Preparation of ultrathin translucent zirconia laminate veneers using 3D printing reduction guides.

Minimizing and controlling the amount of tooth reduction during the preparation of ultrathin laminate veneers is a challenge for minimally invasive dentistry. The use of reduction guides is advised to reach the optimal space required for the definitive restoration without excessive reduction. The digital production of a reduction guide used to control tooth preparation for ultrathin laminate veneers is described.

Randomized clinical trial of zirconia laminate veneers sintered by using conventional versus speed process: 1-year follow-up.

STATEMENT OF PROBLEM: The application of highly translucent multilayered zirconia ceramic in minimally invasive esthetic dentistry allows the achievement of both esthetics and strength with minimal thickness. Clinical studies that have assessed the performance of zirconia ultrathin veneers sintered with the conventional and speed procedure are lacking. PURPOSE: The purpose of this clinical study was to evaluate the effect of speed sintering processes on the translucency and clinical performance of zirconia laminate veneers. MATERIAL AND METHODS: Four participants had their teeth restored with 32 ultratranslucent zirconia laminate veneers. Based on the zirconia sintering procedure, the participants were randomly allocated into 2 groups. The teeth were prepared by selective reduction over trial restorations. Zirconia veneers were milled from multilayer zirconia blanks and sintered either by speed sintering or conventional sintering as specified by the manufacturer's recommendations. The intaglio surface of the veneers were airborne-particle abraded with 50-µm aluminum-oxide, and the veneers were then adhesively bonded to the teeth with translucent light-polymerizing resin cement. The modified California Dental Association (CDA)/Ryge criteria were used to assess participants at baseline and every 3 months for 12 months. The translucency and the color difference of the tooth before and after veneer restoration were evaluated. For statistical analysis, the Pearson chi squared test, independent t test, and paired t test were used (α=.05). RESULTS: Translucency and color differences were significantly higher in the conventionally sintered group (P<.05). No restorations were lost. No significant differences were found between the 2 groups in the CDA/Ryge criteria or color parameter after follow-up intervals (P>.05). The primary qualitative changes observed at the final recall were marginal integrity and marginal discoloration. The color match and zirconia surface were rated Alfa. CONCLUSIONS: After 1 year of follow-up, both conventional and speed sintered ultrathin zirconia laminates showed satisfactory functional, esthetic, and color stability outcomes.

The effects of replacing eggs with chicks on mesotocin, dopamine, and prolactin in the native Thai hen.

The mesotocinergic (MTergic) and dopaminergic (DAergic) systems have been documented to play pivotal roles in maternal behaviors in native Thai chickens. In native Thai chickens, plasma prolactin (PRL) concentrations are associated with maternal behaviors, which are also controlled by the DAergic system. However, the role of MT in conjunction with the roles of DA and PRL on the neuroendocrine regulation of the transition from incubating to rearing behavior has never been studied. Therefore, the aim of this study was to investigate the association of MT, DA, and PRL during the transition from incubating to rearing behavior in native Thai hens. Using an immunohistochemistry technique, the numbers of MT-immunoreactive (-ir) and tyrosine hydroxylase-ir (TH-ir, a DA marker) neurons were compared between incubating hens (INC; n = 6) and hens for which the incubated eggs were replaced with 3 newly hatched chicks for 3 days after 6, 10, and 14 days of incubation (REC; n = 6). Plasma PRL concentrations were determined by enzyme-linked immunosorbent assay. The results revealed that the numbers of MT-ir neurons within the nucleus supraopticus, pars ventralis (SOv), nucleus preopticus medialis (POM), and nucleus paraventricularis magnocellularis (PVN) increased in the REC hens when compared with those of the INC hens at 3 different time points (at days 9, 13, and 17). On the other hand, the number of TH-ir neurons in the nucleus intramedialis (nI) decreased in the REC13 and REC17 hens when compared with those of the INC hens. However, the number of TH-ir neurons in the nucleus mamillaris lateralis (ML) only decreased in the REC13 hens when compared with the INC13 hens. The decrease in the numbers of TH-ir neurons within the nI and ML is associated with the decrease in the levels of plasma PRL. This study suggests that the presence of either eggs or chicks is the key factor regulating the MTergic system within the SOv, POM, and PVN and the DAergic system within the nI and ML during the transition from incubating to rearing behavior in native Thai chickens. The results further indicate that these two systems play pivotal roles in the transition from incubating to rearing behavior in this equatorial species.

