Progress 09/01/04 to 08/31/08
Outputs
OUTPUTS: Brain monoamines are linked to the activity of the reproductive neuroendocrine system in the turkey. Functional changes in dopaminergic, adrenergic, noradrenergic and serotonergic systems appear to have reproductive and behavioural consequences. Recent findings from our laboratory showed the presence of dopamine-melatonin (DA-MEL) neurones in the premammillary nucleus (PMM) of the turkey hypothalamus where DA and MEL are synthesized and co-localized. Circadian activity rhythms generated by DA-MEL neurones are entrained to the day ⁄ night cycle. At this time, it is not known if nonphotic cues mediated by serotonergic and catecholaminergic innervation of DA-MEL neurones in the PMM modulate their response to light. We investigated the monoaminergic systems that innervate the PMM in the hypothalamus of reproductively active hens utilizing a tract-tracing technique, and to determine their neural activity by measuring the expression levels of their rate-limiting enzymes in reproductively active and quiescent female turkeys. For tract tracing, the fluorescent dye DiI was injected unilaterally (0.1 ul) into the PMM using a 30-gauge stainless steel cannula connected by polyethylene tubing to a 0.5- ul syringe. To determine the neurochemical identity of DiI-labelled cell bodies, a procedure for double labeling of the same tissue with serotonin (5-HT), tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), phenylethanolamine- Nmethyltransferase (PNMT) antisera was employed. Changes in the activities of serotonergic, dopaminergic, adrenergic and noradrenergic neuronal systems projecting to the PMM were measured at different reproductive states with in situ hybridization (ISH) techniques, using tryptophan hydroxylase 2 (TPH2) and TH mRNA expression, respectively. The results obtained were presented at national and international meetings and resulted in two publications. One is published in Journal of Neuroendocrinology and one in General and Comparative Endocrinology. PARTICIPANTS: Graduate student and a postdoctoral associate TARGET AUDIENCES: Research professionals in avian neuroendocrinology. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Cells labeled with DiI were found in anatomically discrete areas in or near the hypothalamus and the brainstem. Double immunocytochemistry confirmed that there were serotonin, DBH and PNMT fibres in close apposition to DA-MEL neurones. TPH2 mRNA expression in serotonin neurones was found in several nuclei, and its most abundant mRNA expression was seen in the nucleus Locus ceruleus of laying and incubating hens. TH mRNA expression levels in the six catecholaminegic areas labeled with DiI was measured across the different reproductive states. In the nucleus tractus solitarius (adrenergic), the highest level of TH mRNA expression was found in photorefractory hens and the lowest level in incubating hens. These observed patterns of serotonin ⁄ catecholamine neuronal distribution and their variable interactions with PMM DA-MEL neurones during different reproductive states may offer a significant neuroanatomical basis for understanding the control of avian reproductive seasonality.
Publications
- Leclerc B., Kang, S., Howell, C., Mauro, L., and El Halawani, M.E. 2008. Circadian expression of clock genes in the pineal gland, visual suprachiasmatic nucleus (vSCN) and premammillary nucleus (PMM) in photostimulated turkey hens. Poult. Sci. 87(suppl.1): 14. (abstract)
- El Halawani, M. E., Kang, S. W., Thayananuphat, A., Leclerc, B. 2008 Dopamine-melatonin neurons in the avian hypothalamus and their role as photoperiodic clocks. 9th Int. Symposia. Avian Endocrinol. 48, (abstract).
