Investigations of the regulation of specific 2-[I]iodomelatonin binding sites in Siberian hamsters by endogenous and exogenous melatonin

These studies used quantitative in vitro autoradiography to investigate whether endogenous or exogenous melatonin modulate specific 2-[¹²⁵I]iodomelatonin binding sites in the pars tuberalis or suprachiasmatic nuclei of Siberian hamsters. Saturation analyses were conducted on tissue sections from hamsters that were pinealectomized, exposed to constant illumination (72 h), or injected with melatonin, and from hamsters that were treated as controls. High affinity (K_d approximately 20–75 pM) specific 2-[¹²⁵I]iodomelatonin binding sites were detected in the suprachiasmatic nuclei and pars tuberalis of all animals. Neither pinealectomy nor constant illumination significantly affected either the affinity or the density of the specific 2-[¹²⁵I]iodomelatonin binding sites in either region. Melatonin injections led to a decrease in specific 2-[¹²⁵I]iodomelatonin binding to the pars tuberalis at 3–4 h after the last injection. However, washing the sections before incubation with 2-[¹²⁵I]iodomelatonin eliminated this effect, suggesting that melatonin was occupying the binding sites rather than decreasing their actual number. Furthermore, when hamsters were sacrificed 18 h after the last melatonin injection, no effect of melatonin on either the affinity or density of specific 2-[¹²⁵I]iodomelatonin sites was observed. These data suggest that 2-[¹²⁵I]iodomelatonin binding sites in Siberian hamsters are not regulated by changes in circulating melatonin levels.     

Differences in hypothalamic 2-[125I]iodomelatonin binding in photoresponsive and non-photoresponsive white-footed mice, Peromyscus leucopus.

Photoperiod is an environmental cue used by many temperate-zone species to regulate their reproductive timing. Within species, the degree of reproductive photoresponsiveness can vary widely both among and within populations. The neuroendocrine mechanisms causing this individual variation in photoresponsiveness are unknown. Using selected lines from a population of white-footed mice known to vary genetically in reproductive photoresponsiveness, we tested the hypothesis that variation in the number and/or location of melatonin receptors is the basis for individual differences in reproductive photoresponsiveness. The brains and pars tuberalis of the pituitary from sixteen mice, (eight mice from each of two lines selected for two generations to respond strongly or weakly to photoperiod), were processed for autoradiography using the radioligand 2-[¹²⁵I]-iodomelatonin (IMEL). We found significantly higher specific IMEL binding in the medial preoptic area and the bed nucleus of the stria terminalis of non-responsive mice than responsive mice. There were no differences between groups in specific IMEL binding in the suprachiasmatic and dorsomedial nuclei of the hypothalamus, pars tuberalis, or paraventricular nucleus of the thalamus. These results provide support for the hypothesis that individual variation in photoresponsiveness is due in part to differences in the density or affinity of melatonin receptors.     

Characterization of melatonin binding sites in the pars tuberalis of the European hamster

Melatonin binding sites in the pars tuberalis of the European hamster (Cricetus cricetus) have been characterized using the radioligand 2-[(125) I]iodomelatonin. Specific 2-[(125) I]iodomelatonin binding was assessed using radioreceptor studies of pars tuberalis membrane preparations. Saturation studies revealed a single, high affinity site (K(d) 39.8 (± 7.6 SEM) pM and B(max) 4.1 (± 0.5 SEM) fmol/mg protein, n=4). Kinetic experiments showed the 2-[(125) I]iodomelatonin binding to be rapid, saturable and reversible. The K(d) calculated from the dissociation and association rate constants was 19.4 pM. The order of potency of different indoles for inhibition of 2-[(125) I]iodomelatonin binding was 6-chloromelatonin > melatonin > 6-hydroxymelatonin > N-acetylserotonin > 5-methoxytryptophol > serotonin > 5-methoxytryptamine. GTP caused a dose-dependent inhibition of the 2-[(125) I]iodomelatonin binding. A saturation study showed that GTP reduced the number of binding sites by a third without altering their affinity. These results imply the presence of a G-protein-coupled melatonin receptor in the pars tuberalis of sexually active European hamsters.     

