Linda E. Muske

Evolution of gonadotropin-releasing hormone (GnRH) neuronal systems.

The neuropeptide gonadotropin-releasing hormone (GnRH, LHRH) serves as both a hormone and a neurotransmitter, and it has multiple actions on reproductive physiology and behavior. At least seven different molecular forms of GnRH have evolved, and nearly all vertebrates studied express at least two different forms of GnRH: chicken GnRH II, and a second form that varies across classes. The GnRH cell bodies span a broad region of the forebrain and midbrain, and processes project to virtually every region of the CNS, to the vasculature, and to the cerebrospinal fluid. Comparative evidence supports the model that gnathostomic vertebrates possess two principle GnRH systems, with different embryonic and, probably, evolutionary origins, expressing different molecular forms of GnRH and projecting to different targets. The terminal nerve-septo-preoptic system serves as the principle regulator of gonadotropin release in most vertebrates. Neurons originate in the embryonic olfactory placode and migrate centrally during development, and it is proposed that ontogeny of the TN-septo-preoptic system reflects its evolutionary origins as a peripheral endocrine organ associated with the olfactory system. The second GnRH system, which arises from non-placodal precursors, comprises cell bodies in periventricular regions of the posterior diencephalon and/or midbrain. Although much less is known about the posterior GnRH system, evidence suggests that these cells served as the ancestral brain GnRH system and are the cellular locus of the chicken GnRH peptide. The GnRH system of lampreys does not appear to be homologous to either the TN-septo-preoptic or posterior GnRH system, and it is suggested that GnRH in agnathans represents a third evolutionary event.

Multiple embryonic origins of gonadotropin-releasing hormone (GnRH) immunoreactive neurons

Experiments were conducted to test the hypothesis that gonadotropin-releasing hormone immunoreactive (GnRH-ir) and FMRFamide-ir neurons present in the brain and nervus terminalis originate in the embryonic olfactory placode. The olfactory placodes were bilaterally extirpated in stage 26 or stage 29 embryos of the axolotl, Ambystoma mexicanum, which were then reared for 4-8 months before they were examined immunohistochemically. In experimental subjects with bilateral loss of olfactory epithelia, nerves and bulbs, there was complete absence of GnRH- and FMRFamide-ir neurons in the terminal nerve, and in septal and preoptic areas, and complete absence of large diameter peptidergic fibers associated with the TN-septo-preoptic system. However, GnRH-ir perikarya in the posterior tubercle, and FMRFamide-ir perikarya in the ventral hypothalamus, and small diameter peptidergic fibers were not affected by placodal ablation. These results support the hypothesis that contrary to recent reports, GnRH-ir neurons have more than one embryonic origin. Region-specific patterns of staining with antisera directed against different molecular forms of GnRH support the interpretation that GnRH-ir neurons of placodal origin express mammalian GnRH, whereas GnRH-ir neurons of non-placodal origin, in the posterior tubercle, express chicken GnRH II.

The Nervus terminalis in Amphibians: Anatomy, Chemistry and Relationship with the Hypothalamic Gonadotropin-Releasing Hormone System

The nervus terminalis (TN), a component of the olfactory system, is found in most vertebrates. The TN of some fishes and mammals contains neurons immunoreactive (ir) to gonadotropin-releasing hormone (LHRH), and to several other neuropeptides and neurotransmitter systems, but there is little information on TN chemistry in other vertebrate taxa. Using immunocytochemical techniques, we found LHRH-ir neurons in amphibian TNs. In anurans, but not in a urodele, the TN was also found to contain Phe-Met-Arg-Phe-NH2 (FMRFamide) immunoreactivity. LHRH-ir neurons of the TN and those of the septal-hypothalamic system are morphologically homogeneous and form a distinct anatomical continuum in amphibians. Based upon topographical and cytological criteria, we hypothesize that LHRH-ir systems in vertebrates might derive embryonically from the TN.

Ontogeny of immunoreactive gonadotropin-releasing hormone neuronal systems in amphibians

