Melatonin receptor mRNA and protein expression in Xenopus laevis nonpigmented ciliary epithelial cells.

Melatonin is an output signal of the circadian clock, and may regulate diurnal rhythms in ocular tissues. A role for melatonin has been suggested in the circadian changes in intraocular pressure (IOP). Changes in IOP may be due partially to changes in the rate of aqueous humor secretion, which is produced by the nonpigmented epithelium of the ciliary body. To examine the mechanism by which melatonin may influence ciliary epithelium function and perhaps the IOP diurnal rhythm, immunocytochemistry with an antibody directed against the Mel(1c) melatonin receptor subtype was performed on sections of Xenopus eyes. Melatonin receptor immunoreactivity was observed in the basolateral regions of the nonpigmented epithelial cells of the ciliary body. Receptor immunoreactivity was also observed in cells of the retina, as has been previously reported. Specific immunoreactivity was not observed in the epithelium of the iris or pigmented ciliary epithelium. In situ hybridization of the Xenopus eye revealed expression of Mel(1c) but not Mel(1b) receptor mRNA in the nonpigmented ciliary epithelium. These results provide evidence that the nonpigmented epithelia of the ciliary body are direct targets for melatonin, and supports previous work that melatonin may influence the rate of aqueous humor secretion by ciliary epithelium, and perhaps the circadian rhythm of IOP.

The prairie vole vomeronasal organ is a target for gonadotropin-releasing hormone.

Gonadotropin-releasing hormone (GnRH) is present in nervus terminalis neurons in chemosensory nerve fascicles in vertebrates. In rodents, the majority of GnRH fibers are located within vomeronasal nerves. We have shown that GnRH can alter vomeronasal receptor neuron responses to odors. In this study, using prairie voles, we tested the hypotheses that (i) GnRH-immunoreactive (-ir) neurons project to the vomeronasal organ and accessory olfactory bulb; (ii) a radioactive-labeled GnRH agonist, buserelin, binds to vomeronasal sensory neurons; and (iii) vomeronasal receptor cells express GnRH receptor mRNA as evidenced by reverse transcription-polymerase chain reaction (RT-PCR) combined with Southern blotting. In neonatal voles, GnRH-ir cell bodies and fibers were observed within the vomeronasal epithelium, vomeronasal nerves and accessory olfactory bulbs. In adult voles, GnRH-ir fibers were observed not only in the lamina propria of the vomeronasal mucosa, but also along vomeronasal nerves and in the accessory olfactory bulb. Binding of [(125)I]buserelin was observed specifically over the vomeronasal sensory epithelium, and RT-PCR/Southern blotting demonstrated GnRH receptor expression in the vomeronasal mucosa, as well as in olfactory epithelium and pterygopalatine ganglion, two additional structures containing GnRH-ir neurons of the nervus terminalis. This study supports the hypothesis that GnRH is released from nervus terminalis fibers to modulate chemosensory processes, especially those involving chemoreception in the vomeronasal organ.

A subpopulation of nervus terminalis neurons projects to the olfactory mucosa in Xenopus laevis.

Biocytin application to the normal or zinc sulfate-treated nasal cavity of Xenopus laevis was used to trace retrogradely neurons associated with the terminal nerve (TN). Immunocytochemistry was conducted to identify the relationship of gonadotropin-releasing hormone-immunoreactive (GnRH-ir) TN neurons with biocytin-labeled neurons. Neurons that accumulated biocytin were located in olfactory nerve fascicles close to the olfactory mucosa lining the caudal, medial, and rostral walls of the principal cavity. GnRH-ir fibers were observed only in the olfactory nerve fascicle projecting to the rostral edge of the principal cavity. In addition, GnRH-ir fibers did not contact biocytin-labeled TN neurons. We hypothesize that these two classes of neurons represent separate components of the TN.

Function of gonadotropin-releasing hormone in olfaction.

