Molecular mechanisms controlling the migration of striatal interneurons.

In the developing telencephalon, the medial ganglionic eminence (MGE) generates many cortical and virtually all striatal interneurons. While the molecular mechanisms controlling the migration of interneurons to the cortex have been extensively studied, very little is known about the nature of the signals that guide interneurons to the striatum. Here we report that the allocation of MGE-derived interneurons in the developing striatum of the mouse relies on a combination of chemoattractive and chemorepulsive activities. Specifically, interneurons migrate toward the striatum in response to Nrg1/ErbB4 chemoattraction, and avoid migrating into the adjacent cortical territories by a repulsive activity mediated by EphB/ephrinB signaling. Our results also suggest that the responsiveness of MGE-derived striatal interneurons to these cues is at least in part controlled by the postmitotic activity of the transcription factor Nkx2-1. This study therefore reveals parallel mechanisms for the migration of MGE-derived interneurons to the striatum and the cerebral cortex.

Aspartate-containing neurons of the brainstem and rostral spinal cord of the sea lamprey Petromyzon marinus: distribution and comparison with γ-aminobutyric acid.

The amino acid L-aspartate (ASP) is one of the most abundant excitatory neurotransmitters in the mammalian brain, but its distribution in other vertebrates has not yet been well characterized. We investigated the distribution of ASP in the brainstem and rostral spinal cord of the adult sea lamprey by using ASP immunohistochemistry. Our results indicate that ASP is accumulated in specific neurons, but not in glia (tanycytes). ASP-immunoreactive neuronal populations were rather similar as the glutamatergic populations reported in the adult sea lamprey (Villar-Cerviño et al. [2013] J Comp Neurol 521:522-557), although some important differences were noted. Characteristically, the largest reticular neurons of the lamprey brainstem (Müller cells) showed ASP immunoreactivity in perikarya and processes, in contrast to the absence or faint glutamate immunoreactivity reported in these perikarya. We also compared the distribution of ASP and γ-aminobutyric acid (GABA) in brainstem neurons by using double immunofluorescence methods. In regions such as the midbrain tectum, dorsal isthmus, and motor nuclei, ASP and GABA immunoreactivity was mostly located in different neurons, whereas in other nuclei (torus semicircularis, octavolateralis area, parvocellular reticular formation), many of the ASP-immunonegative neurons displayed colocalization with GABA. These results, together with those of our previous studies of colocalization of glutamate and GABA, suggest that some lamprey neurons may co-release both excitatory and inhibitory neurotransmitters. Further investigation is needed to elucidate the pathways of uptake and release of ASP by ASP-immunoreactive neurons. Our results indicate that ASP is a neurotransmitter in the central nervous system representative of agnathans, the earliest vertebrate group.

Contact repulsion controls the dispersion and final distribution of Cajal-Retzius cells.

Cajal-Retzius (CR) cells play a fundamental role in the development of the mammalian cerebral cortex. They control the formation of cortical layers by regulating the migration of pyramidal cells through the release of Reelin. The function of CR cells critically depends on their regular distribution throughout the surface of the cortex, but little is known about the events controlling this phenomenon. Using time-lapse video microscopy in vivo and in vitro, we found that movement of CR cells is regulated by repulsive interactions, which leads to their random dispersion throughout the cortical surface. Mathematical modeling reveals that contact repulsion is both necessary and sufficient for this process, which demonstrates that complex neuronal assemblies may emerge during development through stochastic events. At the molecular level, we found that contact repulsion is mediated by Eph/ephrin interactions. Our observations reveal a mechanism that controls the even distribution of neurons in the developing brain.

Glutamatergic neuronal populations in the brainstem of the sea lamprey, Petromyzon marinus: an in situ hybridization and immunocytochemical study.

