Neuropeptide Y (NPY) and C-flanking peptide of NPY in the pineal gland of normal and ganglionectomized sheep.

The present immunohistochemical study describes the presence and distribution of nerve fibers containing neuropeptide Y (NPY), and C-Flanking Peptide Of NPY (CPON) in the pineal gland of the sheep. Nerve fibers were detected by using a series of antisera directed against NPY or against CPON. Many positive immunoreactive nerve fibers were identified in the pial capsule of the pineal, in connective septae and in the parenchyma between pinealocytes. The intraparenchymal fibers were particularly evident and created an extensive network throughout the gland. Nerve fibers immunoreactive for all the peptides were also observed in the posterior commissure and in the stria medullaris thalami. No NPY- or CPON-positive neurons were found in the pineal gland. In order to study the site of origin of NPY- and CPON-immunoreactive nerve fibers, the superior cervical ganglia were bilaterally removed in a series of animals. Sympathetic denervation was checked by using an antiserum against tyrosine hydroxylase (TH). Nearly all TH-immunoreactive elements disappeared in the pineal glands of animals sacrificed 15 days after surgery. Also the density of NPY- and CPON-immunoreactive nerve fibers decreased in the animals after the ganglionectomy. However, a number of nerve fibers still remained in the gland. These data indicate that some NPY- and CPON-immunoreactive nerve fibers of the sheep pineal gland derive from an extrasympathetic origin. The very dense innervation of the sheep pineal gland with nerve fibers containing NPY and CPON strongly indicates a functional role for this family of peptides in the pineal gland of this species.

Density of peptide histidine-isoleucine- and vasoactive intestinal peptide-immunoreactive nerve fibers in the sheep pineal gland is not affected by superior cervical ganglionectomy.

Peptide histidine-isoleucine (PHI) is a regulatory peptide, synthesized as part of the same propeptide that includes also vasoactive intestinal peptide (VIP). The present study describes the distribution of PHI-immunoreactive nerve fibers in the sheep pineal organ and compares their location with the distribution of VIP-immunoreactive fibers in both normal and superior cervical ganglionectomized sheep in order to elucidate the origin of the PHI/VIP immunoreactive nerve fibers. Several PHI-immunoreactive nerve fibers were present in the meninges and in the pineal capsule. Numerous positive nerve fibers entered the pineal gland and travelled within connective tissue spaces. Individual PHI-positive nerve fibers were either smooth, without specialization, or varicose. Generally VIP- and PHI-immunoreactive fibers were located close to connective septa and blood vessels. However, many PHIergic and VIPergic fibers possessing varicosities of variable sizes were also dispersed between pinealocytes. The distribution, density, and morphology of PHI- and VIP-immunoreactive fibers in the sheep pineal gland were similar. In superior cervical ganglionectomized animals, intrapineal VIP- and PHI-immunoreactive nerve fibers were present with the same density as in control animals. In agreement, the concentration of immunoreactive VIP and PHI did not change after ganglionectomy. No VIP- and PHI-immunoreactive cell bodies were observed in the superior cervical ganglia. Thus this study shows that the intrapineal VIP- and PHI-immunoreactive nerve fibers do not originate from the sympathetic superior cervical ganglion.

Innervation of the sheep pineal gland by nonsympathetic nerve fibers containing NADPH-diaphorase activity.

We used the NADPH-diaphorase histochemical method as a potential marker for nitric oxide synthase (NOS)-containing nerve fibers innervating the pineal gland of the sheep. Nerve fibers containing NADPH-diaphorase activity provide dense innervation of the sheep pineal gland. The nerve fibers were located in the pineal capsule, in the connective tissue septae separating the lobull of the gland, and penetrating between the pinealocytes. The nerve fibers were either smooth or endowed with boutons en passant. After bilateral removal of the superior cervical ganglion, the dense network of NADPH-diaphorase-positive fibers was still present in the gland. Ganglionectomy affected neither the distribution nor the appearance of the NADPH-diaphorase-positive fibers. Most of the NADPH-diaphorase-positive fibers also contained peptide histidine isoleucine and vasoactive intestinal polypeptide, and a comparatively smaller fraction contained neuropeptide Y. Pinealocytes never exhibited NADPH-diaphorase activity. These results demonstrate a major neural input to the sheep pineal gland with NADPH-diaphorase-positive nerve fibers of nonsympathetic origin.

Pineal microdialysis of the melatonin in conscious sheep: methodology, application to a diurnal rhythm and effect of isoproterenol.