Ovarian steroid withdrawal results in GABAA receptor upregulation in the photoperiodic neuroendocrine pathways of the turkey hen.

The mechanism(s) underlying photorefractoriness in temperate zone seasonally breeding birds remains undetermined. Our recent findings reveal a link between the upregulation of GABAA receptors (GABAARs) in the premammillary nucleus (PMM) and the state of photorefractoriness. Gonadal steroid levels fluctuate during the breeding season; increasing after gonadal recrudescence and declining sharply once gonadal regression begins. Here, we examined the effect of gonadal steroid withdrawal on the expression of GABAARs in the turkey PMM. Exogenous ovarian steroids were administered and then withdrawn from turkey hens to mimic the decline of ovarian steroids levels at the end of a breeding season. The upregulation of GABAAR α3, α4, δ, π, and γ2-subunits was observed in the PMM of the steroid withdrawal group when compared to the non-steroid treatment group. The level of tyrosine hydroxylase, photopigment melanopsin, and circadian clock genes in the PMM of the steroid withdrawal group resembled the levels observed in the natural photorefractory hens and were significantly lower than those of the short-day light stimulated group. A reduction in gonadotropin-releasing hormone-I mRNA expressed within the nucleus commissurae pallii was also observed in hens undergoing steroid withdrawal. These results suggest that the natural decline in circulating ovarian steroid levels may modulate the GABAergic system in the PMM through the upregulation of GABAA receptors. This, in turn, could diminish the reproductive neuroendocrine responses to light and favor a condition resembling the state of photorefractoriness.

GABAergic Neurotransmission in the Premammillary Nucleus of the Turkey Hypothalamus Regulates Reproductive Seasonality and the Onset of Photorefractoriness.

BACKGROUND/AIMS: Photoperiod is a major environmental cue in temperate-zone birds which synchronizes breeding with the time of year that offers the optimal environment for offspring survival. Despite continued long photoperiods, these birds eventually become refractory to the stimulating photoperiod and their reproductive systems regress. In this study, we characterized the role of γ-aminobutyric acid (GABA)ergic neurotransmission in modulating the response of the premammillary nucleus (PMM) to a gonad stimulatory photoperiod and the onset of photorefractoriness. METHODS AND RESULTS: Bilateral ablation of the PMM blocked the light-induced neuroendocrine response from occurring in photosensitive turkeys. Microarray analyses revealed an increase in GABAergic activity in the PMM of photorefractory birds as opposed to photosensitive ones, and this enhanced GABAergic activity appeared to inhibit the photoperiodic signal. Additionally, GABAA and GABAB receptors were expressed by dopamine-melatonin neurons in the PMM, and the administration of the GABA receptor agonist baclofen blocked the photoperiodic reproductive neuroendocrine responses. CONCLUSIONS: Consistent with the present findings, we propose that the long-sought-after mechanism underlying photorefractoriness is linked to the inhibitory actions of GABA. We suggest that (1) GABAergic interference with photoperiodic entrainment in the PMM initiates the photorefractory state and terminates the annual breeding season in temperate-zone birds, and (2) the PMM is a site of photoreception and photorefractoriness that controls the initiation and termination of avian reproductive seasonality.

Enhanced GABAergic inhibition in the premammillary nucleus of photorefractory turkey hens via GABAA receptor upregulation.

