Xesús Manoel Abalo

Ontogeny of γ‐aminobutyric acid‐immunoreactive neuronal populations in the forebrain and midbrain of the sea lamprey

Although brain organization in lampreys is of great interest for understanding evolution in vertebrates, knowledge of early development is very scarce. Here, the development of the forebrain and midbrain γ‐aminobutyric acid (GABA)‐ergic systems was studied in embryos, prolarvae, and small larvae of the sea lamprey using an anti‐GABA antibody. Ancillary immunochemical markers, such as proliferating cell nuclear antigen (PCNA), calretinin, and serotonin, as well as general staining methods and semithin sections were used to characterize the territories containing GABA‐immunoreactive (GABAir) neurons. Differentiation of GABAir neurons in the diencephalon begins in late embryos, whereas differentiation in the telencephalon and midbrain was delayed to posthatching stages. In lamprey prolarvae, the GABAir populations appear either as compact GABAir cell groups or as neurons interspersed among GABA‐negative cells. In the telencephalon of prolarvae, a band of cerebrospinal fluid‐contacting (CSF‐c) GABAir neurons (septum) was separated from the major GABAir telencephalic band, the striatum (ganglionic eminence) primordium. The striatal primordium appears to give rise to most GABAir neurons observed in the olfactory bulb and striatum of early larval stages. GABAir populations in the dorsal telencephalon appear later, in 15–30‐mm‐long larvae. In the diencephalon, GABAir neurons appear in embryos, and the larval pattern of GABAir populations is recognizable in prolarvae. A small GABAir cluster consisting mainly of CSF‐c neurons was observed in the caudal preoptic area, and a wide band of scattered CSF‐c GABAir neurons extended from the preoptic region to the caudal infundibular recess. A mammillary GABAir population was also distinguished. Two compact GABAir clusters, one consisting of CSF‐c neurons, were observed in the rostral (ventral) thalamus. In the caudal (dorsal) thalamus, a long band extended throughout the ventral tier. The nucleus of the medial longitudinal fascicle contained an early‐appearing GABAir population. The paracommissural pretectum of prolarvae and larvae contained a large group of non‐CSF‐c GABAir neurons, although it was less compact than those of the thalamus, and a further group was found in the dorsal pretectum. In the midbrain of larvae, several groups of GABAir neurons were observed in the dorsal and ventral tegmentum and in the torus semicircularis. The development of GABAergic populations in the lamprey forebrain was similar to that observed in teleosts and in mouse, suggesting that GABA is a very useful marker for understanding evolution of forebrain regions. The possible relation between early GABAergic cell groups and the regions of the prosomeric map of the lamprey forebrain (Pombal and Puelles [ 1999 ] J. Comp. Neurol. 414:391–422) is discussed in view of these results and information obtained with ancillary markers. J. Comp. Neurol. 446:360–376, 2002.

Early development of the retina and pineal complex in the sea lamprey: Comparative immunocytochemical study

Lampreys have a complex life cycle, with largely differentiated larval and adult periods. Despite the considerable interest of lampreys for understanding vertebrate evolution, knowledge of the early development of their eye and pineal complex is very scarce. Here, the early immunocytochemical organization of the pineal complex and retina of the sea lamprey was studied by use of antibodies against proliferating cell nuclear antigen (PCNA), opsin, serotonin, and γ‐aminobutyric acid (GABA). Cell differentiation in the retina, pineal organ, and habenula begins in prolarvae, as shown by the appearance of PCNA‐negative cells, whereas differentiation of the parapineal vesicle was delayed until the larval period. In medium‐sized to large larvae, PCNA‐immunoreactive (‐ir) cells were numerous in regions of the lateral retina near the differentiated part of the larval retina (central retina). A late‐proliferating region was observed in the right habenula. Opsin immunoreactivity appears in the pineal vesicle of early prolarvae and 3 or 4 days later in the retina. In the parapineal organ, opsin immunoreactivity was observed only in large larvae. In the pineal organ, serotonin immunoreactivity was first observed in late prolarvae in photoreceptive (photoneuroendocrine) cells, whereas only a few of these cells appeared in the parapineal organ of large larvae. No serotonin immunoreactivity was observed in the larval retina. GABA immunoreactivity appeared earlier in the retina than in the pineal complex. No GABA‐ir perikaryon was observed in the retina of larval lampreys, although a few GABA‐ir centrifugal fibers innervate the inner retina in late prolarvae. First GABA‐ir ganglion cells occur in the pineal organ of 15–17 mm larvae, and their number increases during the larval period. The only GABA‐ir structures observed in the parapineal ganglion of larvae were afferent fibers, which appeared rather late in development. The time sequence of development in these photoreceptive structures is rather different from that observed in teleosts and other vertebrates. This suggests that the unusual development of the three photoreceptive organs in lampreys reflects specialization for their different functions during the larval and adult periods. J. Comp. Neurol. 442:250–265, 2002.