The ontogeny of gonadotropin-releasing hormone (GnRH) systems was investigated in 3 anuran amphibians (genus Rana) by means of immunocytochemical (ICC) techniques and antibodies generated against 3 different forms of GnRH. Antisera that recognize primarily chicken II and mammalian GnRHs revealed two anatomically and developmentally distinct GnRH systems. One system, referred to here as the forebrain-spinal cord system, contained GnRH immunoreactive (ir) fibers extending from the rostral diencephalon through the ventromedial brainstem to the spinal cord. Intensity of labeling was robust in the youngest, premetamorphic tadpoles, but decreased with age. GnRH immunolabeling in the hypothalamic-pituitary tract was not detected until late prometamorphosis and increased with age. Development of GnRHir in the hypothalamic-pituitary tract coincided with first appearance of GnRHir in the terminal nerve in R. catesbeiana, but not in R. cascadae or R. aurora, suggesting species differences. Comparisons of results obtained with antisera to different forms of GnRH support the interpretation that the forebrain-spinal cord system, hitherto undescribed in amphibians, develops first and synthesizes a non-mammalian, chicken II-like GnRH, and that the hypothalamic-pituitary system develops later and synthesizes primarily mammalian GnRH. We speculate that the forebrain-spinal cord system may represent a GnRH innervation of frog sympathetic ganglia, and that the two GnRH systems are chemically and embryonically distinct.

Neuroanatomical distribution of vasotocin in a urodele amphibian (Taricha granulosa) revealed by immunohistochemical and in situ hybridization techniques

Immunohistochemical and in situ hybridization techniques were used to investigate the neuroanatomical distribution of arginine vasotocin‐like systems in the roughskin newt (Taricha granulosa). Vasotocin‐like‐immunoreactive neuronal cell bodies were identified that, based on topographical position, most likely, are homologous to groups of vasopressin‐immunoreactive neuronal cell bodies described in mammals, including those in the bed nucleus of the stria terminalis, medial amygdala, basal septal region, magnocellular basal forebrain—including the horizontal limb of the diagonal band of Broca, paraventricular and supraoptic nuclei, suprachiasmatic nucleus, and dorsomedial hypothalamic nucleus. Several additional vasotocin‐like‐immunoreactive cell groups were observed in the forebrain and brainstem regions; these observations are compared with previous studies of vasotocin‐ and vasopressin‐like systems in vertebrates. Arginine vasotocin‐like‐immunoreactive fibers and presumed terminals also were widely distributed with high densities in the basal limbic forebrain, the ventral preoptic and hypothalamic regions, and the brainstem ventromedial tegmentum. Based on in situ hybridization studies with synthetic oligonucleotide probes for vasotocin and the related neuropeptide mesotocin, as well as double‐labeling studies with combined immunohistochemistry and in situ hybridization, we conclude that the vasotocin immunohistochemical procedures used identify vasotocin‐like, but not mesotocin‐like, elements in the brain of T. granulosa. The distribution of arginine vasotocin‐like systems in T. granulosa is greater than the distribution previously reported for any other single vertebrate species; however, it is consistent with an emerging pattern of distribution of vasotocin‐ and vasopressin‐like peptides in vertebrates. Complexity in the vasotocinergic system adds further support to the conclusion that this peptide regulates multiple neurophysiological and neuroendocrinological functions. J. Comp. Neurol. 385:43–70, 1997.

Segregation of FMRF amide-immunoreactive efferent fibers from NPY-immunoreactive amacrine cells in goldfish retina

SummaryImmunocytochemical studies were conducted on goldfish to determine whether a retinal efferent fiber system, immunoreactive to the tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRFamide), might contain instead a substance similar to one of the 36-amino acid pancreatic polypeptides, the C-terminus of which is similar to FMRFamide.Our results demonstrate the presence of two separate peptidergic systems, one containing FMRFamide-like, and the other pancreatic polypeptide-like peptides. Antisera to FMRFamide reveal the efferent fibers, whose axons exit the optic nerve and terminate in layer 1 of the inner plexiform layer, as previously described. Antisera to porcine neuropeptide Y, and to avian and bovine pancreatic polypeptides label a sparse population of putative amacrine cell bodies and a dense fiber plexus in layers 1, 3, and 5 of the inner plexiform layer. Based on intensity of staining, this amacrine cell peptide appears to be most similar to neuropeptide-Y.Radioimmunoassay and immunocytochemical staining of retinas in which the efferent fiber peptide was depleted by optic nerve crush confirm in large part the observation that the two peptide systems are distinct. However, there is some cross-recognition of the FMRFamide-like tissue antigen by pancreatic polypeptide antibodies.Double-label studies with antisera to tyrosine hydroxylase and neuropeptide-Y indicate that the pancreatic polypeptide antigen is not co-localized with catecholamines.

