S. W. Kang

Melanopsin expression in dopamine-melatonin neurons of the premammillary nucleus of the hypothalamus and seasonal reproduction in birds

Melanopsin (OPN4) is a photoreceptive molecule regulating circadian systems in mammals. Previous studies from our laboratory have shown that co-localized dopamine-melatonin (DA-MEL) neurons in the hypothalamic premammillary nucleus (PMM) are putatively photosensitive and exhibit circadian rhythms in DAergic and MELergic activities. This study investigates turkey OPN4x (tOPN4x) mRNA distribution in the hypothalamus and brainstem, and characterizes its expression in PMM DA-MEL neurons, using in situ hybridization (ISH), immunocytochemistry (ICC), double-label ISH/ICC, and real time-PCR. The mRNA encoding tOPN4x was found in anatomically discrete areas in or near the hypothalamus and the brainstem, including nucleus preopticus medialis (POM), nucleus septalis lateralis (SL), PMM and the pineal gland. Double ICC, using tyrosine hydroxylase (TH, the rate limiting enzyme in DA synthesis)-and OPN4x antibodies, confirmed the existence of OPN4x protein in DA-MEL neurons. Also, tOPN4x mRNA expression was verified with double ISH/ICC using tOPN4x mRNA and TH immunoreactivity. PMM and pineal gland tOPN4x mRNA expression levels were diurnally high during the night and low during the day. A light pulse provided to short day photosensitive hens during the photosensitive phase at night significantly down-regulated tOPN4x expression. The expression level of tOPN4x mRNA in PMM DA-MEL neurons of photorefractory hens was significantly lower as compared with that of short or long day photosensitive hens. The results implicate tOPN4x in hypothalamic PMM DA-MEL neurons as an important component of the photoreceptive system regulating reproductive activity in temperate zone birds.

DOPAMINE-MELATONIN NEURONS IN THE AVIAN HYPOTHALAMUS CONTROLLING SEASONAL REPRODUCTION

Day length cues are used by temperate zone birds to time seasonal changes in reproductive physiology and behavior. However, the neuronal and neurochemical circuits used to measure day length (photoperiodic time measurement; PTM), transduce light information and activate the reproductive neuroendocrine system have not been definitely established. Recent findings from our laboratory provide data showing dopamine (DA) neurons within the premammillary nucleus (PMM) of the caudal turkey hypothalamus are putative photoreceptive neurons. These neurons reach threshold activation when a brief pulse of light is provided during the photo-inducible phase for photosexual stimulation. 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 but not neuronal tryptophan hydroxylase 2 mRNA (TPH 2; the rate limiting enzyme in 5-HT pathway). 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.

Rhythmic Dependent Light Induction of Gonadotrophin‐Releasing Hormone‐I Expression and Activation of Dopaminergic Neurones within the Premammillary Nucleus of the Turkey Hypothalamus

Our previous studies using turkey hens have demonstrated that c‐fos mRNA (a marker of neuronal activation) is expressed in gonadotrophin‐releasing hormone‐I (GnRH‐I), vasoactive intestinal peptide (VIP) and dopamine (DA) neurones following electrical stimulation in the preoptic area. DA has been shown to have both stimulatory and inhibitory effects on the GnRH‐I/luteinising hormone (LH), follicle‐stimulating hormone (FSH) and VIP/prolactin (PRL) systems. To identify the DA neurones that mediate the stimulatory influences of photoperiod on the reproductive system, we examined c‐fos mRNA induction in DA, GnRH‐I, and VIP neurones in the turkey hypothalamus using a dark‐interruption experimental design. A 30‐min light period was provided to short day (6L : 18D) photosensitive turkeys at times when birds were responsive to light (14 h after first light) and at times when birds were unresponsive to light (8 h and 20 h after first light). The only area where DA neurones were activated when the birds were provided with light was in the nucleus premammillaris (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 nucleus commissura pallii (nCPa), as well as an up‐regulation of GnRH‐I mRNA expression. No expression of c‐fos mRNA was observed in VIP neurones in the nucleus infundibularis or up‐regulation of VIP mRNA expression in any of the experimental light treatments. 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.

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.

Regulation of prolactin secretion by dopamine at the level of the hypothalamus in the turkey.