Presence of glutamate, glycine, and γ‐aminobutyric acid in the retina of the larval sea lamprey: Comparative immunohistochemical study of classical neurotransmitters in larval and postmetamorphic retinas

The neurochemistry of the retina of the larval and postmetamorphic sea lamprey was studied via immunocytochemistry using antibodies directed against the major candidate neurotransmitters [glutamate, glycine, γ‐aminobutyric acid (GABA), aspartate, dopamine, serotonin] and the neurotransmitter‐synthesizing enzyme tyrosine hydroxylase. Immunoreactivity to rod opsin and calretinin was also used to distinguish some retinal cells. Two retinal regions are present in larvae: the central retina, with opsin‐immunoreactive photoreceptors, and the lateral retina, which lacks photoreceptors and is mainly neuroblastic. We observed calretinin‐immunostained ganglion cells in both retinal regions; immunolabeled bipolar cells were detected in the central retina only. Glutamate immunoreactivity was present in photoreceptors, ganglion cells, and bipolar cells. Faint to moderate glycine immunostaining was observed in photoreceptors and some cells of the ganglion cell/inner plexiform layer. No GABA‐immunolabeled perikarya were observed. GABA‐immunoreactive centrifugal fibers were present in the central and lateral retina. These centrifugal fibers contacted glutamate‐immunostained ganglion cells. No aspartate, serotonin, dopamine, or TH immunoreactivity was observed in larvae, whereas these molecules, as well as GABA, glycine, and glutamate, were detected in neurons of the retina of recently transformed lamprey. Immunoreactivity to GABA was observed in outer horizontal cells, some bipolar cells, and numerous amacrine cells, whereas immunoreactivity to glycine was found in amacrine cells and interplexiform cells. Dopamine and serotonin immunoreactivity was found in scattered amacrine cells. Amacrine and horizontal cells did not express classical neurotransmitters (with the possible exception of glycine) during larval life, so transmitter‐expressing cells of the larval retina appear to participate only in the vertical processing pathway. J. Comp. Neurol. 499:810–827, 2006.

Calbindin and calretinin immunoreactivity in the retina of adult and larval sea lamprey.

The presence of calretinin and calbindin immunoreactivity is studied in the retina of larval and adult lamprey and their respective distributions are compared. Calretinin distribution is also studied in the retina of transforming stages. Western blot analysis in brain extracts showed a 29-kDa band with both polyclonal anti-calbindin and anti-calretinin antibodies. Calbindin and calretinin immunoreactivity has shown a partially different distribution. In the adult retina large and small bipolar cells, with respectively stratified or diffuse axons, the inner row of horizontal cells and ganglion cells and/or some amacrine cells were labeled with anti-calretinin antibody. The anti-calbindin antibody labels the same cell types except most of ganglion cells, but the label was less conspicuous. Therefore, the possible existence of these two calcium-binding proteins in the central nervous system of the sea lamprey could be discussed. In the differentiated central retina of larval lampreys, numerous calretinin immunoreactive bipolar and ganglion cells were observed, while, in the lateral retina, only ganglion cells were labeled, accordingly with the lack of differentiation of other neural cell types. CR-ir bipolar cells appeared in the retina by the stage 5 of transformation, i.e. about the time when differentiation of photoreceptors occurs. The comparison of the distribution of calretinin and calbindin between adult and larval central retina of lampreys shows striking differences that could be related to the different functionality of eyes in these two stages of the life cycle of lampreys. In addition, this is the first report on the presence of calcium-binding proteins in the larval and transforming lamprey retina, on the presence of calretinin- and calbindin-immunoreactive horizontal cells in adult lamprey retinas and on the differential stratification of bipolar cell terminals.

