Vasoactive Intestinal Peptide Secretion by Turkey Hypothalamic Explants¹

^a Department of Animal Science ^b and Department of Ecology, Evolution, and Behavior, University of Minnesota, St. Paul, Minnesota 55108

	ABSTRACT

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The objective of this study was to culture turkey hypothalami and examine vasoactive intestinal peptide (VIP) release during the turkey reproductive cycle. The release of VIP was studied employing a computer-guided perifusion system. Hypothalami were perifused with Krebs-Ringer bicarbonate medium for 10 or 15 h at a flow rate of 40 µl/min, and perifusate was collected at 5-min intervals. Basal VIP secretion increased (p < 0.05) over time, and no differences in release rate were noted between reproductive stages. Basal VIP release during perifusion was episodic throughout each experimental period. Perifusion with dopamine (DA; 10 and 100 nmol/min) in incubating hens stimulated VIP release in a dose-dependent manner. There were no significant differences (p > 0.05) in VIP release in response to DA stimulation between hypothalamic fragments obtained from nonphotostimulated and incubating birds. The data suggest that 1) a VIP pulse generator appears to be located within the turkey hypothalamus, on the basis of the observed pulsatile release of VIP; 2) hypothalamic secretion of VIP is augmented by removal of inhibitory factors residing outside of the hypothalamus, or by the loss of negative feedback mechanisms that inhibit VIP release; and 3) mechanisms responsible for altering VIP release during different reproductive conditions may lie external to the hypothalamus.

	INTRODUCTION

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The neuroendocrinological relevance of vasoactive intestinal peptide (VIP) in avian prolactin (PRL) secretion is supported by several lines of evidence [1]. Immunoneutralization of endogenous VIP reduces circulating PRL and pituitary PRL mRNA [2]. Immunoneutralization blocks the release of PRL in response to electrical stimulation of the hypothalamus [2] or central infusion of PRL secretagogues (5-HT [3]; dopamine (DA) [4]; dynorphin, unpublished results). In addition, VIP immunoneutralization prevents the induction and maintenance of incubation behavior in turkeys and chickens, and the proliferation of crop sac tissues in pigeons [5–7]. VIP stimulates PRL release and gene expression both in vitro and in vivo [8–11].

Under increasing day length, PRL levels increase during gonadal growth, egg laying, and incubation, peaking at mid and late incubation; they then decrease significantly when eggs hatch and remain low throughout photorefractoriness [12]. The VIP content of the hypothalamus shows a progressive increase exclusively within the median eminence-infundibular nuclear complex region of the hypothalamus throughout the turkey reproductive cycle [13]. Hypothalamic VIP concentration increases throughout the egg-laying phase, reaching a maximum level during the late laying, incubating, and photorefractory stages. These changes in hypothalamic VIP content are associated with coincident increases in circulating PRL, with the notable exception of photorefractory hens. VIP levels in turkey hypophyseal portal blood are significantly greater than in the general circulation and vary according to the reproductive status and circulating PRL levels of the hen [14].

In mammals, gonadotropin releasing hormone (GnRH) is secreted episodically from the hypothalamus, and the frequency and amplitude of GnRH release determines the pattern of gonadotropin secretion [15–17]. Pulsatile release of GnRH in vitro has been reported in birds [18, 19]. At present, nothing is known about hypothalamic VIP release during a photo-induced reproductive cycle in the turkey. The measurement of hypothalamic VIP content is a poor indicator of VIP release dynamics. Therefore, characterization of the alterations in VIP release in conjunction with PRL secretion needs to be investigated. Recently, it has been demonstrated that DA stimulated VIP release from turkey hypothalamic explants and did so in a dose-dependent manner [20]. The objective of this study was to investigate the nature of basal and DA-stimulated VIP release from perifused isolated turkey hypothalami harvested from birds of different reproductive status. The hypothesis was that VIP secretion would be greatest in hyperprolactinemic incubating hens and lowest in hypoprolactinemic nonphotostimulated ones, with laying hens intermediate between the two.

	MATERIALS AND METHODS

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Animals

Nicholas large white female turkeys in their second reproductive cycle were used throughout these studies. The reproductive groups used were 1) reproductively inactive, nonphotostimulated hens that had been maintained under a short day lighting regimen (6L:18D) for a minimum of 8 wk (these birds were completely refeathered, and displayed the hard tight pubic bones and dry cloacae associated with regressed ovaries); 2) laying hens that used the nest box only once or twice a day and regularly laid eggs; and 3) incubating hens, which had stopped laying eggs and were in the nest box at least six times per day for a minimum of 2 wk.