Avian prolactin (PRL) secretion is regulated by vasoactive intestinal peptide (VIP) neurons residing in the infundibular nuclear complex (INF) of the hypothalamus. This VIPergic activity is modulated by stimulatory dopaminergic inputs. Dynorphin, serotonin (5-HT), dopamine (DA) and VIP all appear to stimulate PRL secretion along a hypothalamic pathway, expressing ĸ opioid, serotonergic, dopaminergic and VIPergic receptors in succession, with the VIPergic system as the final mediator. Electrical stimulation (ES) within the turkey hypothalamus at the level of the medial preoptic area (POA), the ventromedial hypothalamic nucleus (VMN), the INF or the median eminence (ME) results in the release of PRL. When the selective D1 DA receptor antagonist SCH-23390 HCl was infused intraventricularly at the rate of 10 nmol/min, ES in the POA or VMN was unable to increase PRL levels, while ES in the INF and ME did increase PRL to the same level as that of controls. These results were interpreted to suggest that the D1 DA receptors involved in PRL release lie caudally to the VMN and dorsally to the INF. Bilateral microinjections (50 ng) of the D1 DA receptor agonist SKF-38393 HCl into the POA or VMN failed to produce any increase in PRL, while similar microinjections in the INF increased PRL significantly within 15 min. Bilateral microinjections of the D1 DA antagonist (50 ng) into the INF blocked the rise in PRL associated with ES in the POA. Bilateral microinjections of a D2 DA antagonist (50 ng) into the INF failed to block PRL secretion induced by ES in the POA. Tract tracing, using double-label immunocytochemistry, revealed the presence of a monosynaptic dopaminergic pathway projecting from the POA to the INF. These data imply that the only hypothalamic D1 DA receptors involved in the regulation of avian PRL secretion are those residing within the INF in the same region as the VIP neurons known to be involved in PRL secretion.

Rhythm-dependent light induction of the c-fos gene in the Turkey hypothalamus

Day length (photoperiod) is a powerful synchroniser of seasonal changes in the reproductive neuroendocrine activity in temperate‐zone birds. When exposed to light during the photoinducible phase, reproductive neuroendocrine responses occur. However, the neuroendocrine systems involved in avian reproduction are poorly understood. We investigated the effect of light exposure at different circadian times upon the hypothalamus and components of the circadian system, using c‐fos mRNA expression, measured by in situ hybridisation, 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. In birds, 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), nucleus premamillaris (PMM), eminentia mediana (ME), and organum vasculosum lamina terminalis (OVLT). Photostimulation increased c‐fos mRNA expression in the pineal gland, nucleus suprachiasmaticus, pars visualis (vSCN) and nucleus inferioris hypothalami compared to that of their corresponding nonphotostimulated controls. However, the magnitudes of the responses in these areas were similar irrespective of where in the dark period the pulses occurred. No c‐fos mRNA was induced in the nucleus infundibulari, in response to the 30‐min light period at any of the circadian times tested. The lack of c‐fos up‐regulation 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, ME, and OVLT support other studies showing that these areas are involved in the onset of reproductive behaviour initiated by long day lengths. The present 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.

Dopamine infusion into the third ventricle increases gene expression of hypothalamic vasoactive intestinal peptide and pituitary prolactin and luteinizing hormone β subunit in the turkey

Turkey prolactin (PRL) secretion is controlled by vasoactive intestinal peptide (VIP) neurons residing in the infundibular nuclear complex (INF) of the hypothalamus. The VIPergic activity is modulated by dopamine (DA) via stimulatory D(1) DA receptors. DA (10 nmol/min for 40 min) was infused into the third ventricle of laying turkey hens to study its effect on circulating PRL, hypothalamic VIP and pituitary PRL and LHbeta subunit mRNA levels. Plasma PRL was significantly elevated after 20 min of DA infusion and remained elevated 30 min after cessation of infusion. Hypothalamic VIP mRNA content was significantly greater in the INF of DA-infused birds than it was in the INF of vehicle-infused control birds. No increase in VIP mRNA due to DA infusion was noted in the preoptic area. Pituitary PRL and LHbeta subunit mRNAs were increased in DA-infused hens as compared to vehicle-infused controls but the rate of increase was more in PRL than LHbeta subunit. This study demonstrates that exogenous DA activates hypothalamic VIP gene expression and this increased expression is limited exclusively to the avian INF. The increased VIP mRNA in the INF is correlated with increased levels of circulating PRL and PRL and LHbeta mRNAs in the anterior pituitary.