Glutamate is the major excitatory neurotransmitter in vertebrates, and glutamatergic cells probably represent a majority of neurons in the brain. Physiological studies have demonstrated a wide presence of excitatory (glutamatergic) neurons in lampreys. The present in situ hybridization study with probes for the lamprey vesicular glutamate transporter (VGLUT) provides an anatomical basis for the general distribution and precise localization of glutamatergic neurons in the sea lamprey brainstem. Most glutamatergic neurons were found within the periventricular gray layer throughout the brainstem, with the following regions being of particular interest: the optic tectum, torus semicircularis, isthmus, dorsal and medial nuclei of the octavolateral area, dorsal column nucleus, solitary tract nucleus, motoneurons, and reticular formation. The reticular population revealed a high degree of cellular heterogeneity including small, medium-sized, large, and giant glutamatergic neurons. We also combined glutamate immunohistochemistry with neuronal tract-tracing methods or γ-aminobutyric acid (GABA) immunohistochemistry to better characterize the glutamatergic populations. Injection of Neurobiotin into the spinal cord revealed that retrogradely labeled small and medium-sized cells of some reticulospinal-projecting groups were often glutamate-immunoreactive, mostly in the hindbrain. In contrast, the large and giant glutamatergic reticulospinal perikarya mostly lacked glutamate immunoreactivity. These results indicate that glutamate immunoreactivity did not reveal the entire set of glutamatergic populations. Some spinal-projecting octaval populations lacked both VGLUT and glutamate. As regards GABA and glutamate, their distribution was largely complementary, but colocalization of glutamate and GABA was observed in some small neurons, suggesting that glutamate immunohistochemistry might also detect non-glutamatergic cells or neurons that co-release both GABA and glutamate.

The glutamatergic neurons in the spinal cord of the sea lamprey: an in situ hybridization and immunohistochemical study.

Glutamate is the main excitatory neurotransmitter involved in spinal cord circuits in vertebrates, but in most groups the distribution of glutamatergic spinal neurons is still unknown. Lampreys have been extensively used as a model to investigate the neuronal circuits underlying locomotion. Glutamatergic circuits have been characterized on the basis of the excitatory responses elicited in postsynaptic neurons. However, the presence of glutamatergic neurochemical markers in spinal neurons has not been investigated. In this study, we report for the first time the expression of a vesicular glutamate transporter (VGLUT) in the spinal cord of the sea lamprey. We also study the distribution of glutamate in perikarya and fibers. The largest glutamatergic neurons found were the dorsal cells and caudal giant cells. Two additional VGLUT-positive gray matter populations, one dorsomedial consisting of small cells and another one lateral consisting of small and large cells were observed. Some cerebrospinal fluid-contacting cells also expressed VGLUT. In the white matter, some edge cells and some cells associated with giant axons (Müller and Mauthner axons) and the dorsolateral funiculus expressed VGLUT. Large lateral cells and the cells associated with reticulospinal axons are in a key position to receive descending inputs involved in the control of locomotion. We also compared the distribution of glutamate immunoreactivity with that of γ-aminobutyric acid (GABA) and glycine. Colocalization of glutamate and GABA or glycine was observed in some small spinal cells. These results confirm the glutamatergic nature of various neuronal populations, and reveal new small-celled glutamatergic populations, predicting that some glutamatergic neurons would exert complex actions on postsynaptic neurons.

Glutamatergic neuronal populations in the forebrain of the sea lamprey, Petromyzon marinus: an in situ hybridization and immunocytochemical study.