Tissue Preparation

Birds were killed either with pentobarbital sodium (Beuthanasia-D Special; Schering-Plough, Kenilworth, NJ; experiments 1 and 2) or by decapitation (experiment 3). The intact brain was immediately dissected from the skull, and the attached pituitary gland was removed under microscopic guidance to prevent any loss of median eminence tissue. The optic chiasma was dissected from the ventral surface of the brain to expose the hypothalamus. The block of tissue removed was limited rostrally by the septomesencephalic tract and caudally by the oculomotor nerve, and it extended laterally to the quintofrontal tract on each side. The tissue block was approximately 4 mm deep and included the median eminence, hypothalamus, and preoptic hypothalamus. The tissue block was sliced longitudinally two times on each side of the midline. The cuts did not extend all the way to the ends of the block, allowing the block of tissue to open in an "accordion" manner without separating into individual pieces. The hypothalami were placed into ice-cold perifusion media before being transferred to the perifusion chambers.

Perifusion

The perifusion procedure has been detailed previously [18, 19, 21, 22]. Briefly, each hypothalamus was transferred to one of six temperature-controlled perifusion chambers (500-µl volume) in a computer-controlled perifusion system (APS-10; Endotronics, Coon Rapids, MN). Perifusate collected during the first 3 h of perifusion (flow rate of 0.5 ml/min) was discarded. After 3 h, the hypothalami were perifused continuously at a flow rate of 40 µl/min. The perifusion medium (41°C) was constantly gassed with CO₂:O₂ (5%:95%). Krebs-Ringer bicarbonate medium was used for perifusion with the following composition: 10 g/L crystalline BSA (fraction V; Sigma, St. Louis, MO), 10 mM D(+)glucose (grade III; Sigma), 20 mM HEPES (Sigma), 0.5 mM ascorbic acid (J.T. Baker Chemical, Phillipsberg, NJ), 0.05 bacitracin mM (Aldrich, Milwaukee, WI), and 0.0056 mM phenol red dye (Sigma). The medium was adjusted to pH 7.4.

Effluent fractions were collected at 5-min intervals into siliconized polypropylene tubes, stored momentarily on ice, and frozen at -80°C until assayed for VIP. Tissue viability was confirmed by adding 50 mM KCl to the perifusion medium to depolarize the hypothalamic neurons. NaCl concentration in the depolarizing medium was reduced by an equimolar amount to maintain medium osmolality. The VIP response to depolarization was tested at the end of each perifusion run. VIP concentrations were expressed as picograms of VIP per 5 min.

Experimental Design and Analysis

Experiment one was designed to determine the nature of the basal release of VIP and compare the differences among reproductive stages. Hypothalami were taken from nonphotostimulated (n = 6), laying (n = 6), and incubating hens (n = 6) and were continuously perifused for 10 h at a flow rate of 40 µl/min.

In experiment 2, the hypothalami were taken from nonphotostimulated (n = 3) and laying hens (n = 3), and were perifused continuously for 15 h at a flow rate of 40 µl/min.

The effect of DA upon VIP release by turkey hypothalamic explants was investigated in experiment 3, using nonphotostimulated (n = 6) and incubating hens (n = 6). Hypothalami were sequentially perifused with medium alone or with medium containing various concentrations of DA (10 and 100 nmol/min). The hypothalami were perifused in the following order: 180 min with medium alone, 30 min with DA at the rate of 10 nmol/min, 180 min with medium alone, 30 min with DA at the rate of 100 nmol/min, and 180 min with medium alone. After every 30-min DA perifusion, the hypothalamic fragments were flushed by perifusion medium alone at the higher flow rate of 0.5 ml/min for 15 min to wash away residual DA. Nontreated control hypothalami (n = 6) were perifused with medium alone throughout the perifusion period.

RIA and Statistics

[(¹²⁵I)Try¹⁰]VIP was prepared by the Iodogen method [23]. VIP measurements were carried out by means of a self-displacement double-antibody RIA [13]. All samples from the same perifusion run were assayed at the same time. The intra- and interassay coefficients of variation were 9% and 11%, respectively. Plasma was assayed for PRL content using the homologous RIA described by Proudman and Opel [24]. The intraassay coefficient of variation was 7%.