The premammillary nucleus (PMM) of the turkey mediobasal hypothalamus, where dopamine-melatonin (DA-Mel) neurons are localized, is a site for photoreception and photoperiodic time measurement, which is essential for the initiation of avian reproductive seasonality. In addition, this area could also be responsible for the onset and maintenance of photorefractoriness at the end of the breeding season due to the enhanced inhibitory effect of γ-aminobutyric acid (GABA). GABA is an inhibitory neurotransmitter in the central nervous system which interferes with the photosexual response in the turkey, a seasonally breeding bird. Here, we further characterized the GABAA receptor subunits in the PMM DA-Mel neurons related to reproductive seasonality and the onset of photorefractoriness. GABAA receptor subunits and GABA synthesis enzymes in the PMM of photosensitive and photorefractory turkey hens were identified using real-time qRT-PCR. The upregulation of GABAA receptor α1-3, ß2-3, γ1-3, ρ1-3, δ, and θ mRNA expression were observed in the PMM of photorefractory birds when compared to those of photosensitive ones while there is no change observed in the GABA synthesis enzymes, glutamate decarboxylase 1 and 2. Those upregulated GABAA receptor subunits were further examined using immunohistochemical staining and they appeared to be co-localized within the PMM DA-Mel neurons. The upregulation of GABAA receptor subunits observed in the PMM of photorefractory birds coincides with a lack of responsiveness to a light stimulus provided during the photosensitive phase. This is supported by the absence of c-fos induction and TH upregulation in the PMM and a subsequence inhibition of c-fos and GnRH-I expression in the nucleus commissurae pallii. The augmented GABAA receptor subunits expression may mediate an enhancement of inhibitory GABAergic neurotransmission and the subsequent interference with the photosexual response. This could contribute to the state of photorefractoriness and the termination of breeding activities in the turkey, a temperate zone bird.

Dopamine and prolactin involvement in the maternal care of chicks in the native Thai hen (Gallus domesticus).

The dopaminergic (DAergic) system plays a pivotal role in incubation behavior via the regulation of prolactin (PRL) secretion in birds, however the role of the DA/PRL system in rearing behavior is poorly understood. The objective of this study was to investigate the relationship between the DA/PRL system and rearing behavior in a gallinaceous bird, the native Thai chicken. Incubating native Thai hens were divided into two groups. In the first group, hens were allowed to care for their chicks (rearing hens; R). In the second group, hens were deprived of their chicks immediately after hatching (non-rearing hens; NR). In both groups, blood samples and brain sections were collected at different time points after the chicks hatched (days 4, 7, 10, 14, 17, 21, 24, and 28; 6 hens/time point/group). In this study, tyrosine hydroxylase (TH) was used as a marker for DAergic neurons. The numbers of TH-immunoreactive (-ir) neurons in the nucleus intramedialis (nI) and in the nucleus mamillaris lateralis (ML), which regulate the vasoactive intestinal peptide (VIP)/PRL system, were determined in R and NR hens utilizing immunohistochemical techniques. Plasma PRL levels were determined by enzyme-linked immunosorbent assays. The results revealed that both the number of TH-ir neurons in the nI and the plasma PRL levels were significantly higher in the R hens compared with the NR hens during the first 14 days of chick rearing (P<0.05). However, there was no significant change in the DAergic activity in the ML in either the R or NR groups throughout the 28-day rearing periods. These results suggest that the DA/PRL system is involved in early rearing behavior. The additional decline in DAergic activity and plasma PRL levels during the disruption of rearing behavior further supports their involvement in rearing behavior in this equatorial precocial species.

Analysis of the Marginal Continuity in Different Designs of Monolithic Zirconia Inlay-Retained Fixed Dental Prostheses: An In Vitro Study.