The Role of Prolactin in Reproductive Failure Associated with Heat Stress in the Domestic Turkey

Abstract Reproductive failure associated with heat stress is a well-known phenomenon in avian species. Increased prolactin (PRL) levels in response to heat stress have been suggested as a mechanism involved in this reproductive malfunction. To test this hypothesis, laying female turkeys were subjected to 40°C for 12 h during the photo-phase daily or maintained at 24–26°C. Birds in each group received oral treatment with parachlorophenyalanine (PCPA; 50 mg/kg BW/day for 3 days), an inhibitor of serotonin (5-HT) biosynthesis, or immunized against vasoactive intestinal peptide (VIP). Both treatments are known to reduce circulating PRL levels. Nontreated birds were included as controls. In the control group, high ambient temperature terminated egg laying, induced ovarian regression, reduced plasma luteinizing hormone (LH) and ovarian steroids (progesterone, testosterone, estradiol) levels, and increased plasma PRL levels and the incidence of incubation behavior. Pretreatment with PCPA reduced (P < 0.05) heat stress-induced decline in egg production, increase in PRL levels, and expression of incubation behavior. Plasma LH and ovarian steroid levels of heat stressed birds were restored to that of controls by PCPA treatment. As in PCPA-treated birds, VIP immunoneutralization of heat-stressed turkeys reduced (P < 0.05) circulating PRL levels and prevented the expression of incubation behavior. But it did not restore the decline in LH, ovarian steroids, and egg production (P > 0.05). The present findings indicate that the detrimental effect of high temperature on reproductive performance may not be related to the elevated PRL levels in heat-stressed birds but to mechanism(s) that involve 5-HT neurotransmission and the induction of hyperthermia.

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

The neural and neurochemical substrates regulating reproduction in birds remain vaguely defined. The findings that electrical stimulation in the medial preoptic area (ES/MPOA) or intracerebroventricular infusion of dopamine (DA) stimulated luteinising hormone (LH) and prolactin (PRL) release in female turkeys, led to the suggestion that ES/MPOA might help to clarify the DA circuitry regulating LH and PRL. We used c‐fos mRNA and tyrosine hydroxylase immunoreactivity as measured by double in situ hybridisation/immunocytochemistry (ISH/ICC) to determine which group/subgroup of DA neurones was activated following unilateral ES/MPOA. To establish that the reproductive neuroendocrine system was activated, double ISH/ICC was also conducted on c‐fos/gonadotrophin‐releasing hormone‐I (GnRH‐I) and c‐fos/vasoactive intestinal peptide (VIP). Changes in circulating LH and PRL were determined by radioimmunoassay. Unilateral ES/MPOA (100 µA, right side) of anaesthetised laying turkeys for 30 min increased circulating LH and PRL levels. It also induced c‐fos mRNA expression on the ipsilateral side 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 were the only VIP group to show c‐fos mRNA expression, suggesting their involvement in ES/MPOA induced PRL release. c‐fos mRNA expression was also observed in a subgroup of DA neurones in the nucleus mamillaris lateralis (ML). To our knowledge, the present study is the first to show that activation of DAergic cells in the ML is associated with the activation of GnRH‐I and VIP neurones and the release of LH and PRL. It is likely that ES/MPOA activated VIP/GnRH‐I neurones via activation of DA neurones in the ML, as this was the only DA subgroup that showed c‐fos mRNA expression.

Dopamine and mesotocin neurotransmission during the transition from incubation to brooding in the turkey.

We investigated the neuroendocrine changes involved in the transition from incubating eggs to brooding of the young in turkeys. Numbers of mesotocin (MT; the avian analog of mammalian oxytocin) immunoreactive (ir) neurons were higher in the nucleus paraventricularis magnocellularis (PVN) and nucleus supraopticus, pars ventralis (SOv) of late stage incubating hens compared to the layers. When incubating and laying hens were presented with poults, all incubating hens displayed brooding behavior. c-fos mRNA expression was found in several brain areas in brooding hens. The majority of c-fos mRNA expression by MT-ir neurons was observed in the PVN and SOv while the majority of c-fos mRNA expression in dopaminergic (DAergic) neurons was observed in the ventral part of the nucleus preopticus medialis (POM). Following intracerebroventricular injection of DA or oxytocin (OT) receptor antagonists, hens incubating eggs were introduced to poults. Over 80% of those injected with vehicle or the D1 DA receptor antagonist brooded poults, while over 80% of those receiving the D2 DA receptor antagonist or the OT receptor antagonist failed to brood the poults. The D2 DA/OT antagonist groups also displayed less c-fos mRNA in the dorsal part of POM and the medial part of the bed nucleus of the stria terminalis (BSTM) areas than did the D1 DA/vehicle groups. These data indicate that numerous brain areas are activated when incubating hens initially transition to poult brooding behavior. They also indicate that DAergic, through its D2 receptor, and MTergic systems may play a role in regulating brooding behaviors in birds.