Gonadotropin-releasing hormone (GnRH) is present within neurons of the nervus terminalis, the zeroeth cranial nerve. In all vertebrate species, except in sharks where it is a separate nerve, the nervus terminalis consists of a chain of neurons embedded within olfactory or vomeronasal nerves in the nasal cavity. The function of the GnRH component of the nervus terminalis is thought to be neuromodulatory. Our research on GnRH effects on olfaction confirms this hypothesis. The processes of GnRH neural cell bodies located within chemosensory nerves project centrally into the ventral forebrain and peripherally into the lamina propria of the nasal chemosensory mucosa. GnRH receptors are expressed by chemosensory neurons as shown by RT-PCR/Southern blotting and GnRH agonist binding studies. Patch-clamp studies have shown that GnRH alters the responses of isolated chemosensory neurons to natural or electrophysiological stimulation through the modulation of voltage-gated and receptor-gated channels. Behavioral experiments demonstrate that interfering with the nasal GnRH system leads to deficits in mating behavior. These studies suggest that the function of the intranasal GnRH system is to modify olfactory information, perhaps at reproductively auspicious times. We speculate that the purpose of this altered olfactory sense is to make pheromones more detectable and salient.

Multiple cell targets for melatonin action in Xenopus laevis retina: distribution of melatonin receptor immunoreactivity.

In the retina of the African clawed frog (Xenopus laevis), melatonin is synthesized by the photoreceptors at night, and binds to receptors that likely mediate paracrine responses. Melatonin appears to alter the sensitivity of the retinal cells to light, and may play a key role in regulating important circadian events that occur in the eye. A polyclonal antibody was raised against a 13 amino acid peptide corresponding to a region of the third cytoplasmic loop of the Xenopus laevis Mel1c melatonin receptor. Western blot analysis revealed a major immunoreactive band of approximately 60 kD in neural retina and retinal pigment epithelium (RPE) membranes. Immunocytochemical labeling of sections of Xenopus eyes demonstrated intense melatonin receptor-like immunoreactivity in the inner plexiform layer (IPL). Immunolabeling with antibodies to glutamate decarboxylase (GAD) or tyrosine hydroxylase (TOH) appeared to co-localize with the melatonin receptor immunoreactivity in different sublaminas of the IPL. This suggests that both GABAergic and dopaminergic amacrine cells express melatonin receptor protein. There were also some melatonin receptor immunoreactive varicose fibers in the IPL that did not co-localize with either TOH or GAD, and may represent efferent fibers, since they could be followed into the optic nerve. Melatonin receptor immunoreactivity was also present on cell soma in the ganglion cell layer. Furthermore, a moderate level of melatonin receptor immunoreactivity was observed in the RPE and rod and cone photoreceptor cells. The presence of melatonin receptor immunoreactivity in these cells supports previous observations of melatonin receptor RNA expression in multiple cell types in the Xenopus retina. Expression of melatonin receptor protein in the photoreceptors suggests that melatonin may have a direct action on these cells.

Neuromodulatory effects of gonadotropin releasing hormone on olfactory receptor neurons.

The terminal nerve is an anterior cranial nerve that innervates the lamina propria of the chemosensory epithelia of the nasal cavity. The function of the terminal nerve is ambiguous, but it has been suggested to serve a neuromodulatory role. We tested this hypothesis by exposing olfactory receptor neurons from mudpuppies (Necturus maculosus) to a peptide, gonadotropin releasing hormone (GnRH), that is found in cells and fibers of the terminal nerve. We used voltage-clamped whole-cell recordings to examine the effects of 0. 5-50 micrometer GnRH on voltage-activated currents in olfactory receptor neurons from epithelial slices. We found that GnRH increases the magnitude, but does not alter the kinetics, of a tetrodotoxin-sensitive inward current. This increase in magnitude generally begins 5-10 min after initial exposure to GnRH, is sustained for at least 60 min during GnRH exposure, and recovers to baseline within 5 min after GnRH is washed off. This effect occurred in almost 60% of the total number of olfactory receptor neurons examined and appeared to be seasonal: approximately 67% of neurons responded to GnRH during the courtship and mating season, compared with approximately 33% during the summer, when the sexes separate. GnRH also appears to alter an outward current in the same cells. Taken together, these data suggest that GnRH increases the excitability of olfactory receptor neurons and that the terminal nerve functions to modulate the odorant sensitivity of olfactory receptor neurons.

Estrogen regulates gonadotropin-releasing hormone in the nervus terminalis of Xenopus laevis.