VIP secretory patterns were analyzed using PC PULSAR software [25]. The cut-off criteria for peak identification were G(1) = 3.80, G(2) = 2.60, G(3) = 1.90, G(4) = 1.50, and G(5) = 1.20. Baseline, mean pulse amplitude, peak length, pulse frequency, and peak interval were determined for each explant.

The effects of time on VIP release were analyzed by repeated-measures analysis using the General Linear Model procedure in the Statistical Analysis System [26]. Duncan's multiple-range test was used to determine differences in VIP release among treatment groups. A p value of less than 0.05 was considered statistically significant.

	RESULTS

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Basal VIP Release from Hypothalamic Explants

VIP release during perifusion of hypothalami was distinctly and consistently pulsatile throughout the entire 10-h experimental period (Fig. 1a). The VIP actual value, baseline, mean pulse amplitude, peak interval, peak length, and pulse frequency did not differ significantly (p > 0.05) between reproductive groups (Table 1). Mean pulse amplitude, pulse frequency, peak length, and peak interval of VIP release did not change throughout the entire experimental period, but the baseline for VIP tended to increase with advancing perifusion time (Fig. 1b). No significant differences (p > 0.05) in baseline VIP release were observed across reproductive stages (Table 1). K⁺ depolarization induced a peak response of 16.7 ± 1.6 pg/5 min from a baseline value of 6.9 ± 1.4 pg/5 min in laying hens.

View larger version (44K): [in this window] [in a new window]   FIG. 1. a) Patterns of VIP secretion from individual perifused turkey hypothalami representing three reproductive stages during 10 h of perifusion. The arrows indicate distinct VIP pulses. The horizontal line represents the calculated baseline generated by the PC PULSAR program. The arrow at K+ indicates the time of depolarization. b) Hourly mean VIP release from perifused turkey hypothalami representing three different reproductive stages during 10 h of perifusion. Each data point represents mean ± SEM (n = 6).

When the perifusion time was extended, VIP release remained episodic throughout the 15-h experimental period (Fig. 2a). There were no differences in mean pulse characteristics between nonphotostimulated and laying hens, but baseline VIP release and mean pulse amplitude of both groups increased significantly (p < 0.05) over time (Fig. 2, a and b). Baseline VIP release increased from 0.2 ± 0.1 pg/5 min during the first hour of perifusion to 43.1 ± 15.5 pg/5 min after the 15-h perifusion period in laying hens.

View larger version (31K): [in this window] [in a new window]   FIG. 2. a) Patterns of VIP secretion from individual perifused turkey hypothalami representing a laying and a nonphotostimulated reproductive stage. The arrows indicate distinct VIP pulses. The arrow at K+ indicates the time of depolarization. b) Hourly mean VIP release from perifused turkey hypothalami during 15 h of perifusion. Each data point represents mean ± SEM (n = 3).

Effects of DA upon VIP Release by Turkey Hypothalamic Explants

As in experiment 1, no significant differences (p > 0.05) in basal VIP release were observed between hypothalamic fragments from nonphotostimulated and incubating birds (33.6 ± 1.7 vs. 37.1 ± 3.3 pg/5 min; n = 6, p > 0.05). Perifusion of hypothalamic fragments from incubating hens with DA for 30 min significantly increased p < 0.05) basal VIP release, from 37.1 ± 3.3 to 137.6 ± 9.2 pg/5 min at the 10 nmol/min DA, and from 42.4 ± 3.9 to 275.4 ± 18.8 pg/5 min at the 100 nmol/min DA (Fig. 3). Challenge of hypothalamic explants from nonphotostimulated birds with 10 and 100 nmol/min DA for 30 min significantly increased (p < 0.05) basal VIP release. There were no significant differences (p > 0.05) in VIP release in response to DA between hypothalamic slices obtained from nonphotostimulated and incubating birds (10 nmol/min: 143.7 ± 9.0 vs. 137.6 ± 9.2; 100 nmol/min: 256.2 ± 20.8 vs. 275.4 ± 17.8 pg/5 min, respectively; n = 6, p > 0.05). The VIP secretory pattern remained unchanged in untreated control hypothalami throughout the perifusion period.

View larger version (22K): [in this window] [in a new window]   FIG. 3. Representative individual plots demonstrating the stimulatory effects of DA (10 and 100 nmol/min) upon VIP release from perifused turkey hypothalami harvested from a) nonphotostimulated hens, b) incubating hens, and c) perifusion with media alone; K+ indicates the time of depolarization.