- Kang, SW., Leclerc, B., Mauro LJ., and Mohamed E. El Halawani, ME. 2008.. Serotonergic and Catecholaminergic Interactions with Co-localized Dopamine-Melatonin Neurons in the Hypothalamus of the Female Turkey. J Neuroendocrinol. doi: 10.1111/j.1365-2826.2008.01804.x
- El Halawani ME., Kang, SW., Leclerc, B., Kosonsiriluk, S., Chaiseha,Y. 2008. Dopamine-melatonin neurons in the avian hypothalamus and their role as photoperiodic clocks. Gen. Comp Endocrinol. http://dx.doi.org/10.1016/j.ygcen.2008.11.030
- Kang, S. W., Leclerc, B., Mauro, L.J., and EI Halawani, M. E. 2008.Serotonergic and catecholaminergic interactions with co- localized dopamine-melatonin neurons in the hypothalamic premammillary nucleus of the female turkey during different reproductive states. Poult. Sci. 87(suppl.1): 15-16. (abstract)
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: We investigated the effect of light exposure at different circadian times upon the hypothalamus and components of the circadian system in female turkeys, using the early gene c-fos mRNA expression, measured by in situ hybridization (ISH), as an indicator of light-induced neuronal activity. Levels of c-fos mRNA in these areas were compared after turkey hens (on a daily 6-h light period) had been exposed to a 30-min period of light occurring at 8, 14, or 20 h after the onset of first light of the day (subjective dawn). Non-photostimulated control birds were harvested at the same times To identify the dopamine (DA) neurones that mediate the stimulatory influences of photoperiod on the reproductive system, we examined c-fos mRNA induction in DA, gonadotropin releasing hormone-I (GnRH-I) and vasoactive intestinal peptide (VIP) neurones in the turkey hypothalamus. To further establish the role of DA neurons within the premammillary nucleus (PMM) of the caudal turkey hypothalamus
as putative photoreceptive neurons that encode light information, and verify the coexistence of DA/melatonin (MEL) within these neurons, we used double-label immunocytochemistry (ICC) or ISH/ICC for, tyrosine hydroxylase (TH; the rate limiting enzyme in DA biosynthesis) and melatonin (MEL) immunoreactivity (ir). Further verification of the coexistence of the DA/MEL system within PMM neurons was established using MEL-ir with TH mRNA, TH-ir with tryptophan hydroxylase-1 (TPH1, the rate limiting enzyme in MEL biosynthesis), mRNA, 5-HT N-acetyltransferase (AANAT; a key regulatory enzyme in MEL synthesis)-ir with TH mRNA and TH-ir with AANAT mRNA. To examine the possibility that DA and MEL might be controlled by a circadian oscillator(s), we measured mRNA expression of TH and TPH1, within the PMM at different circadian times in the day/night cycle under different day length (6h light: 18h dark and 16h L: 9h Dark) To establish that seasonal photoperiodism and its associated changes in
reproductive neuroendocrine activity may be linked to the circadian-like activity phases of DA-MEL co-localized neurons we measured TH and TPH1 mRNA expression at different times in reproductively quiescent adult photosensitive female turkeys. The results obtained were presented at national and international meetings and resulted in three publications. Two are published in Journal of Neuroendocrinology and one in Neuroscience.
PARTICIPANTS: Graduate student and a postdoctoral associate
TARGET AUDIENCES: Research professionals in avian neuroendocrinolgy
Impacts
In female turkeys photostimulated within the photoinducibile phase (14 h), in contrast to before or after, c-fos mRNA was significantly increased in the nucleus commissurae pallii (nCPa) and nucleus premamillaris (PMM) where GnRH-I and DA neurons reside, respectively. However, the magnitudes of the c-fos response in the pineal gland and nucleus suprachiasmaticus, pars visualis (vSCN) were similar irrespective of where in the dark period the pulses occurred. The lack of c-fos upregulation in the pineal gland and vSCN following photostimulation during the photoinducible phase lends credence to the hypothesis that these areas are not involved in the photic initiation of avian reproduction. On the other hand, c-fos mRNA increases in the nCPa support other studies showing that these areas are involved in the onset of reproductive behaviour initiated by long day lengths. This study provides novel data showing that the PMM in the caudal hypothalamus is involved in the
neuronally mediated, light-induced initiation of reproductive activity in the turkey hen. The only area where DA neurones were activated when the birds were provided with light was in the PMM. The number of activated DA neurones was significantly greater when light was provided at 14 h (during the photoinducible phase) than at 8 h or 20 h. At 14 h, there was also an increase in the number of GnRH-I neurones activated in the area of the nCPa, as well as an up-regulation of GnRH-I mRNA expression. These results are the first evidence to demonstrate a relationship between the dopaminergic system in the PMM and the GnRH-I system in the nCPa during the photoinduction of avian reproductive activity. To further clarify the role of PMM neurons in coding daylight information, we showed that by using double-label immunocytochemistry (ICC) these neurons are immunoreactive (ir) to both tyrosine hydroxylase (TH; the rate limiting enzyme in DA biosynthesis) and melatonin (MEL). Moreover, we found
these neurons to express tryptophan hydroxylase 1 (TPH1; the first enzyme in MEL biosynthesis) and 5-HT N-acetyltransferase (AANAT; a key regulatory enzyme in MEL synthesis) mRNAs Both TH and TPH1 mRNAs were shown to cycle rhythmically, and with opposite phases, in PMM neurons of birds kept under a diurnal illumination cycle (12-h light/dark; LD). These neurons could also generate 24 h TH and TPH1 mRNA expression rhythms with the same phase relationship in constant light (LL) and constant dark (DD). In addition, the expression patterns and amplitudes of TH and TPH1 mRNAs were different between long and short photoperiods. These findings may form the basis for an endogenous dual-oscillator circadian system within PMM DA-MEL co-localized neurons controlling reproductive seasonality in birds.
