Manuel Megías

New and Old Thoughts on the Segmental Organization of the Forebrain in Lampreys

Ten years ago, we published the first detailed prosomeric map of the forebrain in lampreys. In the interim, the prosomeric model has been modified and simplified to better explain numerous data on the expression patterns of regulatory genes, as well as data from chemical, hodological and neuroembryological experiments, mostly in amniote vertebrates. In this report we first review the main historical concepts of lamprey forebrain organization, relating them to either columnar- or segmental-influenced models and explicit or implicit axial references. Next, our previous prosomeric model of the lamprey forebrain is updated, postulating some new hypotheses on the organization of the secondary prosencephalon.

Cellular localization and distribution of dopamine D4 receptors in the rat cerebral cortex and their relationship with the cortical dopaminergic and noradrenergic nerve terminal networks

The role of the dopamine D(4) receptor in cognitive processes and its association with several neuropsychiatric disorders have been related to its preferential localization in the cerebral cortex. In the present work we have studied in detail the regional and cellular localization of the dopamine D(4) receptor immunoreactivity (IR) in the rat cerebral cortex and its relationship to the dopaminergic and noradrenergic nerve terminal networks, since both dopamine and noradrenaline have a high affinity for this receptor. High levels of D(4) IR were found in motor, somatosensory, visual, auditory, temporal association, cingulate, retrosplenial and granular insular cortices, whereas agranular insular, piriform, perirhinal and entorhinal cortices showed low levels. D(4) IR was present in both pyramidal and non-pyramidal like neurons, with the receptor being mainly concentrated to layers II/III. Layer I was observed to be exclusively enriched in D(4) IR branches of apical dendrites. Finally, mismatches were observed between D(4) IR and tyrosine hydroxylase and dopamine beta-hydroxylase IR nerve terminal plexuses, indicating that these receptors may be activated at least in part by dopamine and noradrenaline operating as volume transmission signals. The present findings support a major role of the dopamine D(4) receptor in mediating the transmission of cortical dopamine and noradrenaline nerve terminal plexuses.

Epicardial development in lamprey supports an evolutionary origin of the vertebrate epicardium from an ancestral pronephric external glomerulus

SUMMARY The epicardium is the outer layer of the vertebrate heart. Both the embryonic epicardium and its derived mesenchyme are critical to heart development, contributing to the coronary vasculature and modulating the proliferation of the ventricular myocardium. The embryonic epicardium arises from an extracardiac, originally paired progenitor tissue called the proepicardium, a proliferation of coelomic cells found at the limit between the liver and the sinus venosus. Proepicardial cells attach to and spread over the cardiac surface giving rise to the epicardium. Invertebrate hearts always lack of epicardium, and no hypothesis has been proposed about the origin of this tissue and its proepicardial progenitor in vertebrates. We herein describe the epicardial development in a representative of the most basal living lineage of vertebrates, the agnathan Petromyzon marinus (lamprey). The epicardium in lampreys develops by migration of coelomic cells clustered in a paired structure at the roof of the coelomic cavity, between the pronephros and the gut. Later on, these outgrowths differentiate into the pronephric external glomerulus (PEG), a structure composed of capillary networks, mesangial cells, and podocytes. This observation is consistent with the conclusion that the primordia of the most anterior pair of PEG in agnathans have been retained and transformed into the proepicardium in gnathostomes. Glomerular progenitor cells are highly vasculogenic and probably allowed for the vascularization of a cardiac tube primarily devoid of coronary vessels. This new hypothesis accounts for the striking epicardial expression of Wt1 and Pod1, two transcription factors essential for development of the excretory system.

Calbindin and calretinin immunoreactivities identify different types of neurons in the adult lamprey spinal cord

The central pattern generator for locomotion in vertebrates is composed of different spinal neuronal populations that generate locomotor movement. In the lamprey spinal cord, several classes of interneurons have been identified based on morphologic and physiological criteria and integrated in the spinal cord circuits implicated in the generation of locomotion. However, the lack of histochemical markers for most of the interneurons makes it difficult to study whole populations along the spinal cord. We have investigated the immunoreactivity with antibodies raised against calbindin and calretinin. Several types of neurons could be classified: (1) strongly immunoreactive neurons located dorsomedially, (2) moderately immunoreactive neurons located laterally, (3) small weakly immunoreactive neurons, d) ventromedial neurons, (4) liquor contacting cells, and (5) motoneurons. The ventromedial group of calbindin‐immunoreactive neurons also is immunoreactive for serotonin and, therefore, represents the ventromedial group of dopamine/serotonin spinal neurons. Some of the lateral calbindin‐immunoreactive neurons may be CC‐type cells (cells with caudal‐crossed axons), because they are retrogradely labeled by tracer injections into the contralateral spinal cord. Other well‐characterized cell types, such as sensory dorsal cells, lateral interneurons, descending propriospinal edge cells, and spinobulbar giant interneurons are negative for both calbindin and calretinin. Therefore, calbindin and calretinin are useful markers for the study of cell populations that may be integrated in locomotor circuits. J. Comp. Neurol. 455:72–85, 2003.