PURPOSE: To evaluate the marginal integrity of three inlay-retained fixed dental prosthesis (IRFDP) designs fabricated using monolithic zirconia. MATERIALS AND METHODS: In total, 30 IRFDPs were fabricated using 4-YTZP monolithic zirconia and randomly divided into three groups according to the cavity design. Groups ID2 and ID1.5 received an inlay cavity preparation, which includes a proximal box and an occlusal extension with a depth of 2 mm or 1.5 mm, respectively. Group PB received a proximal box cavity preparation without an occlusal extension. The restorations were fabricated and cemented using a dual-cure resin cement (Panavia V5) and subjected to an equivalent of 5 years of aging. The specimens were evaluated under an SEM to measure marginal continuity before and after the aging process. RESULTS: During the whole 5-year aging process, no specimens showed signs of cracking, fracture, or loss of retention in any restorations. In the SEM analysis, most marginal defects observed in the restorations were areas of microgaps at the tooth/cement interface (TC) or zirconia/cement interface (ZC), resulting in loss of adaptation. There was a significant difference between the groups after the aging process at both the TC (F = 4.762, P < .05) and ZC (F = 6.975, P < .05), with Group ID2 presenting the best performance. There was a significant difference between TC and ZC in all groups (P < .05), with ZC presenting more gaps in all groups. CONCLUSIONS: Inlay cavity designs including a proximal box and an occlusal extension exhibited better marginal stability than a proximal box without occlusal extension.

Mesotocin and maternal care of chicks in native Thai hens (Gallus domesticus).

Oxytocin (OT) is known to induce and regulate maternal behaviors in mammals via the supraoptic nucleus and paraventricular nucleus (PVN), whereas the function of mesotocin (MT; the avian homolog of OT) is poorly understood in birds. To elucidate the association of MT and the regulation of maternal behaviors in birds, we studied changes in the number of MT-immunoreactive (ir) neurons in native Thai chickens using immunohistochemistry. We observed that MT-ir neurons and fibers appeared in discrete regions located close to the third ventricle from the level of the preoptic area through the anterior hypothalamus with an abundance observed in the nucleus supraopticus, pars ventralis (SOv), nucleus preopticus medialis (POM), and PVN. The number of MT-ir neurons was low in the SOv, POM, and PVN of non-laying hens, but it increased gradually when the hens entered the laying stage, and peaked in incubating and rearing hens. We compared the number of MT-ir neurons in the SOv, POM, and PVN of native Thai hens rearing chicks (R) with that of non-rearing chicks (NR). The number of MT-ir neurons was high in the R hens, but low in the NR hens in these nuclei. For the first time, these results indicate that the association between the MT neurons and the presence of chicks might, in part, play a role in the neuroendocrine reorganization to establish and maintain maternal behaviors in native Thai chickens. MTergic activity is likely related to the contribution of rearing behavior in this equatorial precocial species.

Photoreceptive oscillators within neurons of the premammillary nucleus (PMM) and seasonal reproduction in temperate zone birds.

The pathway for light transmission regulating the reproductive neuroendocrine system in temperate zone birds remains elusive. Based on the evidence provided from our studies with female turkeys, it is suggested that the circadian clock regulating reproductive seasonality is located in putatively photosensitive dopamine-melatonin (DA-MEL) neurons residing in the premammillary nucleus (PMM) of the caudal hypothalamus. Melanopsin is expressed by these neurons; a known photopigment which mediates light information pertaining to the entrainment of the clock. Exposure to a gonad stimulatory photoperiod enhances the activity of the DAergic system within DA-MEL neurons. DAergic activity encoding the light information is transmitted to the pars tuberalis, where thyroid-stimulating hormone, beta (TSHß) cells reside, and induces the release of TSH. TSH stimulates tanycytes lining the base of the third ventricle and activates type 2 deiodinase in the ependymal which enhances triiodothyronine (T3) synthesis. T3 facilitates the release of gonadotropin-releasing hormone-I which stimulates luteinizing hormone/follicle stimulating hormone release and gonad recrudescence. These data taken together with the findings that clock genes are rhythmically expressed in the PMM where DA-MEL neurons are localized imply that endogenous oscillators containing photoreceptors within DA-MEL neurons are important in regulating the DA and MEL rhythms that drive the circadian cycle controlling seasonal reproduction.

Analysis of the Marginal Continuity in Different Designs of Monolithic Zirconia Inlay-Retained Fixed Dental Prosthesis: (An in vitro Study).