Publications
- Thayananuphat A, Kang SW, Bakken T, Millam JR, El Halawani ME. 2007. Rhythm-Dependent Light Induction of the c-fos Gene in the Turkey Hypothalamus. J Neuroendocrinol.19, 407-417.
- Thayananuphat A, Kang SW, Bakken T, Millam JR, El Halawani ME. 2007. Rhythmic Dependent Light Induction of Gonadotrophin-Releasing Hormone-I Expression and Activation of Dopaminergic Neurones within the Premammillary Nucleus of the Turkey Hypothalamus J Neuroendocrinol. 19, 399-406.
- Kang SW, Thayananuphat A, Bakken T, El Halawani ME. 2007. Dopamine-Melatonin Neurons in the Avian Hypothalamus Controlling Seasonal Reproduction. Neuroscience 150, 223-233.
- Kang S., Thayananuphat A., T. Bakken T., and M. El Halawani M. 2007. Dopamine-melatonin neurons in the turkey hypothalamus controlling seasonal reproduction. Poult. Sci. 86 ( Suppl. 1): 223 (Abstract).
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Progress 01/01/06 to 12/31/06
Outputs
We identified three serotonergic neuronal groups in the turkey brainstem. (1) a ventrorostral mesencephalic group, (2) a dorsal mesencephalo-pontine group and (3) a ventro-caudal ponto-medullary group. Serotonergic activities in groups 1 and 2 are much reduced in reproductively quiescent hens. These findings indicate that the variations in reproductive hormones expression and secretion observed across the turkey reproductive cycle may be explained, in part, by changes in neuronal serotonergic activity in the turkey brainstem. The effects of serotonin on the reproductive neuroendocrine system have been shown to be mediated via two receptor subtypes, a stimulatory 5-HT2A/2C and inhibitory 5-HT1A subtypes. Both receptor subtypes are found in the nucleus preopticus medialis (POM), the nucleus premamillaris (PMM) and nucleus mamillaris lateralis (ML) and the nucleus paraventricularis hypothalami (PVN). A significant difference in 5-HT1A receptor expression is seen in the
ML, PMN, and POM in birds of different reproductive stages, with photorefractory birds showing the highest and incubating birds the lowest expression. In the ML, 5-HT2C receptors density is significantly higher in incubating birds than short day birds. In the ML and PMN, 5-HT1A receptors are colocalized with tyrosine hydroxylase immunoreactive cells. To identify the DAergic neuronal group involved in the reproductive neuroendocrine system activation, electrical stimulation in the medial preoptic area (ES/MPOA) induced LH and PRL secretion as well as c-fos mRNA expression are employed. ES/MPOA of anaesthetised laying turkeys for 30 min increases circulating LH and PRL levels. It also induces c-fos mRNA expression by all GnRH-I neurones within the septopreoptic region, implying that GnRH-I neurones in this region share similar circuitry. VIP neurones within the nucleus infundibularis are the only VIP group to show c-fos mRNA expression, suggesting their involvement in ES/MPOA induced
PRL release. c-fos mRNA expression is also observed in a subgroup of dopamine neurones in the ML.
Impacts
The neuro-chemical circuitry regulating reproductive neuroendocrine system in the turkey is far from clear. The neurotransmitters, serotonin/dopamine and neurohormones, vasoactive intestinal peptide/gonadotrophin releasing hormone-I are key neuro-chemicals regulating the reproductive system. However, their anatomical locations have not been identified. We have been using several techniques including immunocytochemistry, electrophysiology, in situ hybridization to identify the group/subgroup of these neuronal systems which are involved in the regulation of reproductive system. The findings will allow us to understand the mechanism(s) concerned with the initiation and termination of reproductive activity. This will aide establishing methods to enhance reproductive efficiency.
Publications
- Al-Zailaie KA, Kang SW, Youngren OM, Thayananuphat A, Bakken T, Chaiseha Y, Millam JR, Proudman JA, El Halawani ME. 2006. Identification of dopamine, gonadotrophin-releasing hormone-I, and vasoactive intestinal Peptide neurones activated by electrical stimulation to the medial preoptic area of the Turkey hypothalamus: a potential reproductive neuroendocrine circuit. J Neuroendocrinol. 18(7):514-525.