This paper describes the development of a new technique to measure melatonin contents in the pineal gland of moving sheep: the microdialysis. A dialysis probe was used to collect extracellular fluid in the sheep pineal gland, but also to inject directly into it different drugs such as isoproterenol at a very low concentration. The probe was implanted the day before the beginning of the experiment in order to obtain low levels of melatonin. This technique makes it possible to measure melatonin in the dialysate and plasma of rams submitted to 8L:16D. No melatonin either in the dialysate or in the plasma was found during the light phase. Shortly after lighting off, the melatonin concentration increased in the dialysate and plasma and remained stable during the dark phase. Melatonin concentrations began to decrease before lighting on and no detectable levels were found during the following light phase. The secretion of melatonin is, at least, under adrenergic regulation. Local infusion of isoproterenol (90 microliters at 10(-6) M), an agonist of beta adrenergic receptor, through the probe, increased melatonin levels during 2 h, even when infusions were repeated 3 times. This demonstrates the presence of beta adrenergic receptors. The technique presented in this paper could be of considerable interest for studying sheep pineal gland and its main secretion, melatonin, for example during diurnal rhythms or for studying its regulation.

Regulation of melatonin release and N-acetyltransferase activity in ovine pineal cells.

Abstract A suspension culture of ovine pineal cells was developed to investigate the regulation of melatonin synthesis and release. Dosedependent stimulation of melatonin release by a series of adrenergic agonists yielded a typical beta-adrenergic profile. Serotonin N-acetyltransferase (NAT), the rate-limiting enzyme in melatonin biosynthesis, was also stimulated by a beta-adrenergic mechanism. However, NAT activity appeared less sensitive than melatonin release to beta-adrenergic stimulation. No evidence was obtained for a contribution of alpha(1)-adrenergic receptors to the regulation of NAT activity and melatonin release. Activation of adenylate cyclase by forskolin or addition of a cyclic AMP analogue increased both melatonin release and NAT activity. In contrast, the Ca(2+) ionophore A(23187) stimulated melatonin release without a detectable increase in NAT activity. Together, the present data argue for a beta-adrenergic regulation of both NAT activity and melatonin release in ovine pinealocytes. The evidence also suggests that two intracellular mechanisms may control melatonin release in ovine pinealocytes: a cyclic AMP-dependent mechanism, associated with an increase in NAT activity and a Ca(2+)-dependent mechanism, independent of NAT activity.

Pineal photoreceptor cells: photoperiodic control of melatonin production after cell dissociation and culture.

Trout pineal cells were dissociated using a trypsin-DNase digestion technique. An enriched population of photoreceptor cells was selected from a Percoll gradient centrifugation. The ability of cultured photoreceptor cells (selected or not on a Percoll gradient) to produce melatonin rhythmically was investigated during seven 24 h light/dark cycles. During each cycle, trout pineal photoreceptor cells released low amounts of melatonin during daytime and high amounts during night-time. Under continuous darkness, melatonin release was continually high. The profile of its rhythm and that of the activity of the hydroxyindole-O-methyltransferase-the last enzyme of the melatonin biosynthetic pathway-depended on the substrates and on the culture media used. Some of them appear suitable for short- or long-term culture of photoreceptor cells permitting the study of their neuroendocrine properties.

Pineal photoreceptor cells in culture: fine structure, and light/ dark control of cyclic nucleotide levels and melatonin secretion.

Trout pineal photoreceptor cells were dissociated by trypsin-DNase digestion and further purified by a Percoll gradient centrifugation. Total cells or purified photoreceptor cells were then embedded in a collagen gel, or layered on culture-treated polycarbonate membranes, or maintained in suspension, with RPMI 1640 medium or BGjb medium. It has been shown that cells maintain a rhythmic production of melatonin for at least seven 24 h light/dark cycles under these conditions. In this complementary study, the morphofunctional state of the photoreceptor cells was examined 1) by electron (transmission, scanning) microscopy, and 2) by pharmacological tests under different lighting conditions. Using polycarbonate membranes together with RPMI 1640 medium appeared the most suitable. The segmented organization of photoreceptor cells was well preserved when using the culture-treated membranes. It tended to disappear in cells embedded in the collagen gel and was lost after passage through the Percoll gradient. However, this one allowed obtention of an homogeneous population of photoreceptors, as recognized by their intracellular components. Intracellular organelles were rather well preserved in the embedded photoreceptors. The study also provides novel information on the nature of second messengers involved in the photoperiodic control of melatonin production in photoreceptor cells. From the effects of an adenylyl cyclase activator and a phosphodiesterase inhibitor it appeared that 1) total cells and Percoll-selected cells behaved similarly, 2) the nocturnal rise in melatonin secretion was associated with an increase in cAMP content, and 3) a fall in cAMP may be a mechanism through which light reduces melatonin secretion by photoreceptor cells. Cyclic GMP, the metabolism of which also appeared to be controlled by light, did not seem involved in the photoperiodic control of melatonin production. The method proposed herein offers interesting perspectives for the study of the photoneuroendocrine properties of isolated photoreceptor cells.

The chemical neuroanatomy of the mammalian pineal gland: neuropeptides.