Development of the serotonergic system in the central nervous system of the sea lamprey.

Lampreys belong to the most primitive extant group of vertebrates, the Agnathans, which is considered the sister group of jawed vertebrates. Accordingly, characterization of neuronal groups and their development appears useful for understanding early evolution of the nervous system in vertebrates. Here, the development of the serotonergic system in the central nervous system of the sea lamprey, Petromyzon marinus, was investigated by immunohistochemical analysis of specimens ranging from embryos to adults. The different serotonin-immunoreactive (5-HT-ir) neuronal populations that are found in adults were observed between the embryonic and metamorphic stages. The earliest serotonergic neurons were observed in the basal plate of the isthmus region of late embryos. In prolarvae, progressive appearance of new serotonergic cell groups was observed: firstly in the spinal cord, then in the pineal organ, tuberal region, zona limitans intrathalamica, rostral isthmus, and the caudal part of the rhombencephalon. In early larvae a new group of serotonergic cells was observed in the mammillary region, whereas in the pretectal region and the parapineal organ the first serotonergic cells were seen in the middle and late larval stages, respectively. The first serotonergic fibres appeared in early prolarvae, with fibres that ascend and descend from the isthmic cell group, and the number of immunoreactive fibres increased progressively until the adult stage. The results reveal strong resemblances between lampreys and other vertebrates in the spatio-temporal pattern of development of brainstem populations. This study also reveals a shared pattern of early ascending and descending serotonergic pathways in lampreys and jawed vertebrates.

Development of the dopamine-immunoreactive system in the central nervous system of the sea lamprey.

The development of dopamine-immunoreactive (DAir) populations in the central nervous system of the sea lamprey, a modern representative of the earliest vertebrates, was studied to achieve further understanding of dopaminergic systems in vertebrates. The first DAir cell groups appeared in the spinal cord, the posterior tubercle nucleus and the dorsal hypothalamic nucleus of prolarval stages. In larvae, new DAir cell groups were observed in the caudal preoptic nucleus, the postoptic commissure nucleus, the postinfundibular commissure nucleus and the caudal rhombencephalon. All these DAir cell groups observed in larvae were also DA-positive in adults, which showed only one new DAir cell group found in the ventral hypothalamic nucleus. Occasional DAir cells were observed in the telencephalon, the ventral thalamus and/or the isthmus of large larvae and adults. From medium-sized larvae to adults, the regions most richly innervated by DAir fibers were the neurohypophysis, the striatum, the pretectum and the midbrain tegmentum. Striking differences are observed between lampreys and other vertebrates in regard to the relative time of appearance of DAir cells, which is probably related with the complex life cycle of the sea lamprey.

