Blanca Fernández-López

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. J. Comp. Neurol. 521:522–557, 2013.

Traumatic injury induces changes in the expression of the serotonin 1A receptor in the spinal cord of lampreys.

After spinal cord injury (SCI) in mammals, the loss of serotonin coming from the brainstem reduces the excitability of motor neurons and leads to a compensatory overexpression of serotonin receptors. Despite the key role of the serotonin receptor 1a in the control of locomotion, little attention has been put in the study of this receptor after SCI. In contrast to mammals, lampreys recover locomotion after a complete SCI, so, studies in this specie could help to understand events that lead to recovery of function. Here, we showed that in lampreys there is an acute increase in the expression of the serotonin 1A receptor transcript (5-ht1a) after SCI and a few weeks later expression levels go back to normal rostrally and caudally to the lesion. Overexpression of the 5-ht1a in rostral levels after SCI has not been reported in mammals, suggesting that this could be part of the plastic events that lead to the recovery of function in lampreys. The analysis of changes in 5-ht1a expression by zones (periventricular region and horizontally extended grey matter) showed that they followed the same pattern of changes detected in the spinal cord as a whole, with the exception of the caudal periventricular layer, where no significant differences were observed between control and experimental animals at any time post lesion. This suggests that different molecular signals act on the periventricular cells of the rostral and caudal regions to injury site and thus affecting their response to the injury in terms of expression of the 5-ht1a.

Neuronal release and successful astrocyte uptake of aminoacidergic neurotransmitters after spinal cord injury in lampreys.

In contrast to mammals, the spinal cord of lampreys spontaneously recovers from a complete spinal cord injury (SCI). Understanding the differences between lampreys and mammals in their response to SCI could provide valuable information to propose new therapies. Unique properties of the astrocytes of lampreys probably contribute to the success of spinal cord regeneration. The main aim of our study was to investigate, in the sea lamprey, the release of aminoacidergic neurotransmitters and the subsequent astrocyte uptake of these neurotransmitters during the first week following a complete SCI by detecting glutamate, GABA, glycine, Hu and cytokeratin immunoreactivities. This is the first time that aminoacidergic neurotransmitter release from neurons and the subsequent astrocytic response after SCI are analysed by immunocytochemistry in any vertebrate. Spinal injury caused the immediate loss of glutamate, GABA and glycine immunoreactivities in neurons close to the lesion site (except for the cerebrospinal fluid‐contacting GABA cells). Only after SCI, astrocytes showed glutamate, GABA and glycine immunoreactivity. Treatment with an inhibitor of glutamate transporters (DL‐TBOA) showed that neuronal glutamate was actively transported into astrocytes after SCI. Moreover, after SCI, a massive accumulation of inhibitory neurotransmitters around some reticulospinal axons was observed. Presence of GABA accumulation significantly correlated with a higher survival ability of these neurons. Our data show that, in contrast to mammals, astrocytes of lampreys have a high capacity to actively uptake glutamate after SCI. GABA may play a protective role that could explain the higher regenerative and survival ability of specific descending neurons of lampreys. GLIA 2014;62:1254–1269

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.

Inhibitory descending rhombencephalic projections in larval sea lamprey.

Lampreys are jawless vertebrates, the most basal group of extant vertebrates. This phylogenetic position makes them invaluable models in comparative studies of the vertebrate central nervous system. Lampreys have been used as vertebrate models to study the neuronal circuits underlying locomotion control and axonal regeneration after spinal cord injury. Inhibitory inputs are key elements in the networks controlling locomotor behaviour, but very little is known about the descending inhibitory projections in lampreys. The aim of this study was to investigate the presence of brain-spinal descending inhibitory pathways in larval stages of the sea lamprey Petromyzon marinus by means of tract-tracing with neurobiotin, combined with immunofluorescence triple-labeling methods. Neurobiotin was applied in the rostral spinal cord at the level of the third gill, and inhibitory populations were identified by the use of cocktails of antibodies raised against glycine and GABA. Glycine-immunoreactive (-ir) neurons that project to the spinal cord were observed in three rhombencephalic reticular nuclei: anterior, middle and posterior. Spinal-projecting GABA-ir neurons were observed in the anterior and posterior reticular nuclei. Double glycine-ir/GABA-ir spinal cord-projecting neurons were only observed in the posterior reticular nucleus, and most glycine-ir neurons did not display GABA immunoreactivity. The present results reveal the existence of inhibitory descending projections from brainstem reticular neurons to the spinal cord, which were analyzed in comparative and functional contexts. Further studies should investigate which spinal cord circuits are affected by these descending inhibitory projections.