The nervus terminalis or terminal nerve (TN) is a neuronal plexus found in the nasal cavity and rostral forebrain of most vertebrates. The hormone gonadotropin-releasing hormone (GnRH) is found in a population of TN neurons as well as hypothalamic neurons which regulate pituitary secretion of the gonadotropins. The GnRH-containing neurons of the TN appear to represent a rostral continuation of the hypothalamic population since they both originate from the olfactory placode and are frequently anatomically continuous. Previous studies have shown that the hypothalamic GnRH neurons are regulated by circulating estrogen levels. Ovariectomy decreases while estrogen administration increases GnRH content in these neurons. It is not known whether the GnRH-containing TN neurons are also regulated in a similar manner. This study demonstrates that ovariectomy and estrogen readministration alters GnRH-immunoreactive (ir) levels in the TN of female Xenopus laevis in a manner similar to that seen in the hypothalamus. One week after ovariectomy, the density of TN GnRH-ir fibers in the olfactory bulb region (one site of TN termination) is significantly decreased. In contrast, a significant increase in GnRH-ir TN fiber density is observed following estrogen readministration to ovariectomized frogs. These findings demonstrate that estrogen regulates GnRH metabolism in neurons of the TN.

Nervus terminalis lesions: II. Enhancement of lordosis induced by tactile stimulation in the hamster.

The involvement of the nervus terminalis in lordosis, induced by manual tactile stimulation, was investigated in the female hamster. Lordosis latencies were measured in response to manual lumbosacral tactile stimulation, which was performed at 5 different delay times after the previous lordotic response. Nervus terminalis lesion (TNX), control forebrain lesion (FBX), and sham (SH) surgeries were performed after preoperative data was collected, and animals were tested again postoperatively. Latencies to lordose were compared separately for each delay time between preoperative and postoperative tests. The TNX group showed small, but significant, decreases in lordosis latencies postoperatively. Lordosis latencies for the SH and FBX groups did not change. These results suggest that the nervus terminalis plays a minor role in mediating sensory processing during reproductive encounters.

Biocytin: a neuronal tracer compatible with rapid decalcification procedures.

The compatibility of neuronal tract-tracing and decalcification procedures was examined in salamander nasal chemosensory systems. Biocytin, but not horseradish peroxidase, retained its labeling capacity following rapid decalcification of the cranial bone. The combination of biocytin tract-tracing and decalcification procedures allows the visualization of labeled neurons and/or their projections within bony regions of intact specimens.

Melatonin receptor distribution in the brain and retina of a lizard, Anolis carolinensis.

Melatonin binding sites were identified in the brain and retina of the lizard Anolis carolinensis using in vitro autoradiography. Radioactive labeling was observed in areas which receive primary, secondary, and tertiary visual input: the superficial layers of the optic tectum, lateral geniculate nucleus, nucleus rotundus, dorsal ventricular ridge, and striatum. Other areas that demonstrated binding included the left medial habenular nucleus, the interpeduncular nucleus, medial cortex, dorsal cortex, mammillary nucleus, and septum. In the retina, melatonin binding was localized in the inner plexiform layer. Radioactive melatonin binding to the optic tectum was reduced in the presence of a nonhydrolyzable cyclic GMP analog, indicating that the melatonin receptor in the brain of this lizard is associated with a G-protein. These results suggest that melatonin receptor binding sites are widely distributed in the forebrain and midbrain of the iguanid lizard, and are prominent in areas of the nervous system that are associated with visual processing. The highest degree of melatonin binding appeared in the left medial habenular nucleus, interpeduncular nucleus, and dorsal ventricular ridge. This suggests that these brain regions may be important targets for the actions of melatonin, such as its effects on circadian rhythmicity, thermoregulation and photoperiodic reproduction.

Gonadotropin-releasing hormone agonist binding in tiger salamander nasal cavity.

Binding of the iodinated gonadotropin-releasing hormone (GnRH) agonist, buserelin, was examined in the nasal cavities of tiger salamanders using in vitro autoradiography. Binding of [125I]buserelin was seen within the chemosensory epithelium of the main nasal cavity and Jacobson's (vomeronasal) organ. Highest levels of binding were observed over the chemosensory neuron dendrites. Given the apparent lack of GnRH-immunoreactive fibers within the chemosensory epithelium as we have observed in a previous study, these observations suggest that GnRH may diffuse from fibers in the lamina propria of the chemosensory mucosa into the sensory epithelium to modulate chemosensory reception.