- Bakken T, Kang SW, Thayananuphat A, Proudman JA, and El Halawani ME. 2006. Expression of 5-HT1A and 5-HT2C receptors in the hypothalamus during the turkey reproductive cycle: Colocalization with tyrosine hydroxylase. Poultry Sci 85(suppl.1):42 (Abstract)
- Thayananuphat A, Kang SW, Bakken T, Millam JR, and El Halawani ME. 2006. Dopamine and gonadotrophin releasing hormone-I neuronal activation following photostimulation in the turkey. Poultry Sci 85(suppl.1):43 (Abstract)
- Kang SW, Bakken T, and El Halawani ME. 2006. Characterization of the serotonergic system in the brainstem during the turkey reproductive cycle. Poultry Sci 85(suppl.1): 43 (Abstract). Poultry Sci 85(suppl.1):43 (Abstract)
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Progress 01/01/05 to 12/31/05
Outputs
This study investigated the role of Ca2+ signaling in mediating the regulatory effects of vasoactive intestinal peptide (VIP) and dopamine (dopamine) on PRL (PRL) mRNA levels and prolactin release in cultured turkey anterior pituitary cells. VIP or the L-type Ca2+ channel activator increased [Ca2+]i ,an effect abolished by preincubating the cells with a D2 dopamine receptor agonist (D2AG) or an L-type Ca2+ channel blocker. Similarly, either L-type Ca2+ channel blocker or D2Ag diminished the VIP/ L-type Ca2+ channel activator stimulatory effect on prolactin expression and release. On the other hand, depleting the intracellular Ca2+ stores, showed no effect on basal or VIP-stimulated prolactin mRNA levels. These results suggest that intracellular Ca2+ represents a common signal transduction pathway through which VIP and dopamine can exert antagonistic control on prolactin synthesis and release in avian lactotrophs. The induction of a neuronal activation marker, c-fos
mRNA, was observed in dopamine (DA) and Mesotocin (MT) neurons during the transition from incubating eggs to brooding of poults in turkeys. This finding suggested the implication of DA and MT with this transition. Hens receiving D2 DA or oxytocin (OT) receptor antagonist showed partial or no brooding behavior within the 30-minute experimental period. C-fos mRNA induction was markedly less in the preoptic nucleus, the critical nucleus involved in the neural control of maternal behavior in hens received D2 DA or OT receptor antagonist as compared to that in vehicle treated control hens. These data demonstrated that DA and MT might be two of the key neurotransmitter/neurohormone involved in the transition from incubating to brooding behavior 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 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 D1 dopamine (DA) receptors in the infundibular area (INF).
Impacts
The neuro-chemical circuitry regulating reproductive hormones in the turkey is far from clear. Serotonin, dopamine and vasoactive intestinal peptide stimulate PRL secretion. However, their anatomical location has not been identified. We have been using several techniques including immunocytochemistry, electrophysiology, in situ hybridization to identify the group/subgroup of these neuronal systems which are involved in the regulation of reproductive hormones. The findings will allow us to understand the mechanism(s) concerned with the initiation and termination of reproductive activity. This will aide establishing methods to enhance reproductive efficiency.
Publications
- Chaiseha, Yupaporn and El Halawani, Mohamed. 2005. Neuroendocrinology of the female turkey reproductive cycle. J Poult Sci. 42: 87-100.
- Al Kahtane A, Kang SW, Kannan M, El Halawani ME. 2005. Regulation of Prolactin Gene Expression by Vasoactive Intestinal Peptide and Dopamine: Role of Ca2+ Signaling. J. Neuroendocrinol. 17:649-655.
- El Halawani M, Youngren O, Proudman J, Chaiseha Y. 2005. Serotonin receptor subtypes influence prolactin secretion in the turkey. Poult Sc. 84 (Suppl. 1), 45.
- Thayananuphat A,Youngren O, Kang S, Bakken T, Proudman J, El Halawani M. 2005. Effects of dopamine and oxytocin receptor antagonists on the transition to brooding of young in turkeys. Poult Sc. 84 (Suppl. 1), 31.
- Kang S,Youngren O, Bakken T, El Halawani M. 2005. Distribution of serotonergic neurons and fibers and interaction with dopaminergic neurons in the turkey hypothalamus and brainstem. Poult Sc. 84 (Suppl. 1), 74.
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Progress 01/01/04 to 12/31/04
Outputs
Studies have just started on this project. One study is designed to investigate the interaction between the dopaminergic and serotonergic systems in the regulation of PRL and LH in breeder hen turkeys. The second study investigates the distribution of 5-HT 2A/2C and 5-HT1A receptors in the turkey hypothalamus as measured by in situ hybridization.
Impacts
Understanding the mechanism(s) by which the DAergic and the 5-HTergic systems interact in regulating the turkey reproductive cycle will aide in developing tools to enhance reproductive efficiency.
Publications
- No publications reported this period
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