PURPOSE: To evaluate the marginal integrity of three inlay-retained fixed dental prosthesis (IRFDP) designs fabricated using monolithic zirconia. MATERIALS AND METHODS: Thirty inlay-retained fixed dental prostheses were fabricated using a 4-YTZP monolithic zirconia, and divided randomly into 3 groups according to the cavity design. Group ID2 and ID1.5 received an inlay cavity preparation which includes a proximal box and an occlusal extension with a depth of 2 mm or 1.5 mm respectively. Group PB received a proximal box cavity preparation without an occlusal extension. The restorations were fabricated and cemented using a dual cure resin cement (Panava V5) and subjected to an equivalent of 5 years of ageing. The specimens were evaluated under a SEM to measure marginal continuity before and after the ageing process. RESULTS: During the whole 5-year ageing process, no specimens showed signs of cracking,fracture or loss of retention in any of the restorations. In the SEM analysis, most of the marginal defects observed in the restorations were areas of micro-gaps at the TC (tooth/cement interface) or ZC (zirconia/cement interface) resulting in loss of adaptation. There was a significant difference between the groups after the ageing process at both the TC (F=4.762, p<.05) and ZC (F=6.975, p<.05), with group ID2 presenting the best performance. There was a significant difference between TC and ZC in all groups (p<.05) with ZC presenting more gaps in all groups. CONCLUSION: Inlay cavity design including a proximal box and an occlusal extension exhibited better marginal stability than a proximal box without occlusal extension.

Effect of sintering time on microstructure and optical properties of yttria-partially stabilized monolithic zirconia.

OBJECTIVES: To evaluate the impact of speed sintering on the microstructure and optical properties of 3 and 5 mol% yttria-partially stabilized monolithic zirconias. METHODS: 120 plate-shaped zirconia specimens (10x10x 0.4 mm) were prepared from three commercial 5 mol% yttria-partially stabilized zirconia blocks (5Y-PSZs); Katana UTML (Kuraray Noritake), Cercon xtML (Dentsply Sirona), and Zolid FX white (Amann Girrbach), and two commercial 3 mol% yttria-partially stabilized zirconia blocks (3Y-PSZs); Lava Plus (3 M ESPE) and InCoris (Sirona, GmbH). Specimens were either conventional sintered (CS) using a 7-hour program or speed sintered (SS) using a quick 90-minute program. The microstructure was inspected with a scanning electron microscope (SEM), and phase fractions were detected using x-ray diffraction analysis (XRD). Translucency (TP00), and contrast ratio (CR) were obtained using a spectrophotometer (VITA Easyshade V). Color difference (ΔE00) between both sintering processes was calculated with the CIEDE2000 formula. ΔE00 up to 1.8 was set as the acceptability threshold. Data were analyzed using two-way ANOVA, Krusakll-Wallis, and Mann-Whitney U tests. (n = 12, α = .05). RESULTS: Grain size was significantly decreased after SS for all tested materials (P < .0001). The average grain sizes of 5Y-PSZs were significantly larger than those of 3Y-PSZs. The atomic structure, microstructure, and transparency of CS and SS were all affected by the amount of yttria, the size of the crystals, and tetragonality. SS significantly reduced TP00 (F = 7135.95, P < 0.0001) and increased CR (F = 453.21, P < 0.0001). The CS Katana presented the highest TP00 and lowest CR value. ΔE00 between the CS and SS groups were clinically acceptable except for Lava, which had values above the set threshold (1.89). SIGNIFICANCE: SS altered the grain size and internal structure of the tested materials, which was reflected in translucency.

Changes in vasoactive intestinal peptide and tyrosine hydroxylase immunoreactivity in the brain of nest-deprived native Thai hen.