Despite the importance of glutamate as a major excitatory neurotransmitter in the brain, the distribution of glutamatergic populations in the brain of most vertebrates is still unknown. Here, we studied for the first time the distribution of glutamatergic neurons in the forebrain of the sea lamprey (Petromyzon marinus), belonging to the most ancient group of vertebrates (agnathans). For this, we used in situ hybridization with probes for a lamprey vesicular glutamate transporter (VGLUT) in larvae and immunofluorescence with antiglutamate antibodies in both larvae and adults. We also compared glutamate and γ-aminobutyric acid (GABA) immunoreactivities in sections using double-immunofluorescence methods. VGLUT-expressing neurons were observed in the olfactory bulb, pallium, septum, subhippocampal lobe, preoptic region, thalamic eminence, prethalamus, thalamus, epithalamus, pretectum, hypothalamus, posterior tubercle, and nucleus of the medial longitudinal fascicle. Comparison of VGLUT signal and glutamate immunoreactivity in larval forebrain revealed a consistent distribution of positive cells, which were numerous in most regions. Glutamate-immunoreactive cell populations were also found in similar regions of the adult forebrain. These include mitral-like cells of the olfactory bulbs and abundant cells in the lateral pallium, septum, and various diencephalic regions, mainly in the prethalamus, thalamus, habenula, pineal complex, and pretectum. Only a small portion of the glutamate-immunoreactive cells showed colocalization with GABA, which was observed mainly in the olfactory bulb, telencephalon, hypothalamus, ventral thalamus, and pretectum. Comparison with glutamatergic cells observed in rodent forebrains suggests that the regional distribution of glutamatergic cells does not differ greatly in lampreys and mammals.

A vesicular glutamate transporter in lampreys: cDNA cloning and early expression in the nervous system.

Vesicular glutamate transporters (VGLUTs) accumulate glutamate into synaptic vesicles of glutamatergic neurons, and thus are considered to define the phenotype of these neurons. Glutamate also appears to play a role in the development of the nervous system of vertebrates. Here we report the characterization of a vesicular glutamate transporter of lamprey (lVGluT), a novel member of the VGluT gene family. Phylogenetic analysis indicates that lVGLUT cannot be assigned to any of the three VGLUT isoforms characterized in teleosts and mammals, suggesting that these classes may have been fixed after the splitting between cyclostomes and gnathostomes. Expression pattern analysis during lamprey embryogenesis and prolarval stages shows that lVGluT expression is restricted to the nervous system. The first structure to express lVGluT was the olfactory epithelium of late embryos. In the brain of early prolarvae, lVGluT was expressed in most of the neuronal populations that generate the early axonal scaffold. lVGluT expression was also observed in neuronal populations of the rhombencephalon and spinal cord and in ganglia of the branchiomeric, octaval and posterior lateral line nerves. In the rhombencephalon, lVGluT expression appears to be spatially restricted in dorsal and ventral longitudinal domains. Comparison of the early expression of VGluT genes between the lamprey and some anamniotan gnathostomes (frog, zebrafish) reveals a conserved expression pattern, likely to reflect ancestral vertebrate characteristics.

D-serine is distributed in neurons in the brain of the sea lamprey.

The amino acid D-serine is an endogenous coagonist of N-methyl-D-aspartate (NMDA) receptors in mammals that has been shown to play an important role in synaptic function, behavior, learning, and memory. The distribution and cellular location of D-serine in the brain of the sea lamprey was investigated by using immunofluorescence methods. One major finding of our study, unlike early studies of mammals, was the localization of D-serine immunoreactivity in perikarya and dendrites of neurons, whereas D-serine immunoreactivity was not generally observed in the lamprey glia. D-serine-immunoreactive neurons were observed in different brain regions, including the olfactory bulb, medial pallium, thalamus, torus semicircularis, isthmus, and reticular formation. The colocalization of D-serine with gamma-aminobutyric acid (GABA) was also studied with a double-immunofluorescence technique. The relationship between D-serine and glycine immunoreactivities was studied in alternate parallel series of sections stained for either D-serine/GABA or glycine/GABA. Colocalization with GABA was observed in various D-serine-immunoreactive populations, and codistribution and possible colocalization with glycine was also observed in some populations, mainly in the dorsal isthmic gray, medial octavolateral nucleus, dorsal column nucleus, interpeduncular nucleus, and reticular formation. Although numerous fibers were strongly GABA- and glycine-immunoreactive, D-serine immunoreactivity was observed mostly in cell perikarya and dendrites. The present results indicate that the D-serine immunoreactive cells are small to medium-sized neurons, some exhibiting classical inhibitory neurotransmitters, in which D-serine might be acting as a modulator. The neuronal distribution of D-serine and its frequent colocalization and/or codistribution with the two main inhibitory neurotransmitters appeared early in vertebrates.