Spatiotemporal Pattern of Doublecortin Expression in the Retina of the Sea Lamprey

Despite the importance of doublecortin (DCX) for the development of the nervous system, its expression in the retina of most vertebrates is still unknown. The key phylogenetic position of lampreys, together with their complex life cycle, with a long blind larval stage and an active predator adult stage, makes them an interesting model to study retinal development. Here, we studied the spatiotemporal pattern of expression of DCX in the retina of the sea lamprey. In order to characterize the DCX expressing structures, the expression of acetylated α-tubulin (a neuronal marker) and cytokeratins (glial marker) was also analyzed. Tract-tracing methods were used to label ganglion cells. DCX immunoreactivity appeared initially in photoreceptors, ganglion cells and in fibers of the prolarval retina. In larvae smaller than 100 mm, DCX expression was observed in photoreceptors, in cells located in the inner nuclear and inner plexiform layers (IPLs) and in fibers coursing in the nuclear and IPLs, and in the optic nerve (ON). In retinas of premetamorphic and metamorphic larvae, DCX immunoreactivity was also observed in radially oriented cells and fibers and in a layer of cells located in the outer part of the inner neuroblastic layer (INbL) of the lateral retina. Photoreceptors and fibers ending in the outer limitans membrane (OLM) showed DCX expression in adults. Some retinal pigment epithelium cells were also DCX immunoreactive. Immunofluorescence for α-tubulin in premetamorphic larvae showed coexpression in most of the DCX immunoreactive structures. No cells/fibers were found showing DCX and cytokeratins colocalization. The perikaryon of mature ganglion cells is DCX negative. The expression of DCX in sea lamprey retinas suggests that it could play roles in the migration of cells that differentiate in the metamorphosis, in the establishment of connections of ganglion cells and in the development of photoreceptors. Our results also suggest that the radial glia and retinal pigment epithelium cells of lampreys are neurogenic. Comparison of our observations with those reported in gnathostomes reveals similarities and interesting differences probably due to the peculiar development of the sea lamprey retina.

Cloning of the GABAB Receptor Subunits B1 and B2 and their Expression in the Central Nervous System of the Adult Sea Lamprey

In vertebrates, γ-aminobutyric acid (GABA) is the main inhibitory transmitter in the central nervous system (CNS) acting through ionotropic (GABAA) and metabotropic (GABAB) receptors. The GABAB receptor produces a slow inhibition since it activates second messenger systems through the binding and activation of guanine nucleotide-binding proteins [G-protein-coupled receptors (GPCRs)]. Lampreys are a key reference to understand molecular evolution in vertebrates. The importance of the GABAB receptor for the modulation of the circuits controlling locomotion and other behaviors has been shown in pharmacological/physiological studies in lampreys. However, there is no data about the sequence of the GABAB subunits or their expression in the CNS of lampreys. Our aim was to identify the sea lamprey GABAB1 and GABAB2 transcripts and study their expression in the CNS of adults. We cloned two partial sequences corresponding to the GABAB1 and GABAB2 cDNAs of the sea lamprey as confirmed by sequence analysis and comparison with known GABAB sequences of other vertebrates. In phylogenetic analyses, the sea lamprey GABAB sequences clustered together with GABABs sequences of vertebrates and emerged as an outgroup to all gnathostome sequences. We observed a broad and overlapping expression of both transcripts in the entire CNS. Expression was mainly observed in neuronal somas of the periventricular regions including the identified reticulospinal cells. No expression was observed in identifiable fibers. Comparison of our results with those reported in other vertebrates indicates that a broad and overlapping expression of the GABAB subunits in the CNS is a conserved character shared by agnathans and gnathostomes.

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. J. Comp. Neurol. 522:1209–1231, 2014.