Autoradiographic localization of binding sites for 2-[125I]iodomelatonin in the pars tuberalis of the western spotted skunk (Spilogale putorius latifrons).

Pineal melatonin secretion mediates photoperiodic regulation of implantation of blastocysts in the female western spotted skunk. Autoradiography studies demonstrated that high affinity 2-[125I]iodomelatonin binding sites are present in the pars tuberalis but not in any other hypophyseal region or in the hypothalamus or thalamus of this species. This restricted localization of 2-[125I]iodomelatonin binding, which was characteristic of spotted skunks sacrificed at various times during their annual reproductive cycles, suggests that the pars tuberalis mediates photoperiodic responses in this species.     

Melatonin Receptors in the Brain and Pituitary Gland of Hypothalamo-Pituitary Disconnected Soay Rams

In Soay rams in which the pituitary gland has been surgically separated from the hypothalamus, blood prolactin concentrations vary in response to changes in photoperiod and the administration of melatonin, as in intact animals, providing evidence that melatonin acts within the pituitary gland to control prolactin secretion. In this study the presence of potentially functional melatonin receptors in the pars tuberalis and zona tuberalis (PT/ZT) of hypothalamo-pituitary disconnected (HPD) Soay rams is confirmed using both in vitro autoradiography with the ligand 2-(¹²⁵I)- iodomelatonin and in situ hybridization for the melatonin receptor. There was no effect of the HPD operation on the pattern and quantity of 2-(¹²⁵I)iodomelatonin binding in the brain demonstrating that this binding is independent of hypothalamic regulation. The possibility that melatonin may control prolactin secretion directly via specific receptors on lactotrophs was investigated using dual in situ hybridization with a (³⁵S) labelled probe for the ovine melatonin receptor (Mel1a_b) and a Digoxigenin labelled probe for ovine prolactin. Melatonin receptor gene expression was observed in the PT/ZT in both intact and HPD rams, however, there was no colocalization with prolactin gene expression; only in the ZT was there a close association between cells expressing the melatonin receptor and lactotrophs. The results provide strong support for the view that melatonin acts via the PT/ZT to mediate the effects of photoperiod on the seasonal cycle in prolactin secretion.     

Autoradiographic localization of binding sites for 2-[I]iodomelatonin in the pars tuberalis of the western spotted skunk (Spilogale putorius latifrons)

Pineal melatonin secretion mediates photoperiodic regulation of implantation of blastocysts in the female western spotted skunk. Autoradiography studies demonstrated that high affinity 2-[¹²⁵I]iodomelatonin binding sites are present in the pars tuberalis but not in any other hypophyseal region or in the hypothalamus or thalamus of this pieces. This restricted localization of 2-[¹²⁵I]iodemelatonin binding, which was characteristic of spotted skunks sacrificed at various times during their annual reproductive cycles, suggests that the pars tuberalis mediates photoperiodic responses in this species.     

Optic nerve transection decreases 2-[I]iodomelatonin binding in the chick optic tectum

The distribution of specific 2-[¹²⁵I]iodomelatonin binding sites in the various layers of the chick optic tectum was analyzed using quantitative receptor autoradiography. Following unilateral optic nerve transection, binding in the optic fiber layer and superficial retinorecipient layers of the contralateral tectum was significantly decreased at 7 and 14 days, but not at 1 day, following transection. The results are consistent with the presence of presynaptic melatonin receptors on axon terminals of retinotectal fibers.     