Gonadotropin-releasing hormones in microdissected brain regions of an amphibian: concentration and anatomical distribution of immunoreactive mammalian GnRH and chicken GnRH II

Mammalian and chicken II gonadotropin-releasing hormones (mGnRH, cGnRH II) were extracted from 350 microns diameter punches from brains of a urodele amphibian, Taricha granulosa, and measured by means of radioimmunoassay (RIA) with specific antisera. Measurable quantities of both peptides were found in the lateral pallium, the subpallium (along the course of the nervus terminalis), the preoptic area, habenula, optic tectum, infundibulum, paraventricular organ/posterior tubercle of the caudal diencephalon, medulla, and cerebrospinal fluid. Highest concentrations of both peptides were in the preoptic area and infundibulum, suggesting a role in gonadotropin release. In most extrahypothalamic regions, cGnRH II concentrations exceeded those of mGnRH, suggesting that cGnRH II may function as a neurotransmitter in many sites, perhaps to control reproductive behaviors. Results are largely consistent with immunocytochemical (ICC) analyses, except that RIA revealed small amounts of both peptides not found by ICC in some areas of the brain. Results from this microdissection/RIA study and prior ICC studies in amphibians support the conclusions that GnRH cell bodies in the terminal nerve and preoptic area, which project mainly to the median eminence and habenula, express mGnRH, and that GnRH cell bodies in the caudal diencephalon, which project widely throughout the CNS, express cGnRH II. Comparative data support the view that cGnRH II, and the neural systems in which it is expressed, evolved early in vertebrate phylogeny and have been highly conserved.

Luteinizing Hormone‐Releasing Hormone‐Immunoreactive Neurons in the Amphibian Brain Are Distributed along the Course of the Nervus Terminalis

The nervus terminalis ( T N ) is a system of cell bodies and fibers associated with the olfactory and vomeronasal systems of many vertebrates.’-’ In certain fishesand mammals,’-’ some T N neurons contain gonadotropin-releasing hormone ( L H R H ) immunoreactivity, which suggests a role in reproduction. Furthermore, lesions of T N fibers in olfactory pathways disrupt mating behaviors in goldfish” and hamsters,” two species that use olfactory signals during reproduction. On the basis of these and other observations, Demski and Northcutt” have proposed that the T N may modulate reproductive behaviors by responding to pheromones. Because information on T N chemistry and function has been limited mainly to fishes and mammals, it has been unclear whether LHRH immunoreactivity in the T N is a generalized feature of vertebrates or is a specialization, restricted to those animals that rely on pheromonal signals during mating. Comparative studies of other vertebrates are needed, and for this purpose amphibians can provide useful information. As descendants of the group that gave rise to all land vertebrates, amphibians can provide insights into the evolutionary history of the TN. Extant amphibians comprise both primitive and relatively specialized groups. Despite the diversity in amphibian reproductive patterns, brain organization in all species is similar and is much less complex than that of most other vertebrates. Moreover, it is believed that this comparatively simple organization reflects the primitive vertebrate condition.“ Thus, studies of amphibians can provide insights in the basic organization of more complex neural structures in other vertebrates. In amphibians,’4*’’ as well as in other vertebrates,I6 LHRH injections can activate reproductive behaviors. Apparently, LHRH acts within the brain, independently of the pituitary-gonad axis. Recently, we showed that neurons immunoreactive ( i r ) to LHRH are present in the T N of the newt Taricha granulosu,” confirming similar observations on two other amphibian species.” In the newt, concentrations of irLHRH

Evolution of Gonadotropin-Releasing Hormone (GnRH) Neuronal Systems (Part 2 of 2)

The neuropeptide gonadotropin-releasing hormone (GnRH, LHRH) serves as both a hormone and a neurotransmitter, and it has multiple actions on reproductive physiology and behavior. At least seven different molecular forms of GnRH have evolved, and nearly all vertebrates studied express at least two different forms of GnRH: chicken GnRH II, and a second form that varies across classes. The GnRH cell bodies span a broad region of the forebrain and midbrain, and processes project to virtually every region of the CNS, to the vasculature, and to the cerebrospinal fluid. Comparative evidence supports the model that gnathostomic vertebrates possess two principle GnRH systems, with different embryonic and, probably, evolutionary origins, expressing different molecular forms of GnRH and projecting to different targets. The terminal nerve-septo-preoptic system serves as the principle regulator of gonadotropin release in most vertebrates. Neurons originate in the embryonic olfactory placode and migrate centrally during development, and it is proposed that ontogeny of the TN-septo-preoptic system reflects its evolutionary origins as a peripheral endocrine organ associated with the olfactory system. The second GnRH system, which arises from non-placodal precursors, comprises cell bodies in periventricular regions of the posterior diencephalon and/or midbrain. Although much less is known about the posterior GnRH system, evidence suggests that these cells served as the ancestral brain GnRH system and are the cellular locus of the chicken GnRH peptide. The GnRH system of lampreys does not appear to be homologous to either the TN-septo-preoptic or posterior GnRH system, and it is suggested that GnRH in agnathans represents a third evolutionary event.