Hyperprolactinemia is associated with incubation behavior and ovarian regression in birds. To investigate the association of prolactin (PRL), vasoactive intestinal peptide (VIP), and dopamine (DA) with the neuroendocrine regulation of incubation behavior, changes in the number of visible VIP-immunoreactive (VIP-ir) neurons in the nucleus inferioris hypothalami (IH) and nucleus infundibuli hypothalami (IN) and tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the nucleus intramedialis (nI) and nucleus mamillaris lateralis (ML) of incubating native Thai hens were compared with those of nest-deprived hens. TH was used as a marker for dopaminergic (DAergic) neurons. Blood samples were collected to determine PRL levels. The localization and the number of visible VIP-ir and TH-ir neurons were determined by immunohistochemistry. Disruption of incubation behavior was accompanied by a precipitous decline in plasma PRL levels. The number of visible VIP-ir neurons in the IH-IN and TH-ir neurons in the nI and ML were high during incubation and decreased when hens were deprived of their nests. This study indicated an association between VIP neurons in the IH-IN and DA neurons in the nI and ML with the degree of hyperprolactinemia, suggesting that the expression of incubation behavior in birds might be, in part, regulated by the DAergic input from the nI and ML to VIP neurons in the IH-IN and subsequent PRL release.

Evaluating the marginal integrity and clinical outcome of posterior zirconia inlay-retained fixed dental prostheses: A randomized clinical trial.

PURPOSE: To assess the short-term outcomes of two different conservative preparation designs for posterior zirconia inlay-retained fixed dental prostheses (IRFDPs) and to analyze the marginal integrity of such restorations. MATERIALS AND METHODS: Thirty patients with a missing maxillary first molar participated in this study and were divided randomly into two groups: one that received abutment preparations for inlay retainers (ID group, n = 20), including a proximal box and an occlusal extension, and one that received only proximal box preparations (PB group, n = 10). Zirconia IRFDPs were bonded in position and followed up after 2 weeks, 6 months, 1 year, and then annually. The in situ restorations were duplicated utilizing a single-step putty/wash impression technique at the 2-week and 2-year follow-up visits. Restoration margins were evaluated under scanning electron microscopy, and marginal continuity was calculated as a percentage of the whole margin at the tooth-cement (TC) interface and the zirconia-cement (ZC) interface. RESULTS: After a mean observation period of 26.2 months, all restorations were still in function. The only complication encountered was debonding of restorations; however, they were re-bonded back in place without issue. The cumulative Kaplan-Meier 2-year survival rate for the IRFDPs was 86.6%. There was a significant difference between the two groups regarding percentage of continuous margins (P < .05) after 2 years, with the percentage of continuous margins in the ID group at 92.8% and 91.5% at the TC and ZC interfaces, respectively, and in the PB group at 84.1% and 78.6% at the TC and ZC interfaces, respectively. CONCLUSION: Zirconia-based monolithic IRFDPs can be recommended for short-term replacement of a single maxillary molar.

Serotonin receptor subtypes influence prolactin secretion in the turkey.

Serotonin (5-HT) stimulation of prolactin (PRL) secretion is mediated through the dopaminergic (DAergic) system, with 5-HT ligands having no direct effect on pituitary PRL release. Infusion of 5-HT into the third ventricle (ICV) or electrical stimulation (ES) of the medial preoptic area (POM) or the ventromedial nucleus (VMN) induces an increase in circulating PRL in the turkey. These increases in PRL do not occur when a selective antagonist blocks the D(1) dopamine (DA) receptors in the infundibular area (INF). In this study, the ICV infusion of (R)(-)-DOI hydrochloride (DOI), a selective 5-HT(2A) eceptor agonist, caused PRL to increase. Pretreatment with Ketanserin tartrate salt (KETAN), a selective 5-HT(2A) receptor antagonist, blocked DOI-induced PRL secretion, attesting to the specificity of the response. DOI-induced PRL secretion was prevented when the D(1) DA receptors in the INF were blocked by the D(1) DA receptor antagonist, R(+)-SCH-23390 hydrochloride microinjection, suggesting that the DAergic activation of the vasoactive intestinal peptide (VIP)/PRL system is mediated by a stimulatory 5-HT(2A) receptor subtype. The DOI-induced PRL increase did not occur when (+/-)-8-OH-DPAT (DPAT) was concurrently infused. DPAT is a 5-T(1A) receptor agonist which appears to mediate the inhibitory influence of 5-HT on PRL secretion. When DPAT was microinjected directly into the VMN, it blocked the PRL release affected by ES in the POM. These data suggested that when 5-HT(2A) receptors are activated, they influence the release of DA to the INF. When 5-HT(1A) receptors are stimulated, they somehow inhibit the PRL-releasing actions of 5-HT(2A) receptors. This inhibition could take place centrally, or it could occur postsynaptically at the pituitary level. It is known that D(2) DA receptors in the pituitary antagonize PRL-releasing effect of VIP. A release of DA to the pituitary, initiated by 5-HT(1A) receptors, could effectively inhibit PRL secretion.