The area of 2-[I]iodomelatonin binding in the pars tuberalis of the ground squirrel is decreased during hibernation

Quantitative receptor autoradiography was used to analyze 2-[¹²⁵I]iodomelatonin binding sites in the brains of golden-mantled ground squirrels (Citellus lateralis). Specific binding appeared to be discretely localized to the pars tuberalis region of the pituitary that surrounds the medial basal hypothalamus. The total area of binding was significantly decreased in brains of hibernating squirrels as compared to those of awake, euthermic animals. These findings support a role for melatonin receptors of the pars tuberalis in seasonal behavior.     

Photoperiodic regulation of prolactin gene expression in the Syrian hamster by a pars tuberalis-derived factor

Syrian hamsters exhibit a marked seasonal variation in prolactin secretion. The aim of this study was to analyse the nature of the photoperiodic regulation of prolactin gene expression, and to define the role of melatonin and the pars tuberalis of the anterior pituitary in this process. Pituitary prolactin gene expression, restricted to the pars distalis, was increased in hamsters maintained in long daylengths (16 h : 8 h, light : dark) compared to hamsters exposed to short daylengths (8 h : 16 h, light : dark) for 8-12 weeks. Analysis of single cells by in situ hybridization showed that photoperiod had no effect on the percentage of pars distalis cells expressing prolactin mRNA, but shifted the frequency distribution of prolactin mRNA expression per cell, such that in long photoperiods a greater proportion of cells were recruited to a higher expressing population. In vitro coculture of hamster pars tuberalis fragments increased prolactin promoter-driven luciferase activity in stably transfected GH3 cells in a dose- and duration-dependent manner. Conditioned medium from hamster and ovine pars tuberalis also activated the prolactin promoter. Furthermore, basal and forskolin-stimulated conditioned medium from hamster pars tuberalis increased prolactin mRNA expression in primary cultures of pars distalis cells. Melatonin attenuated the activity of pars tuberalis-conditioned medium but had no direct effect on either prolactin mRNA expression or secretion in pars distalis cell cultures. Finally, pars tuberalis fragments from long photoperiod hamsters stimulated prolactin gene promoter activity to a greater extent than those from short photoperiod hamsters. In conclusion, this study provides the first evidence in a seasonal mammal that the synthesis of prolactin depends on photoperiodic modulation of a pars tuberalis-derived factor. Our data support further the hypothesis that seasonal modulation of prolactin gene expression depends upon a melatonin-dependent paracrine action of the pars tuberalis on pars distalis lactotrophic cells.     

Melatonin binding sites in the ovine brain and pituitary: characterization during the oestrous cycle

The distribution of putative melatonin receptors in the sheep has been investigated using in vitro autoradiography and the high affinity, high specific activity ligand 2-[(125) l]iodomelatonin. A wide distribution of specific labelling was found in both the ovine brain and pituitary gland as previously reported. Several novel areas of binding were also identified in the present study, including a fine layer of labelling at the medial edge of the diagonal band of Broca, the trigeminal nucleus, laminae II and III of the substantia gelatinosa, the molecular layer of the cerebellum as well as a scattered labelling in the pars distalis of the pituitary. There was no evidence of specific labelling in any of the peripheral tissues examined. Characterization studies performed on both neuronal and pituitary melatonin binding sites revealed that binding was time- and temperature-dependent and reversible on addition of 1 μM melatonin. The binding of 2-[(125) l]iodomelatonin was also competitively inhibited by increasing concentrations of 2-iodomelatonin and melatonin. The inhibition constants (K(i) ) estimated for each of these substances were similar for both neuronal and pituitary sites. Saturation studies also revealed similarities between neuronal and pituitary tissues with 2-[(125) l]iodomelatonin binding specifically to a single class of high affinity binding sites. Values for equilibrium constants (K(d) ) were within a range of 28 to 48 pM, and values were found to be not significantly different amongst the four regions of the brain investigated and the pars tuberalis of the pituitary. In contrast, the concentration of 2-[(125) l]iodomelatonin binding sites (B(max) ) ranged from 3 to 218fmol/mg protein and were maximal for the pars tuberalis. Saturation studies on brain and pituitary tissues taken from ewes killed either on the day of oestrus or during the luteal phase of the oestrous cycle, indicated that no differences exist in the affinity or concentration of 2-[(125) l]iodomelatonin binding in any region between the two times of the cycle investigated.     