Cell Proliferation in the Forebrain and Midbrain of the Sea Lamprey

Cell proliferation in the forebrain and midbrain of the sea lamprey (Petromyzon marinus L.) was investigated by proliferation cell nuclear antigen (PCNA) immunocytochemistry, with BrdU labeling as a complementary technique. Correspondence between proliferation regions and areas of early neuronal differentiation was also assessed using antibodies against HNK‐1 early differentiation marker. The brain of late embryos shows a homogeneously thick ventricular zone (VZ) containing PCNA‐immunoreactive (PCNA‐ir) nuclei. In early prolarvae, several discontinuities formed by PCNA‐negative cells, and differences among regions in VZ thickness, become apparent. In late prolarvae and early larvae, these differences in VZ thickness and appearance, as well as the presence of PCNA‐negative discontinuities, allowed us to correlate proliferation domains and neuroanatomical regions. In larvae, the number of PCNA‐ir cells in the VZs diminish gradually, although a few PCNA‐ir cells are present in the ependyma of most regions. In late larvae, proliferation becomes confined to a few ventricular areas (medial pallium, caudal habenula, ventral preoptic recess near the optic nerve, and tuberal portion of the posterior hypothalamic recess). During metamorphosis there appears to be no proliferation, but in upstream adults a few PCNA‐ir cells are observed in the most caudal habenula. The characteristics of the proliferative regions revealed in lamprey with PCNA immunocytochemistry show notable differences from those observed in other vertebrates, and these differences may be related to the peculiar life cycle of lampreys. J. Comp. Neurol. 494:986–1006, 2006.

Choline acetyltransferase-immunoreactive neurons in the retina of adult and developing lampreys.

The presence of choline acetyltransferase-immunoreactive (ChATir) amacrine cells is reported for the first time in the retinas of three species of lamprey (Lampetra fluviatilis, Ichthyomyzon unicuspis, and Petromyzon marinus). In the three species, the ChATir cells were mainly distributed in the inner plexiform layer (IPL), which in lampreys extends from the inner nuclear layer (INL) to the inner limiting membrane. These cells had a bipolar, triangular or stellate appearance, and gave rise to processes coursing in the inner plexiform layer. In transforming lampreys, ChATir processes formed two asymmetrical inner and outer subplexuses in the inner plexiform layer, which is reminiscent of the distribution of processes of ChATir cells in the On and Off sublaminae reported in jawed vertebrates. The larval retina lacked ChAT immunoreactivity, and ChATir cells and processes appeared at early metamorphosis throughout the retina, exhibiting in late transforming stages an organization similar to that of adults. This first report of ChATir cells in the lamprey retina indicates that the appearance of cholinergic circuits in the retina of vertebrates occurred before the divergence of the agnathan and gnathostome lines.

Late proliferation and photoreceptor differentiation in the transforming lamprey retina

Lamprey eyes exhibit dual retinal development, with highly different larval and adult phases. Here, cell proliferation and photoreceptor differentiation was investigated in late larvae and during transformation (occurring several years after egg hatching) by using immunohistochemistry against the proliferating cell nuclear antigen (PCNA) and opsins. In large larvae proliferating cells are mainly located in the lateral retina, a wide undifferentiated region, whereas opsin immunoreactivity revealed only a single type of photoreceptors in the very small central retina. In premetamorphic larvae, retinal cell proliferation increases considerably, but at metamorphosis it becomes progressively restricted to the periphery of the lateral retina. Proliferating (PCNA-immunoreactive) cells were mainly observed in the inner nuclear layer but also in the outer plexiform layer and outer nuclear layer, suggesting that the latter proliferating cells migrate to the outer nuclear layer and differentiate into photoreceptors. In the lateral retina, first photoreceptors expressing opsins were observed at middle metamorphic stages, and outer and inner segments were present at latter stages. Some immature photoreceptors were also observed in postmetamorphic retina. Unlike teleost and amphibian retinas, no proliferating cells were observed in the retina after metamorphosis, indicating that the retinal growth after this period is due to cellular reorganization and increase in cell size.

Proliferating cell nuclear antigen (PCNA) immunoreactivity and development of the pineal complex and habenula of the sea lamprey

The development of the pineal complex and the habenula of the sea lamprey was studied with proliferating cell nuclear antigen (PCNA) immunocytochemistry. The pineal organ and the habenula primordia appeared in late embryos, and neuron differentiation began in prolarvae, as indicated by the presence of PCNA-negative cells. The parapineal primordium could not be distinguished until early prolarval stages, and cell differentiation was delayed to the larval period. Although the number of cycling (PCNA-immunoreactive) cells gradually decreased during the larval period in the three organs studied, their patterns of differentiation were different. We conclude that the unusual developmental pattern observed is related with the complex life cycle of lampreys.