Dopamine-melatonin neurons in the avian hypothalamus and their role as photoperiodic clocks.

A timing mechanism in the brain governs reproduction in seasonally breeding temperate zone birds by triggering gonad development in response to long days in the spring. The neural mechanism(s) responsible for the timing and induction of reproductive activity by this clock are unknown. Utilizing in situ hybridization, immunocytochemistry and reverse transcriptase-polymerase chain reaction techniques, a group of dopamine (DA) neurons in the premammillary nucleus (PMM) of the caudal turkey hypothalamus that synthesize and colocalize both DA and melatonin (MEL) were identified. In addition, these neurons are found to express clock genes and the circadian photoreceptor melanopsin. DA-MEL neurons reach threshold activation (c-fos expression) when a light pulse is given during the photosensitive phase. This is associated with increases in the number of gonadotropin releasing hormone-I (GnRH-I) neurones activated, as well as an up-regulation of GnRH-I mRNA expression. The expression of tyrosine hydroxylase (TH; the rate limiting enzyme in DA biosynthesis) and tryptophan hydroxylase 1, (TPH1; the first enzyme in MEL biosynthesis) and consequently DAergic-MELergic activities are associated with the daily light-dark cycle. TPH1 mRNA expression shows low levels during the light phase and high levels during the dark phase of the light/dark illumination cycle and is 180 degrees out of phase with the rhythm of TH mRNA expression. Hypothalamic DA-MEL neurons may constitute a critical cellular process involved in the generation and expression of seasonal reproductive rhythms and suggests a previously undescribed mechanism(s) by which light signals gain access to neural targets.

Vasoactive intestinal peptide and its role in continuous and seasonal reproduction in birds.

Native Thai chicken, an equatorial species breeds throughout the year, whereas turkeys are seasonal temperate zone breeder whose reproductive cycle is terminated by the onset of photorefractoriness. This study investigated VIPergic activity throughout a reproductive cycle in both species, hypothesizing that the differential expression of vasoactive intestinal peptide (VIP) would provide an insight into the differing reproductive strategies of the two species. Distribution of VIP neurons in the native Thai chicken and a comparison of VIPergic activity in the nucleus inferioris hypothalami (IH) and nucleus infundibuli hypothalami (IN) were investigated. VIP immunoreactivity was found throughout the native Thai chicken brain, predominantly located within the IH-IN. The pattern of VIP distribution in the native Thai chicken supports the findings reported in temperate zone species. Unlike the turkey, where there is a dissociation between VIPergic activity and prolactin levels during photorefractoriness, in the native Thai chicken, which do not express photorefractoriness, changes in VIP immunoreactive (VIP-ir) neurons within the IH-IN were directly correlated with prolactin throughout the reproductive cycle. VIPergic activity reached its lowest level after hatching of the chicks in the native Thai chicken, while in the turkey VIPergic activity was lowest only after exposure to a short day photoperiod and the acquisition of photosensitivity. This suggests that VIP neurons in the IH-IN may play a pivotal role in regulating the reproductive cycle and its differential expression following hatching of the young may, in part, account for the difference in reproductive mode between equatorial, continually breeding, non-photoperiodic birds and seasonally breeding, photoperiodic birds.