Vasopressin binds to microvessels from rat hippocampus

Recent evidence suggests that vasopressin may influence the permeability of the endothelium of brain capillaries. We measured the binding of [¹²⁵I]arginine-8-vasopressin ([¹²⁵I]AVP) to microvessels isolated from different regions of the rat brain. The study revealed saturable and specific binding of [¹²⁵I]AVP to microvessels isolated from hippocampus. Scatchard analysis confirmed a single class of high affinity sites with an equilibrium dissociation constant,K_d, of 3.2 nM and an apparent maximal binding capacity of 205 fmol/mg protein. No binding was observed to microvessels from neocortex and striatum.     

The area of 2-[125I] iodomelatonin binding in the pars tuberalis of the ground squirrel is decreased during hibernation.

Quantitative receptor autoradiography was used to analyze 2-[125I]iodomelatonin binding sites in the brains of golden-mantled ground squirrels (Citellus lateralis). Specific binding appeared to be discretely localized to the pars tuberalis region of the pituitary that surrounds the medial basal hypothalamus. The total area of binding was significantly decreased in brains of hibernating squirrels as compared to those of awake, euthermic animals. These findings support a role for melatonin receptors of the pars tuberalis in seasonal behavior.     

Melatonin Receptors are Present in Non-Optic Regions of the Brain of a Deep-Sea Fish Living in the Absence of Solar Light

Pineal melatonin hormonally transduces photoperiod to influence daily and seasonal cycles in most vertebrates (1,  2). Evidence of melatonin receptors throughout the brain of several fish species (3–5), particularly in retinorecipient structures, also indicates a role in visual processing. Despite the absence of solar light many deep-sea organisms show seasonality (6–8). The presence of central melatonin receptors was investigated by quantitative in vitro autoradiography in the deep-sea fish Coryphaenoides (Nematonurus) armatus . Specific, time-dependent, saturable, high affinity and guanine nucleotide sensitive, 2-[¹²⁵I]iodomelatonin binding was found over the mid-brain tegmentum and hindbrain. Competing ligand potency was iodomelatonin > melatonin 5-HT. Although C.(N.) armatus has well developed eyes no 2-[¹²⁵I]iodomelatonin binding occurred in optic tectum, cerebellum or hypothalamus. Thus melatonin involvement in processing of visual information and control of seasonal physiology via hypothalamic areas appears to be absent in this species. The presence of central G-protein coupled receptors indicates a function for melatonin unrelated to solar light.     

Localization and quantification of the dopamine transporter: comparison of [H]WIN 35,428 and [I]RTI-55

Transport into the presynaptic terminal by the dopamine transporter is the primary mechanism for removing dopamine from the synaptic cleft. This transporter is a specific marker for dopamine terminals and is a primary site for CNS actions of cocaine. Several radioligands have been developed for analysis of the dopamine transporter. The ligands vary in affinity and specificity, leading to differences in reported transporter density in brain regions. We compared two of the most commonly used ligands, [³H]WIN 35,428 and [¹²⁵I]RTI-55, analyzing the localization and density of sites in the rat brain using serial sections and quantitative autoradiography. Citalopram at 50 nmol/1 was used to block [¹²⁵I]RTI-55 binding to serotonin transport sites. Transporter density was highest in the striatum and both ligands labeled equivalent numbers of sites, with lateral to medial and anterior to posterior gradients. In most areas the density of sites measured with the two ligands was similar. However, [¹²⁵I]RTI-55 binding was significantly higher than [³H]WIN 35,428 binding in the substantia nigra zona compacta, ventral tegmental area, subthalamic nucleus and a number of other subcortical nuclear groups while [³H]WIN 35,428 binding was higher in lateral striatum and in olfactory tubercle. These differences could reflect different forms of the transporter, perhaps due to post-translational modifications, and they may provide a basis for differential pharmacological regulation of transporter function in discrete brain regions and disease states.     