Dopamine and gamma-aminobutyric acid are colocalized in restricted groups of neurons in the sea lamprey brain: insights into the early evolution of neurotransmitter colocalization in vertebrates.

Since its discovery, the possible corelease of classic neurotransmitters from neurons has received much attention. Colocalization of monoamines and amino acidergic neurotransmitters [mainly glutamate and dopamine (DA) or serotonin] in mammalian neurons has been reported. However, few studies have dealt with the colocalization of DA and gamma-aminobutyric acid (GABA) in neurons. With the aim of providing some insight into the colocalization of neurotransmitters during early vertebrate phylogeny, we studied GABA expression in dopaminergic neurons in the sea lamprey brain by using double-immunofluorescence methods with anti-DA and anti-GABA antibodies. Different degrees of colocalization of DA and GABA were observed in different dopaminergic brain nuclei. A high degree of colocalization (GABA in at least 25% of DA-immunoreactive neurons) was observed in populations of the caudal rhombencephalon, ventral isthmus, postoptic commissure nucleus, preoptic nucleus and in granule-like cells of the olfactory bulb. A new DA-immunoreactive striatal population that showed colocalization with GABA in about a quarter of its neurons was observed. In the periventricular hypothalamus, colocalization was observed in only a few cells, despite the abundance of DA- and GABA-immunoreactive neurons, and no double-labelled cells were observed in the paratubercular nucleus. The frequent colocalization of DA and GABA reveals that the dopaminergic populations of lampreys are more complex than previously reported. Double-labelled fibres or terminals were observed in different brain regions, suggesting possible corelease of DA and GABA by these lamprey neurons. The present results suggest that colocalization of DA and GABA in neurons appeared early in vertebrate evolution.

A monoclonal antibody as a tool to study the subcommissural organ and Reissner's fibre of the sea lamprey: an immunofluorescence study before and after a spinal cord transection.

Lampreys are vertebrate animal models in spinal cord regeneration studies. In order to gain knowledge on the mechanisms that provide to the lamprey spinal cord its capacity of regeneration we decided to compare the expression patterns of the growth-associated protein 43 (GAP-43) in the CNS of the sea lamprey before and after a complete spinal transection by immunocytochemical methods using an anti-GAP-43 antibody. Surprisingly, in the brain/spinal cord of both normal and injured animals, anti-GAP-43-like labeling was only observed in the subcommissural organ (SCO) and Reissner's fibre (RF). In injured larvae, a dotted labeling was also observed in the meninges and in the blood the vessels of the neighbouring tissues at the site of lesion. The experiments in injured animals showed that after complete spinal cord transection the SCO seems to continue to produce the Reissner's substance (RS), which is accumulated at the proximal site of spinal transection. The dotted labeling observed in the neighbouring tissues could correspond to RS that was released from the site of injury. In Western blot experiments done using protein extracts of the lamprey brain, the anti-GAP-43 antibody did not recognize any protein band of the expected GAP-43 molecular weight, indicating that the secreted material is not this protein. An anti-serotonin antibody was also used as a marker of some brain structures. Serotonergic afferent fibres innervated the SCO. Here we show a new tool that can be used as a highly specific marker in further studies of the SCO/RF system of lampreys.

Development of glycine immunoreactivity in the brain of the sea lamprey: comparison with gamma-aminobutyric acid immunoreactivity.