The mammalian pineal gland contains multiple afferent peptidergic nerve fibres. Sympathetic nerve fibres, with their origin in the superior cervical ganglia, contain neuropeptide Y colocalized with norepinephrine. Other pinealopetal nerve fibres, probably originating in the pterygopalatine ganglion, contain vasoactive intestinal peptide and peptide histidine isoleucine. Fibres containing substance P and calcitonin gene-related peptide have also been demonstrated in pinealopetal nerve fibres. These fibres might originate in the trigeminal ganglion. The neurotransmitter content of the fibres of the central innervation, innervating the gland from the brain via the pineal stalk, has not been elucidated. However, strong indications for the presence of neuropeptide Y, substance P, somatostatin, and vasopressin in these fibres have been presented. Recent immunohistochemical studies have further shown the presence of subtypes of pinealocytes containing neuropeptides. Thus, pinealocytes containing beta-endorphin, leu-enkephalin, and somatostatin have been demonstrated in the gland. Immunohistochemistry at the electron microscopical level has shown, that in some species, leu-enkephalin containing pinealocytes make synaptic contacts with other pinealocytes indicating of paracrine regulation of the pineal gland. It must however be emphasized that large interspecies variations exist with regard to the peptidergic pineal innervation and its content of peptidergic cells.

Binding of an adenosine A1 receptor agonist and adenosine A1 receptor antagonist to sheep pineal membranes.

The pineal organ of vertebrates produces melatonin and adenosine. In lower vertebrates, adenosine modulates melatonin production. We report herein that 2-chloro-cyclopentyl-[3H]-adenosine ([3H]CCPA: adenosine A1 receptor agonist) and [3H]-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX: adenosine A1 receptor antagonist), bind specifically to sheep pineal membranes. Binding of [3H]CCPA reached equilibrium at 90 min and dissociation revealed the presence of two components. Saturation analysis suggested the presence of a single population of binding sites (Kd = 1.67 +/- 0.06 nM, Bmax = 2386 fmol/mg protein). Binding was sensitive to GTP and GTPgammaS. Binding of [3H]DPCPX reached equilibrium at 60 min and dissociation was monophasic. Saturation analysis revealed a single population of binding sites (Kd = 5.8 +/- 1.12 nM, Bmax = 1116 fmol/mg protein). The specificity of the [3H]-analogues used and the rank order potency of the competitors tested in the competition experiments suggested the presence of A1 receptors. Future investigations are necessary to elucidate the significance of the differences observed between the binding properties of the adenosine A1 receptor agonist and adenosine A1 receptor antagonist.

Melatonin binding sites in the sheep pars tuberalis.

The localization of [125I]melatonin binding sites has been studied by autoradiography on frozen unfixed sections of the pituitary stalk, the suprachiasmatic area, the pineal and the pituitary glands in sheep. Dense specific labelling has been found exclusively in the pars tuberalis of the pituitary stalk but not in the part of the median eminence surrounded by the pars tuberalis. The labelling was completely excluded by a 200-fold excess of cold melatonin. No comparable labelling was found in the suprachiasmatic nucleus, the pineal and the pituitary glands. These results constitute the first report of melatonin-specific labelling in the ovine species.

Circadian and circahemidian rhythms in plasma prolactin of the ram: seasonal changes.

The existence of a circadian rhythm in plasma prolactin of the ram is controversial. Differences among authors can be related to both data sampling (e.g. interindividual changes, time of day, time of year, sampling interval among others) and statistical analyses. To test this hypothesis six adult "Préalpes du Sud" rams were studied individually during 72 hr in January (8 hr of light-16 hr of darkness), April (13L-11D), June (16L-8D) and September (13L-11D). Blood was sampled (vacutainer) from a jugular vein every hour, centrifuged and plasma samples stored at -20 degrees C until prolactin determinations (radioimmunoassay) were made. Individual time series were analysed according to three complementary methods: display of raw data (chronogram), best fitting cosine functions with different period tau (iterative cosinors) and power spectra. Seasonal changes in the 24 hr mean (peak time in June) were confirmed. A circahemidian rhythm (tau = 12 hr) and a circadian rhythm (tau = 24 hr) were validated, respectively in January and April while time series documented in June and September exhibited no rhythmic organization. It seems, therefore, that animals adjusted their rhythmic patterns of prolactin secretion to the increasing (January, April) rather than decreasing (June, September) photofraction (duration of the light span/24 hr).

Entrainment of melatonin rhythms in rams by symmetrical light-dark cycles of different period length.