Seasonal regulation of melatonin receptors in rodent pars tuberalis: correlation with reproductive state

Summary Using quantitative autoradiography, we have studied the relationship between melatonin receptor density in the pars tuberalis (PT) and photoperiodic changes in sexual activity in a photoperiodic (Syrian hamster), and a non photoperiodic (rat) rodent. Syrian hamsters exposed to short photoperiod (SP) for 13 weeks or kept in long photoperiod (LP) with daily melatonin injections for 8 weeks, had both gonadal regression and a significant decrease in 2-¹²⁵I-melatonin binding site density in the PT when compared to controls. In contrast, when the animals were sexually active, photorefractory Syrian hamsters exposed to SP for 27 weeks, or rats kept for 13 weeks in SP, the PT melatonin receptor density was similar to that of control animals kept in LP. These results show clearly that a correlation exists between gonadal status and density of melatonin receptors in the PT and suggest that the PT could be the site where melatonin mediates its effects on seasonal function.     

Seasonal variation of melatonin binding sites in the pars tuberalis of the male mink (Mustela vison)

The seasonal changes of 2-[125I]iodomelatonin binding were studied using quantitative autoradiography in the pars tuberalis (PT) of the mink, a short-day breeder, kept out of doors. Studies were performed at 7 times of the year (July, September, October, January, February, and May), corresponding to different states of responsiveness of the gonadal system to the photoperiod. Melatonin binding was observed in the PT and on the ventral border of the pars distalis. Histological staining revealed that the binding on the border of the pars distalis corresponded to the zona tuberalis, a ventral extension of the PT. The binding was specific and saturable. The density of melatonin binding varied significantly with the time of year. The lowest density of binding was found in July, when animals experienced a long daylength and sexual rest, increased from July to reach a maximum in October, when animals experienced decreasing daylength and the hypothalamo-pituitary activity resumed, then slightly decreased and remained constant from November to May. The saturation study demonstrated that the decrease in melatonin binding density between October and February resulted from a change in the number (Bmax: October 70.6 +/- 4.0 vs February 49.6 +/- 2.8 fmol/mg protein; P < 0.01) but not in the affinity (Kd: October 33.6 +/- 7.1 vs February 20.8 +/- 5.1 pM; P > 0.05) of the binding sites. These results are discussed according to the different phases of mink reproductive cycle and to reported data on the sites of action of melatonin on seasonal reproduction and prolactin secretion.     

Melatonin receptors and signal transduction during development in Siberian hamsters (Phodopus sungorus).

Maternal melatonin communicates daylength information to the fetus in Siberian hamsters. Fetal sensitivity to melatonin declines near birth. In this report, we describe melatonin receptor distribution and a second messenger response to melatonin in Siberian hamsters during the perinatal period. The sites of high-affinity 2-[125I]iodomelatonin ([125I]MEL) binding were generally similar throughout the perinatal period. The non-hydrolyzable GTP analog, guanosine-5'-O-(3-thiotriphosphate) (100 microM) inhibited [125I]MEL binding at each age, suggesting the melatonin receptors are associated with guanine nucleotide binding proteins (G proteins). Furthermore, melatonin (10 nM) inhibited forskolin-stimulated cAMP accumulation in median eminence/pars tuberalis (ME/PT) explants as early as 4 days before birth, when sensitivity to melatonin in vivo is high. The cAMP regulatory system appeared disrupted on the day of birth, in that forskolin (10 microM) stimulation of cAMP accumulation was reduced, and melatonin did not inhibit cAMP accumulation stimulated by forskolin. A higher forskolin dose (100 microM) elevated cAMP levels more clearly on the day of birth, and melatonin inhibited forskolin-stimulated cAMP accumulation. These results suggest that the decreased physiological responsiveness to melatonin at the end of gestation may be due to alterations in the cAMP regulatory system.     