Immunocytochemical localization of calretinin in the olfactory system of the adult lamprey, Lampetra fluviatilis

The distribution of calretinin immunoreactive (CR-ir) structures in the adult lamprey (Lampetra fluviatilis) olfactory system was studied by using immunocytochemical techniques. In the olfactory epithelium, a subpopulation of olfactory receptor cells was CR-ir. In the olfactory bulbs, three different cell populations were observed. Large CR-ir cells (mitral cells) were located medially to the olfactory glomeruli and occasionally between them. In the inner cellular layer, two types of CR-ir perikarya were found: numerous small CR-ir cells (granule cells) and some medium-sized CR-ir cells (putative displaced periglomerular cells). In addition, different intensities of CR-ir fibers were present in particular rootlets of the olfactory nerves, as well as in particular glomeruli. The presence of CR-ir cells and fibers in all layers of the lamprey olfactory bulbs supports the idea that this protein is present in pathways underlying the processing of sensory information throughout evolution.

Development and organization of the lamprey telencephalon with special reference to the GABAergic system.

Lampreys, together with hagfishes, represent the sister group of gnathostome vertebrates. There is an increasing interest for comparing the forebrain organization observed in lampreys and gnathostomes to shed light on vertebrate brain evolution. Within the prosencephalon, there is now a general agreement on the major subdivisions of the lamprey diencephalon; however, the organization of the telencephalon, and particularly its pallial subdivisions, is still a matter of controversy. In this study, recent progress on the development and organization of the lamprey telencephalon is reviewed, with particular emphasis on the GABA immunoreactive cell populations trying to understand their putative origin. First, we describe some early general cytoarchitectonic events by searching the classical literature as well as our collection of embryonic and prolarval series of hematoxylin-stained sections. Then, we comment on the cell proliferation activity throughout the larval period, followed by a detailed description of the early events on the development of the telencephalic GABAergic system. In this context, lampreys apparently do not possess the same molecularly distinct subdivisions of the gnathostome basal telencephalon because of the absence of a Nkx2.1-expressing domain in the developing subpallium; a fact that has been related to the absence of a medial ganglionic eminence as well as of its derived nucleus in gnathostomes, the pallidum. Therefore, these data raise interesting questions such as whether or not a different mechanism to specify telencephalic GABAergic neurons exists in lampreys or what are their migration pathways. Finally, we summarize the organization of the adult lamprey telencephalon by analyzing the main proposed conceptions, including the available data on the expression pattern of some developmental regulatory genes which are of importance for building its adult shape.

Development of GABA-immunoreactive cells in the spinal cord of the sea lamprey, P. marinus

The lamprey spinal cord increases in length and size during all its life cycle; thus, it is expected that new cells will be generated. This expectation suggests that the locomotor circuits must be continuously remodeled. Key elements in the cellular network controlling locomotor behavior are inhibitory cells. Here, we studied the γ‐aminobutyric acid–immunoreactive (GABA‐ir) cells in the lamprey spinal cord during postembryonic development. Three major populations of GABA‐ir cells were identified according to their distribution: those located in the gray matter, those contacting the cerebrospinal liquid (LC cells), and those located in the white matter. The results show (1) the number of GABA‐ir cells per segment increase from prolarvae (<10 mm) to adulthood; (2) the lower number of GABA‐ir cells in 100 μm of spinal cord is 66 ± 7, found in premetamorphic larvae, and the highest is 107 ± 6, found in postmetamorphic animals; (3) the gray matter and LC GABA‐ir cells show different variations in number depending on the developmental period. Thus, in the 10‐mm larvae, the gray matter GABA‐ir cells are more abundant than LC cells, whereas in the young postmetamorphic specimens, the contrary occurs. Most of the GABA‐ir cells located in the white matter were classified as edge cells. They increase in number from the beginning of the prolarval period, where there are not white matter–positive cells, to the middle larval period, where there are 9 ± 4 GABA‐ir edge cells per segment. This value was unaltered in later periods, where GABA‐ir edge cells represent 20–30% of the total number of edge cells per segment. The increase in number of GABA‐ir cells in these populations during a specific point of the lamprey life cycle may indicate different inhibitory requirements of the locomotor circuit at different developmental periods. J. Comp. Neurol. 470:151–163, 2004.