The objective of this study was to investigate the entrainment of melatonin rhythms in rams using symmetrical light-dark cycles of different period length. Five groups of six Ile de France rams were kept in 12L:12D for 7 weeks and then (i) 12L:12D, (ii) 11L:11D, (iii) 10L:10D, (iv) 13L:13D and (v) 14L:14D for a further 3 weeks. Environmental factors other than the light dark cycle were not controlled. The onset and offset of the plasma melatonin rhythm in DD after 3 weeks of the respective light treatments was assessed for 48 hr, immediately after transferring to DD. The duration of secretion in DD was positively related to the length of the previous dark phase. The phase of the melatonin rhythm with respect to the anticipated dark phase suggested entrainment with no change in phase-relationship to the zeitgeber by 12L:12D and 13L:13D. Entrainment with a phase-delay or a phase-advance was apparent after 11L:11D and 14L:14D, but the individual rhythms were not all synchronized with respect to each other after 10L:10D. Activity recordings for 2-3-week periods during 12L:12D, 10L:10D and 14L:14D all showed a major 24-hr component at all times, with activity during the light phase in 12L:12D. It appears that melatonin may be readily desynchronized from overt activity-rest cycles in sheep. The upper and lower entrainment limits are probably greater than 28 hr and close to 20 hr cycles, respectively.

Photoperiodic control of growth hormone secretion and body weight in rams.

This study was undertaken to assess the influence of photoperiod on growth hormone (GH) secretion in rams and its possible influence on body weight. Twenty young adult rams were divided into two groups. One was subjected to an annual (AR) and the other to a semestral (SR) light regime during the same 18-month period. In both groups, daylength (DL) varied gradually between 8 to 17 hr. Plasma prolactin (PRL) and GH profiles consisting of 6 hr samples were determined and animals were weighed throughout the course of the experiment. Maximal PRL secretion was observed with largest DL. In contrast, GH secretion increased during increasing DL but it began to decrease before maximal DL was reached in both light regimes. Mean GH secretion was maximal when the DL was about 11 hr in SR and between 8 to 12 hr in AR. Similarly, body weight increased when DL increased and plateaued during decreasing DL in both AR and SR animal groups. Significant (P less than 0.05) differences were observed throughout the course of the experiment according to the effects of decreasing or increasing DL in each group. Analysis of variance showed that the effect of DL on plasma PRL and GH levels and weight velocity (WV) was significant (P less than 0.05) in both light regimes. This suggests that in SR, plasma PRL and GH levels and WV vary according to a six month period.(ABSTRACT TRUNCATED AT 250 WORDS)

Circadian profile and production rate of melatonin in the cow.

Five adult pasture-bred French Friesian cows were used to qualify the circadian profile and characterized pulsatility of plasma melatonin, and to estimate melatonin secretion rate, around the summer solstice. Plasma concentrations of melatonin were low (5 pg/ml) during the photophase, began to rise at sunset (light intensity less than 20 lx) and reached a maximum (about 90 pg/ml) in the middle of the scotophase. The mean amplitude of peaks was 48.67 +/- 23.01 pg/ml, their mean duration was 32.30 +/- 21.50 min and the frequency was 1.5 +/- 0.3 peak/hr during the secretory period (537 +/- 42.3 min). The plasma clearance (ClB) was 0.0247 +/- 0.0013 1/kg per min, the steady state volume of distribution (Vss) was 1.404 +/- 0.225 1/kg, the elimination half life (t1/2 beta) was 66.66 +/- 11.30 min, the mean residence time was 51.37 +/- 9.92 min and the mean production rate was 399.9 +/- 57.37 ng/kg per 24 hr. These results support the concept of linearity for melatonin kinetics in cattle and the plasma clearance value suggest a first-pass hepatic effect.

Prolactin in the ram: seasonal variations in the concentration of blood plasma from birth until three years old.

Peripheral prolactin levels were measured by radioimmunoassay in nine Ile de France rams, born in autumn and reared under natural conditions of light and temperature until three years old. During December, when the rams were 80-100 days old, a brief and transitory peak of prolactin occurred (70 ng PS6/ml plasma +/- 10 ng). During each following year, the prolactin level fell in winter to approximately 20 ng/ml. However, in summer the prolactin was 4 times higher than the winter level during the first year and 11 times higher during the 2nd and 3rd years. A possible role of prolactin variation throughout the breeding season is discussed.

Suppression of melatonin secretion in Ile-de-France rams by different light intensities.

The intensity of cool, white, fluorescent light required to suppress melatonin secretion in Ile-de-France rams was investigated. Animals were conditioned to 12L:12D, lights on 0600 hours, 104 microW/cm2 (350 lux) at eye level and subjected to a 1-hour light pulse beginning 3 hours after lights off. Plasma melatonin measurements indicated that secretion was significantly suppressed by 0.30, 7.46, and 26.32 microW/cm2 (1.02, 25.10, and 88.60 lux, respectively) but not by 0.043 microW/cm2 (0.15 lux). A clear dose-response relationship was apparent between light intensity and degree of melatonin suppression.