Effects of nest-deprivation on hypothalamic mesotocin in incubating native Thai hens (Gallus domesticus).

Avian mesotocin (MT) is homologous to oxytocin in mammals. Native Thai chickens (Gallus domesticus) strongly express maternal behaviors including incubation and rearing. However, the role of MT during incubation behavior has never been studied. The objective of this study was to determine the physiological function(s) of the MTergic system in incubation behavior in native Thai chickens. The brains were collected from incubating (INC) and nest-deprived (ND) hens at different time points (days 3, 6, 8, 10, 14, 18, and 21; n=6). Immunohistochemistry technique was used to compare the numbers of MT-immunoreactive (-ir) neurons between the INC and ND hens within the Nucleus supraopticus, pars ventralis (SOv), Nucleus preopticus medialis (POM), and Nucleus paraventricularis magnocellularis (PVN). The results revealed that the numbers of MT-ir neurons within the SOv, POM, and PVN remained high during the incubating stage. The number of MT-ir neurons in the SOv was lower than that of the POM and PVN. Disruption of incubation behavior by nest deprivation caused the numbers of MT-ir neurons within the SOv, POM, and PVN to decrease throughout the observation periods. For the first time, this study demonstrates that the MTergic system within the SOv, POM, and PVN may be involved with incubation behavior. In addition, these results further suggest that the MTergic neurons in these nuclei are not only regulated by rearing behavior but also might have a role in the initiation and maintenance of incubation behavior in this tropical species.

Distribution of mesotocin-immunoreactive neurons in the brain of the male native Thai chicken.

Mesotocin (MT), a homolog of oxytocin (OT) in mammals, is a nonapeptide neurohypophysial hormone that is mainly synthesized in specific neuronal groups within the hypothalamus and released from the posterior pituitary gland in amphibian, reptilian, and avian species. MT is associated with the neuroendocrine regulation of reproductive cycle and maternal behaviors in female native Thai chickens. Male birds exhibit parental behaviors as well. However, there are limited data regarding the role(s) of the MTergic system in males. Thus, the objective of this study was to elucidate the localization of the MT neuronal groups in the brain of male native Thai chickens. The distributions of MT-immunoreactive (-ir) neurons and fibers in the brain were studied utilizing immunohistochemistry technique. The results revealed that MT-ir neurons and fibers were distributed throughout the brain and extensively in the diencephalon. MT-ir neurons and fibers were predominantly located within the nucleus supraopticus, pars ventralis (SOv), nucleus preopticus medialis (POM), nucleus ventrolateralis thalami (VLT), nucleus paraventricularis magnocellularis (PVN), and regio lateralis hypothalami (LHy), suggesting that MT neurons in these nuclei might be involved in the reproductive activities and/or parental behavior in the male chickens. In addition, the numbers of MT-ir neurons within the SOv and POM were significantly higher than those of the VLT, PVN, and LHy. More importantly, the number of MT-ir neurons in the SOv was high in the male brain when compared with the female brain, indicating that the MTergic system in the SOv might play a significant role in male reproductive activities in this equatorial species.

Normalization of transfection efficiency using the beta-lactamase gene of the pGL3 luciferase vector in primary anterior pituitary cells.

The beta-galactosidase reporter gene is commonly used as a control for transfection efficiency in the promoter reporter assay system. While investigating vasoactive intestinal peptide response elements in the promoter of the prolactin gene, we found that primary pituitary cells from turkey hens highly expressed endogenous beta-galactosidase. Therefore, we developed a new protocol for determining transfection efficiency using the beta-lactamase gene, which is present on many expression vectors. Transcript levels of beta-lactamase were measured by RT-PCR after transfection of different amounts of the pGL3-basic and pGL3-control vectors. A high correlation was observed between the amount of plasmid transfected and beta-lactamase mRNA levels. Although no eukaryotic promoter was present, there was apparently leaky expression of the beta-lactamase gene. Expression of beta-lactamase was independent of expression from the simian virus 40 or turkey prolactin promoters cloned upstream of the luciferase gene.