) axis reported to date. 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. Therefore, the pattern of kisspeptin-induced intracellular signaling and the role of PKC in the intracellular signaling cascade were investigated by fluorescence calcium imaging using the immortalized GnRH-secreting GT1-7 hypothalamic neurons. Kisspeptin-10 caused a triphasic change characterized by an initial small increase followed by a significant decrease and increase in intracellular free calcium concentrations ([Ca 2+] i ). The changes in [Ca 2+] i were significantly attenuated by pre-treatment with protein kinase C inhibitor. The compatibility of appeared mirrored-patterns of kisspeptin-10-induced changes in [Ca 2+] i concentrations in these neurons and GnRH secretion confirm the importance of intracellular calcium flux downstream from GPR54 through PKC signaling pathway.