Differences in hypothalamic 2-[I]iodomelatonin binding in photoresponsive and non-photoresponsive white-footed mice, Peromyscus leucopus

Photoperiod is an environmental cue used by many temperate-zone species to regulate their reproductive timing. Within species, the degree of reproductive photoresponsiveness can vary widely both among and within populations. The neuroendocrine mechanisms causing this individual variation in photoresponsiveness are unknown. Using selected lines from a population of white-footed mice known to vary genetically in reproductive photoresponsiveness, we tested the hypothesis that variation in the number and/or location of melatonin receptors is the basis for individual differences in reproductive photoresponsiveness. The brains and pars tuberalis of the pituitary from sixteen mice, (eight mice from each of two lines selected for two generations to respond strongly or weakly to photoperiod), were processed for autoradiography using the radioligand 2-[¹²⁵I]-iodomelatonin (IMEL). We found significantly higher specific IMEL binding in the medial preoptic area and the bed nucleus of the stria terminalis of non-responsive mice than responsive mice. There were no differences between groups in specific IMEL binding in the suprachiasmatic and dorsomedial nuclei of the hypothalamus, pars tuberalis, or paraventricular nucleus of the thalamus. These results provide support for the hypothesis that individual variation in photoresponsiveness is due in part to differences in the density or affinity of melatonin receptors.     

Galanin Receptor Binding Sites in Adult Rat Spinal Cord Respond Differentially to Neonatal Capsaicin, Dorsal Rhizotomy and Peripheral Axotomy

The discrete distribution and possible changes in specific [¹²⁵I]galanin binding sites were evaluated in the rat spinal cord following neonatal capsaicin treatment, dorsal rhizotomy and sciatic nerve section. The highest density of [¹²⁵I]galanin binding sites in the normal rat spinal cord was particularly evident in the superficial layers of the dorsal horn whereas moderate to low amounts of labelling were associated with the deeper dorsal horn, areas around the central canal and the ventral horn. Capsaicin-treated rats, compared to littermate controls, showed a significant bilateral increase in [¹²⁵I]galanin binding in the superficial laminae of the dorsal horn. Similarly, unilateral dorsal rhizotomy evoked a significant increase in the density of [¹²⁵I]galanin binding sites in the superficial dorsal horn ipsilateral to surgery. Section of the sciatic nerve, on the other hand, induced a significant depletion in [¹²⁵I]galanin binding in laminae I and II of the ipsilateral dorsal horn. These results, in parallel to those reported for galanin immunoreactivity under similar conditions, suggest that [¹²⁵I]galanin binding sites are preferentially located postsynaptically to the primary afferent fibre terminals in the dorsal horn of the spinal cord. Thus it seems that galanin, at the level of the dorsal spinal cord, regulates the processing of nociceptive information by acting on its own class of specific receptors located postsynaptically to primary sensory terminals.     

Loss of muscarinic M4 receptors in hippocampus of Alzheimer patients

We assessed muscarinic M₁, M₂ and M₄ receptor subtypes in the hippocampus of Alzheimer’s and control brains by receptor autoradiography using ligands such as [¹²⁵I]muscarinic toxin-1 ([¹²⁵I]MT-1, M₁ selective), [³H]AFDX-384 (M₂ partially selective) and [¹²⁵I]muscarinic toxin 4 ([¹²⁵I]M₄ toxin-1, M₄ selective). Our results revealed a significant decrease in muscarinic M₄ receptor binding in the dentate gyrus and CA4 regions of brain sections from Alzheimer’s patients compared to controls. No changes in the density of M₁ or M₂ receptor binding were observed. Our findings suggest that, relative to other muscarinic receptor subtypes, the M₄ receptor could be the subtype which is selectively compromised in Alzeheimer’s disease (AD).