	DISCUSSION

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The results of the present study show that VIP was released in a pulsatile pattern from turkey hypothalamic explants. The amplitude of the VIP pulse was increased during DA challenge, and the amount of VIP released was dependent upon the DA concentration. No significant differences in basal VIP release, VIP pulse characteristics, or response to DA stimulation were observed between perifused isolated hypothalami harvested from hyper- and hypoprolactinemic turkeys.

In this study, the hypothalami displayed a synchronized pattern of episodic VIP release. To our knowledge, this is the first demonstration of pulsatile VIP secretion from the hypothalamus, certainly the first for an avian species. The significance of VIP pulsatility in the regulation of PRL secretion remains to be determined. Continuous intracranial infusion of VIP down-regulated turkey pituitary VIP receptors and PRL secretion [27]; and PRL secretion has been shown to occur in a pulsatile manner in the turkey [28]. It is of interest that, in the rat, the secretion of pituitary gonadotropins, and consequently the activation of gonadal function, are dependent on the pattern of episodic stimulation of the pituitary gland by GnRH [15–17]. The results showed that the pattern of pulsatile release of VIP differed from bird to bird. Similar individual variations of in vitro release of neurotransmitters and neuropeptides have been reported for mammals [16, 29] and are thought to be due to innate variability in the neurosecretory system, including a random pattern of release [30]. Furthermore, our data showed that neighboring VIP pulses were often of markedly different amplitudes and pulse durations. These changes in the characteristics of the VIP pulses may be postulated to alter subsequent PRL release by the pituitary.

The stimulatory effect of DA upon in vitro VIP release from the turkey hypothalamus agrees with our earlier results [20] and corresponds nicely with the in vivo finding that DA stimulated PRL secretion [4]. In the turkey, active immunization against VIP blocked PRL release induced by intracranial infusion of DA, suggesting that DA stimulated PRL release via VIPergic mechanisms [4]. Moreover, VIP concentrations in the hypothalamus and in hypophysial portal blood varied according to the reproductive condition of the hen and reflected plasma PRL levels [13, 14]; VIP concentrations in hyperprolactinemic incubating hens were greater than those in laying or reproductively quiescent birds. The failure of basal and DA-stimulated VIP secretion from isolated hypothalami to reflect in vivo VIP concentrations reported in the aforementioned studies is far from clear. Isolating the hypothalamus may disrupt the VIP-releasing mechanism(s), thus masking any differences between reproductive states. In the present study, VIP secretion increased constantly over time in all isolated turkey hypothalami, but no significant differences were observed between differing reproductive stages. This suggests that hypothalamic secretion of VIP may be enhanced because of the removal of some endogenous, extra-hypothalamic inhibitory factor(s) and/or the loss of regulatory feedback mechanisms. PRL has been shown to have a negative feedback action upon VIP release in mammals [31]. Systemic PRL administration reduced the hypothalamic VIP content in turkeys [32], and intracranial PRL injection reduced the number of VIP-like neurons in the infundibulum of ring doves [33], indicating a negative feedback loop for PRL in the avian hypothalamus.

It is of interest that VIP release by hypothalamic explants harvested from turkeys killed with pentobarbital sodium (experiments 1 and 2) was suppressed compared to turkeys killed by decapitation (experiment 3). The baseline level for VIP and the mean pulse amplitude were considerably greater in decapitated birds than in pentobarbital-killed ones [34]. Nevertheless, the same lack of difference between reproductive groups regarding basal VIP secretion and VIP pulse characteristics was observed in both groups of hens.

Based on the observed pulsatile release of VIP, a VIP pulse generating mechanism appears to be located within the hypothalamus of the turkey. This pulsatile mode of VIP release may be essential for the maintenance of PRL synthesis and release. DA increased the amplitude of the VIP pulse and the amount of VIP released. The increase in basal VIP secretion over time may be due to a loss of negative feedback mechanism(s) and/or removal of endogenous, extra-hypothalamic inhibitory inputs. This may be the reason why there was no observed difference in hypothalamic VIP release between hyper- and hypoprolactinemic turkeys.

	FOOTNOTES

 
¹ This is Scientific Journal Series Paper 22,534 of the Minnesota Agriculture Experiment Station. Research supported by USDA Grant No. 97-35203-4960.

² Correspondence: Mohamed El Halawani, 495 Animal Science/Veterinary Medicine, 1988 Fitch Avenue, University of Minnesota, St. Paul, MN 55108. FAX: (612) 625-2743; elhal001{at}maroon.tc.umn.edu

Accepted: April 30, 1998.

Received: February 24, 1998.

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