Peripheral projections of nervus terminalis LHRH-containing neurons in the tiger salamander, Ambystoma tigrinum.

The peripheral projections of the nervus terminalis (NT) have been difficult to examine due to the weak immunoreactivity of the processes to various antibodies. We performed two experimental manipulations in the tiger salamander in an attempt to increase the luteinizing hormone-releasing hormone-immunoreactive (LHRH-ir) labelling in the peripheral processes of the NT:1) the NT was sectioned centrally, or 2) a 100 mg melatonin pellet was embedded subcutaneously for 3 days prior to sacrifice. Following these manipulations, animals were sacrificed and tissue was processed with standard immunocytochemical techniques for the analysis of the distribution of LHRH-ir processes. In the nasal cavity, LHRH-ir fibers were observed projecting 1) into the rostral olfactory epithelium, 2) to Bowman's glands in the lamina propria of the rostromedial olfactory mucosa and ventrolateral mucosa between the main nasal cavity and Jacobson's organ, 3) into the naris constrictor muscle, and 4) along the palatine nerves and ganglia. These lesion and hormone manipulations have enabled the detection of peripheral projections of the NT not observed previously with immunocytochemical procedures alone. The wide distribution of LHRH-ir NT processes in the nasal cavity and cranium suggests that this nerve may influence many different cranial structures during appropriate pheromonal or neuroendocrine events.

Nervus terminalis lesions: I. No effect on pheromonally induced testosterone surges in the male hamster.

The involvement of the nervus terminalis or terminal nerve in the pheromonally induced testosterone surge in the male hamster was investigated. Blood was collected from male hamsters not exposed to odor (baseline), and then a week later from the same hamsters exposed to the odor of vaginal discharge from an estrous female. Terminal nerve lesions, forebrain lesions, or sham surgeries were performed, and blood was collected again with and without odor stimulation. Serum testosterone levels were assessed by radioimmunoassay. None of the surgical procedures interrupted the ability of the male hamsters to demonstrate an increase in serum testosterone following exposure to the odor of an estrous female. We conclude that the terminal nerve is not necessary for this pheromonally mediated neuroendocrine reflex.

Distribution of melatonin receptors in the brain of the frog Rana pipiens as revealed by in vitro autoradiography.

Melatonin binding sites were identified in the brain of the frog Rana pipiens using in vitro autoradiography. Coronal sections were incubated for 1 h in 100 pM 2-125I-iodomelatonin. Specific binding was displaced with 1 microM nonradioactive melatonin. Autoradiographic labeling of 3H-Hyperfilm was observed in areas that receive primary, secondary, and tertiary visual input: the superficial layers of the optic tectum, anterior and posterior thalamic nuclei, striatum, medial pallium, and interpeduncular nucleus. Other areas that demonstrated binding included the medial and lateral septal nuclei, medial preoptic area, suprachiasmatic region, and anterodorsal tegmental nucleus. Binding was also apparent in the distribution of the lateral olfactory tract (lateral pallium), and in tracts associated with visual pathways: optic nerve, chiasm and tract, and lateral and medial forebrain bundles. A high degree of melatonin binding was observed in the left habenular nucleus, but not in the habenulum of the right side of the brain. Radioreceptor binding assays of frog whole-brain homogenate demonstrated specific saturable melatonin binding (Kd = 70 pM, Bmax = 0.80 fmol/mg protein). Melatonin and 6-chloromelatonin were potent displacers of 2-125I-iodomelatonin, while 5-hydroxytryptamine, 5-methoxytryptamine, and N-acetylserotonin were much less potent. Melatonin inhibited the forskolin-stimulated increase in cAMP synthesis in optic tectum explants. These results suggest that high-affinity melatonin receptor binding sites are widely distributed in the telencephalon, diencephalon, and mesencephalon and are very prominent in areas of the frog brain that are associated with visual processing.

Asymmetric distribution of melatonin receptors in the brain of the lizard Anolis carolinensis.