The development of glycine immunoreactivity in the brain of the sea lamprey was studied by use of immunofluorescence techniques at embryonic to larval stages. Glycine distribution was also compared with that of gamma-aminobutyric acid (GABA) by use of double immunofluorescence. The first glycine-immunoreactive (ir) cells appeared in the caudal rhombencephalon of late embryos, diencephalon of early prolarvae, and mesencephalon of late prolarvae, in which glycine-ir cells were observed in several prosencephalic regions (preoptic nucleus, hypothalamus, ventral thalamus, dorsal thalamus, pretectum, and nucleus of the medial longitudinal fascicle), mesencephalon (M5), isthmus, and rhombencephalon. In larvae, glycine-ir populations were observed in the olfactory bulbs, preoptic nucleus and thalamus (prosencephalon), M5 and oculomotor nucleus (mesencephalon), dorsal isthmic gray, isthmic reticular formation, and various alar and basal plate rhombencephalic populations. No glycine-ir cells were observed in the larval optic tectum or torus semicircularis, which contain glycine-ir populations in adults. A wide distribution of glycine-ir fibers was observed, which suggests involvement of glycine in the function of most lamprey brain regions. Colocalization of GABA and glycine in prolarvae was found mainly in cell groups of the diencephalon, in the ventral isthmic group, and in trigeminal populations. In larvae, colocalization of GABA and glycine was principally observed in the M5 nucleus, the reticular formation, and the dorsal column nucleus. The present results reveal for the first time the complex developmental pattern of the glycinergic system in lamprey, including early glycine-ir populations, populations transiently expressing glycine, and late-appearing populations, in relation to maturation changes that occur during metamorphosis.

Descending brain-spinal cord projections in a primitive vertebrate, the lamprey: cerebrospinal fluid-contacting and dopaminergic neurons.

We used Neurobiotin as a retrograde tract tracer in both larval and adult sea lampreys and observed a number of neuronal brainstem populations (mainly reticular and octaval populations and some diencephalic nuclei) that project to the spinal cord, in agreement with the results of previous tracer studies. We also observed small labeled neurons in the ventral hypothalamus, the mammillary region, and the paratubercular nucleus, nuclei that were not reported as spinal projecting. Notably, most of the labeled cells of the mammillary region and some of the ventral hypothalamus were cerebrospinal fluid-contacting (CSF-c) neurons. Combined tract tracing and immunocytochemistry showed that some of the labeled neurons of the mammillary and paratubercular nuclei were dopamine immunoreactive. In addition, some CSF-c cells were labeled in the caudal rhombencephalon and rostral spinal cord, and many were also dopamine immunoreactive. Results with other tracers (biotinylated dextran amines, horseradish peroxidase, and the carbocyanine dye DiI) also demonstrated that the molecular weight or the molecular nature of the tracer was determinant in revealing diencephalic cells with very thin axons. The results show that descending systems afferent to the spinal cord in lampreys are more varied than previously reported, and reveal a descending projection from CSF-c cells, which is unknown in vertebrates. The present results also reveal the existence of large differences between agnathans and gnathostomes in the organization of the dopaminergic cells that project to the spinal cord.

Neurochemical characterization of sea lamprey taste buds and afferent gustatory fibers: presence of serotonin, calretinin, and CGRP immunoreactivity in taste bud bi-ciliated cells of the earliest vertebrates.

Neuroactive substances such as serotonin and other monoamines have been suggested to be involved in the transmission of gustatory signals from taste bud cells to afferent fibers. Lampreys are the earliest vertebrates that possess taste buds, although these differ in structure from taste buds in jawed vertebrates, and their neurochemistry remains unknown. We used immunofluorescence methods with antibodies raised against serotonin, tyrosine hydroxylase (TH), gamma-aminobutyric acid (GABA), glutamate, calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), calretinin, and acetylated alpha-tubulin to characterize the neurochemistry and innervation of taste buds in the sea lamprey, Petromyzon marinus L. For localization of proliferative cells in taste buds we used bromodeoxyuridine labeling and proliferating cell nuclear antigen immunohistochemistry. Results with both markers indicate that proliferating cells are restricted to a few basal cells and that almost all cells in taste buds are nonproliferating. A large number of serotonin-, calretinin-, and CGRP-immunoreactive bi-ciliated cells were revealed in lamprey taste buds. This suggests that serotonin participates in the transmission of gustatory signals and indicates that this substance appeared early on in vertebrate evolution. The basal surface of the bi-ciliated taste bud cells was contacted by tubulin-immunoreactive fibers. Some of the fibers surrounding the taste bud were calretinin immunoreactive. Lamprey taste bud cells or afferent fibers did not exhibit TH, GABA, glutamate, or NPY immunoreactivity, which suggests that expression of these substances evolved in taste buds of some gnathostomes lines after the separation of gnathostomes and lampreys.