Epigenetic regulation of sex ratios may explain natural variation in self-fertilization rates

Self-fertilization (selfing) favours reproductive success when mate availability is low, but renders populations more vulnerable to environmental change by reducing genetic variability. A mixed-breeding strategy (alternating selfing and outcrossing) may allow species to balance these needs, but requires a system for regulating sexual identity. We explored the role of DNA methylation as a regulatory system for sex-ratio modulation in the mixed-mating fish Kryptolebias marmoratus. We found a significant interaction between sexual identity (male or hermaphrodite), temperature and methylation patterns when two selfing lines were exposed to different temperatures during development. We also identified several genes differentially methylated in males and hermaphrodites that represent candidates for the temperature-mediated sex regulation in K. marmoratus. We conclude that an epigenetic mechanism regulated by temperature modulates sexual identity in this selfing species, providing a potentially widespread mechanism by which environmental change may influence selfing rates. We also suggest that K. marmoratus, with naturally inbred populations, represents a good vertebrate model for epigenetic studies.

Distribution of neuropeptide FF-like immunoreactive structures in the lamprey central nervous system and its relation to catecholaminergic neuronal structures

The neuropeptide FF (NPFF) is an octapeptide of the RFamide-related peptides (FaRPs) that was primarily isolated from the bovine brain. Its distribution in the CNS has been reported in several mammalian species, as well as in some amphibians. Therefore, in order to gain insight in the evolution on the expression pattern of this neuropeptide in vertebrates, we carried out an immunohistochemical study in the sea lamprey, Petromyzon marinus. The distribution of NPFF-like-immunoreactive (NPFF-ir) structures in the lamprey brain is, in general, comparable to that previously described in other vertebrate species. In lamprey, most of the NPFF-ir cells were found in the hypothalamus, particularly in two large populations, the bed nucleus of the tract of the postoptic commissure and the tuberomammillary area. Numerous NPFF-ir cells were also observed in the rostral rhombencephalon, including a population in the dorsal isthmic gray and the reticular formation. Additional labeled neurons were found inside the preoptic region, the parapineal vesicle, the periventricular mesencephalic tegmentum, the descending trigeminal tract, the nucleus of the solitary tract, as well as in the gray matter of the spinal cord. The NPFF-ir fibers were widely distributed in the brain and the spinal cord, being, in general, more concentrated throughout the basal plate. The presence of NPFF-ir fibers in the lamprey neurohypophysis suggests that the involvement of NPFF-like substances in the hypothalamo-hypophyseal system had emerged early during evolution.

Distribution of a Y1 receptor mRNA in the brain of two lamprey species, the sea lamprey (Petromyzon marinus) and the river lamprey (Lampetra fluviatilis)

The neuropeptide Y system consists of several neuropeptides acting through a broad number of receptor subtypes, the NPY family of receptors. NPY receptors are divided into three subfamilies (Y1, Y2, and Y5) that display a complex evolutionary history due to local and large‐scale gene duplication events and gene losses. Lampreys emerged from a basal branch of the tree of vertebrates and they are in a key position to shed light on the evolutionary history of the NPY system. One member of the Y1 subfamily has been reported in agnathans, but the phylogenetic tree of the Y1 subfamily is not yet clear. We cloned the sequences of the Y1‐subtype receptor of Petromyzon marinus and Lampetra fluviatilis to study the expression pattern of this receptor in lampreys by in situ hybridization and to analyze the phylogeny of the Y1‐subfamily receptors in vertebrates. The phylogenetic study showed that the Y1 receptor of lampreys is basal to the Y1/6 branch of the Y1‐subfamily receptors. In situ hybridization showed that the Y1 receptor is widely expressed throughout the brain of lampreys, with some regions showing numerous positive neurons, as well as the presence of numerous cerebrospinal fluid–contacting cells in the spinal cord. This broad distribution of the lamprey Y1 receptor is more similar to that found in other vertebrates for the Y1 receptor than that of the other members of the Y1 subfamily: Y4, Y8, and Y6 receptors. Both phylogenetic relationship and expression pattern suggest that this receptor is a Y1 receptor. J. Comp. Neurol. 521:426–447, 2013.