The pineal hormone melatonin may regulate seasonal reproduction and entrainment of circadian rhythms by binding to specific brain receptors. An analysis of melatonin receptor distribution in the lizard brain revealed an asymmetry of melatonin binding in the diencephalon. A high degree of melatonin binding was present in the left habenular nucleus, but no binding was observed in the habenulum of the right brain hemisphere. It is intriguing that the left habenular nucleus, in contrast to the right habenulum, both possesses a high density of melatonin receptors and receives primary photic input from the parietal eye. Similarly, the optic tectum, which receives primary visual input from the retina, is also rich in melatonin receptors. These observations suggest that the left habenulum is under dual control (neuronal and hormonal) of the parietal eye/pineal complex, and that melatonin may play a significant role in neural processing of visual information.

LHRH-immunoreactive neurons in the pterygopalatine ganglia of voles: a component of the nervus terminalis?

Cranial tissue from adult and neonatal voles was examined with immunocytochemical techniques to determine the distribution of luteinizing hormone-releasing hormone-immunoreactive (LHRH-ir) neurons in extracerebral structures. The overall distribution of the LHRH-ir portion of the nervus terminalis in the nasal and extracranial cavities was comparable to that of other rodents. However, we observed a unique association of LHRH-ir neurons with the pterygopalatine ganglia of neonatal and adult voles. We also found LHRH-ir fibers in nasopalatine nerves, and trigeminal nerves and ganglia of neonatal voles. We speculate that these neurons may influence the autonomic control of the vascular pump in the vomeronasal organ.

Localization and quantification of high-affinity melatonin binding sites in Rana pipiens retina.

Melatonin binding was localized to the inner plexiform layer (IPL) of the frog retina by in vitro autoradiography, using 2-125I-melatonin as the radioligand. Radioreceptor binding assays of frog retinal homogenate demonstrated saturable melatonin binding. Scatchard analysis revealed a single population of binding sites with an apparent dissociation constant (Kd) of 125 pM, with a Bmax of 0.138 fmoles/mg of protein. These results suggest that high-affinity melatonin binding sites are present in the IPL of the frog retina, which may reflect the presence of melatonin receptors in this synaptic layer.

The nervus terminalis in the chick: a FMRFamide-immunoreactive and AChE-positive nerve.

The chick terminal nerve (TN) was examined by immunocytochemical and histochemical methods. Molluscan cardioexcitatory peptide-immunoreactive (FMRFamide-ir) and acetylcholinesterase (AChE)-positive TN perikarya and fibers were distributed along olfactory and trigeminal nerves. FMRFamide-ir TN fibers terminated in the olfactory lamina propria and epithelium and in ganglia along the rostroventral nasal septum. This initial description of several populations of avian TN neurons should provide the foundation for future developmental studies of this system.

FMRFamide-immunoreactive retinopetal fibers in the frog, Rana pipiens: demonstration by lesion and immunocytochemical techniques.

Retinopetal fibers, immunoreactive to a molluscan cardioexcitatory-like peptide (FMRFamide-ir), were examined in Rana pipiens with the use of immunocytochemical and lesion techniques. In intact frogs, FMRFamide-ir retinopetal fibers were found in the optic nerve, optic nerve head, and nerve fiber, ganglion cell and inner plexiform layers of the retina. Presumptive monostratified amacrine cells were also labeled. As observed in flat-mounted retinas, the retinopetal fibers radiated from the optic disc toward the peripheral retina, branched many times along their course and were more prevalent in the dorsal retina. Crushing the optic nerve eliminated retinopetal fibers from all regions except the cerebral stump of the optic nerve, indicating that this projection was of central origin. Bilateral prechiasmatic lesions completely eliminated retinopetal fibers from both retinas, indicating that the fibers arose from the rostral forebrain. Within the rostral brain, FMRFamide-ir perikarya were found in olfactory bulb, diagonal band, medial septum, anterior commissure area, and two regions of the posterior preoptic area. Olfactory bulbectomy and midforebrain lesions equally reduced the numbers of these fibers in the retina, implicating the nervus terminalis as a possible source for some of the retinopetal projection. These data will serve as a foundation for future studies on the function of retinopetal fibers in the frog retina.