Development and organization of the descending serotonergic brainstem-spinal projections in the sea lamprey.

The organization and development of the descending spinal projections from serotonergic rhombencephalic neurons in the larval sea lamprey were investigated by double labeling, tract-tracing methods and immunocytochemistry against serotonin. The results showed that two serotonergic populations of the isthmic and vagal reticular regions present reticulospinal neurons from the beginning of the larval period. Of the three serotonergic subpopulations recognized in the isthmic reticular group [Abalo, X.M., Villar-Cheda, B., Meléndez-Ferro, M., Pérez-Costas, E., Anadón, R., Rodicio, M.C., 2007. Development of the serotonergic system in the central nervous system of the sea lamprey. J. Chem. Neuroanat. 34, 29-46], only two - the medial and ventral subpopulations - project to the spinal cord, with most of the projecting cells in the caudal part of the medial isthmic subpopulation. Occasional cells projecting to the spinal cord were observed in the ventral subpopulation. The vagal reticular serotonergic nucleus situated in the caudal rhombencephalon also presents cells with descending projections. The early development of the brainstem serotonergic projections to the spinal cord appears to be a conserved trait in all vertebrates studied. Although a serotonergic hindbrain-spinal projection system appears to have been present before the divergence of agnathans and gnathostomes, no serotonergic cells were observed in the raphe region in lamprey. Moreover, proportionally more rostral hindbrain serotonergic cells contribute to the spinal serotonergic projections in the sea lamprey than in jawed vertebrates.

Colocalization of dopamine and GABA in spinal cord neurones in the sea lamprey.

In this study, double immunofluorescence methods were used to investigate possible colocalization of the neurotransmitters dopamine [DA] and GABA in rostral spinal cord neurones in the upstream migrating adult sea lamprey (Petromyzon marinus). Double immunofluorescence revealed that all the DA-immunoreactive (ir) cerebrospinal fluid-contacting (CSF-c) cells, approximately 30% of the medioventral DA-ir cells, and most of the DA-ir cells located in the grey lateral to the central canal were also GABA-ir. The results also revealed some DA-ir cells located dorsally to the central canal, which increases the number of dopaminergic cell types known in lamprey. Double-labelled fibres were mainly distributed in the ventral column, and double-labelled boutons contacted some dorsal GABA-ir CSF-c cells, as well as some non-CSF-c GABA-ir cells and ventromedial dendrites of motoneurones. The findings reveal colocalization of dopamine and GABA in some cells and fibres, which suggests co-release of these substances in some synaptic terminals. Although dopaminergic/GABAergic CSF-c cells have been reported in some other vertebrates, the other double-labelled spinal populations appear exclusive to lampreys.

Aspartate immunoreactivity in the telencephalon of the adult sea lamprey: comparison with GABA immunoreactivity.

The excitatory amino acid l-aspartate (Asp) plays a number of roles in neuronal function. We studied the distribution of Asp-immunoreactive (ir) cells in the telencephalon of young and upstream migrating adult sea lamprey, Petromyzon marinus, and compared it with the distribution of gamma-aminobutyric acid (GABA) immunoreactivity, by using double immunofluorescence methods. Our results reveal for the first time the existence of Asp-ir neuronal populations in the lamprey forebrain. In the olfactory bulbs, Asp-ir neurons were observed in the mitral cell layer and in the inner cellular layer. Many granule-like cells were both Asp-ir and GABA-ir. In the pallium, Asp-ir cells were abundant in the lateral pallium and most of them were also GABA-ir. In the septum/terminal lamina nucleus, some cerebrospinal fluid-contacting type (CSF-c) cells were either Asp-ir or GABA-ir, and a few were double-labeled. Some non-CSF-c septal cells were both Asp-ir and GABA-ir. In the striatum, Asp-ir and/or GABA-ir cells were either subependymal or located in the characteristic arched cell row. In the lateral preoptic region, a few small Asp-ir/GABA-ir neurons were observed. In the caudal preoptic recess nucleus, numerous CSF-c cells were Asp-ir and/or GABA-ir. This study also reveals that colocalization of GABA and Asp immunoreactivities in telencephalic neurons is partial. Further investigation is required to establish whether Asp is a neurotransmitter and/or an intermediate in GABA synthesis in lamprey telencephalon.