Anatomical recovery of the GABAergic system after a complete spinal cord injury in lampreys

ABSTRACT Lampreys recover locomotion spontaneously several weeks after a complete spinal cord injury. Dysfunction of the GABAergic system following SCI has been reported in mammalian models. So, it is of great interest to understand how the GABAergic system of lampreys adapts to the post‐injury situation and how this relates to spontaneous recovery. The spinal cord of lampreys contains 3 populations of GABAergic neurons and most of the GABAergic innervation of the spinal cord comes from these local cells. GABAB receptors are expressed in the spinal cord of lampreys and they play important roles in the control of locomotion. The aims of the present study were to quantify: 1) the changes in the number of GABAergic neurons and innervation of the spinal cord and 2) the changes in the expression of the gabab receptor subunits b1 and b2 in the spinal cord of the sea lamprey after SCI. We performed complete spinal cord transections at the level of the fifth gill of mature larval lampreys and GABA immunohistochemistry or gabab in situ hybridization experiments. Animals were analysed up to 10 weeks post‐lesion (wpl), when behavioural analyses showed that they recovered normal appearing locomotion (stage 6 in the Ayers scale of locomotor recovery). We observed a significant decrease in the number of GABA‐ir cells and fibres 1 h after lesion both rostral and caudal to the lesion site. GABA‐ir cell numbers and innervation were recovered to control levels 1 to 2 wpl. At 1, 4 and 10 wpl the expression of gabab1 and gabab2 transcripts was significantly decreased in the spinal cord compared to control un‐lesioned animals. This is the first study reporting the quantitative long‐term changes in the number of GABAergic cells and fibres and in the expression of gabab receptors in the spinal cord of any vertebrate following a traumatic SCI. Our results show that in lampreys there is a full recovery of the GABAergic neurons and a decrease in the expression of gabab receptors when functional recovery is achieved. HighlightsWe analysed the recovery of the spinal GABAergic system after spinal cord injury in lampreys.There is an initial and acute loss of GABA immunoreactivity.The number of GABAergic cells and fibres is recovered completely between 1 and 2 weeks after the injury.The expression of the GABAB receptor was reduced even when the animals had recovered normal appearing locomotion.

Organization of alpha-transducin immunoreactive system in the brain and retina of larval and young adult Sea Lamprey (Petromyzon marinus), and their relationship with other neural systems

We employed an anti‐transducin antibody (Gαt‐S), in combination with other markers, to characterize the Gαt‐S‐immunoreactive (ir) system in the CNS of the sea lamprey, Petromyzon marinus. Gαt‐S immunoreactivity was observed in some neuronal populations and numerous fibers distributed throughout the brain. Double Gαt‐S‐ and opsin‐ir neurons (putative photoreceptors) are distributed in the hypothalamus (postoptic commissure nucleus, dorsal and ventral hypothalamus) and caudal diencephalon, confirming results of García‐Fernández et al. (Cell and Tissue Research, 288, 267–278, 1997). Singly Gαt‐S‐ir cells were observed in the midbrain and hindbrain, increasing the known populations. Our results reveal for the first time in vertebrates the extensive innervation of many brain regions and the spinal cord by Gαt‐S‐ir fibers. The Gαt‐S innervation of the habenula is very selective, fibers densely innervating the lamprey homologue of the mammalian medial nucleus (Stephenson‐Jones et al., Proceedings of the National Academy of Sciences of the United States of America, 109, E164–E173, 2012), but not the lateral nucleus homologue. The lamprey neurohypophysis was not innervated by Gαt‐S‐ir fibers. We also analyzed by double immunofluorescence the relation of this system with other systems. A dopaminergic marker (TH), serotonin (5‐HT) or GABA do not co‐localize with Gαt‐S‐ir neurons although codistribution of fibers was observed. Codistribution of Gαt‐S‐ir fibers and isolectin‐labeled extrabulbar primary olfactory fibers was observed in the striatum and hypothalamus. Neurobiotin retrograde transport from the spinal cord combined with immunofluorescence revealed spinal‐projecting Gαt‐S‐ir reticular neurons in the caudal hindbrain. Present results in an ancient vertebrate reveal for the first time a collection of brain targets of Gαt‐S‐ir neurons, suggesting they might mediate non‐visual modulation by light in many systems.