Neurochemical differentiation of horizontal and amacrine cells during transformation of the sea lamprey retina.

The sea lamprey is a modern representative of the earliest vertebrates (the agnathans) in which development of the eye and retina shows unique patterns. In larval stages the retina is poorly developed, and although a small central region has developed glutamatergic vertical pathways, there is no evidence of chemical differentiation of amacrine and horizontal cells in the central or lateral larval retina [Villar-Cerviño, V., Abalo, X.M., Villar-Cheda, B., Meléndez-Ferro, M., Pérez-Costas, E., Holstein, G.R., Martinelli, G.P., Rodicio, M.C., Anadón, R., 2006. Presence of glutamate, glycine, and gamma-aminobutyric acid in the retina of the larval sea lamprey: comparative immunohistochemical study of classical neurotransmitters in larval and postmetamorphic retinas. J. Comp. Neurol. 499, 810-827.]. However, in adults all the retina was differentiated and both amacrine and horizontal cells are well developed. Present immunocytochemical results show that the horizontal and amacrine cells of the retina begin their neurochemical differentiation during metamorphosis, when they start to express GABA, glycine, serotonin and dopamine; this occurs several years after the onset of development. Immunoreactivity for GABA, glycine and serotonin was found at early metamorphic stages, while expression of the markers of catecholaminergic amacrine cells, dopamine and tyrosine hydroxylase, was found to be delayed until intermediate metamorphic stages. GABA, which is found in some amacrine and horizontal cells of adults, was first observed in amacrine cells during early stages of transformation and then in horizontal cells during middle stages. All cells immunoreactive to serotonin or tyrosine hydroxylase/dopamine were amacrine cells. Interestingly, all these markers began expression before the appearance of opsin-immunoreactive photoreceptors in the lateral retina. The pattern of chemical differentiation of amacrine and horizontal cells was compared with that of other vertebrates and their significance was discussed.

Glycine-immunoreactive neurons in the developing spinal cord of the sea lamprey: comparison with the gamma-aminobutyric acidergic system.

The development and cellular distribution of the inhibitory neurotransmitter glycine in the spinal cord of the sea lamprey were studied by immunocytochemistry and double immunofluorescence and compared with the distribution of gamma-aminobutyric acid (GABA). Results in lamprey embryos and prolarvae reveal that the appearance of glycine-immunoreactive (-ir) spinal neurons precedes that of GABA-ir neurons. Throughout development, glycine-ir cells in the lateral and dorsomedial gray matter of the spinal cord are more numerous than the GABA-ir cells. Only a subset of these neurons shows colocalization of GABA and glycine, suggesting that they are primarily disparate neuronal populations. In contrast, most cerebrospinal fluid (CSF)-contacting neurons of the central canal walls are strongly GABA-ir, and only a portion of them are faintly glycine-ir. Some edge cells (lamprey intraspinal mechanoreceptors) were glycine-ir in larvae and adults. The glycine-ir and GABA-ir neuronal populations observed in the adult spinal cord were similar to those found in larvae. Comparison of glycine-ir and GABA-ir fibers coursing longitudinally in the spinal cord of adult lamprey revealed large differences in diameter between these two types of fiber. Commissural glycine-ir fibers appear in prolarvae and become numerous at larval stages, whereas crossed GABA-ir are scarce. Taken together, results in this primitive vertebrate indicate that the spinal glycinergic cells do not arise by biochemical shift of preexisting GABAergic cells but instead suggest that glycine is present in the earliest circuitry of the developing lamprey spinal cord, where it might act transiently as an excitatory transmitter.

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.